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Sequestration of Proteins in Stress Granules Relies on the In-Cell but Not the In Vitro Folding Stability
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  • Nirnay Samanta
    Nirnay Samanta
    Institute of Physical and Theoretical Chemistry, TU Braunschweig, Rebenring 56, D-38106 Braunschweig, Germany
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  • Sara S. Ribeiro
    Sara S. Ribeiro
    Institute of Physical and Theoretical Chemistry, TU Braunschweig, Rebenring 56, D-38106 Braunschweig, Germany
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    Orcidhttps://orcid.org/0000-0001-6033-8853
  • Mailin Becker
    Mailin Becker
    Institute of Physical and Theoretical Chemistry, TU Braunschweig, Rebenring 56, D-38106 Braunschweig, Germany
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  • Emeline Laborie
    Emeline Laborie
    CNRS Laboratoire de Biochimie Théorique, Institut de Biologie Physico-Chimique, Université Paris Denis Diderot, Sorbonne Paris Cité, PSL Research University, 13 rue Pierre et Marie Curie, Paris 75005, France
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  • Roland Pollak
    Roland Pollak
    Institute of Physical and Theoretical Chemistry, TU Braunschweig, Rebenring 56, D-38106 Braunschweig, Germany
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  • Stepan Timr*
    Stepan Timr
    CNRS Laboratoire de Biochimie Théorique, Institut de Biologie Physico-Chimique, Université Paris Denis Diderot, Sorbonne Paris Cité, PSL Research University, 13 rue Pierre et Marie Curie, Paris 75005, France
    J. Heyrovský Institute of Physical Chemistry, Czech Academy of Sciences, Dolejskova 2155/3, Prague 8 182 23, Czech Republic
    *Email: [email protected]
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  • Fabio Sterpone*
    Fabio Sterpone
    CNRS Laboratoire de Biochimie Théorique, Institut de Biologie Physico-Chimique, Université Paris Denis Diderot, Sorbonne Paris Cité, PSL Research University, 13 rue Pierre et Marie Curie, Paris 75005, France
    *Email: [email protected]
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    Orcidhttps://orcid.org/0000-0003-0894-8069
  • Simon Ebbinghaus*
    Simon Ebbinghaus
    Institute of Physical and Theoretical Chemistry, TU Braunschweig, Rebenring 56, D-38106 Braunschweig, Germany
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Journal of the American Chemical Society

Cite this: J. Am. Chem. Soc. 2021, 143, 47, 19909–19918
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https://doi.org/10.1021/jacs.1c09589
Published November 17, 2021

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Abstract

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Stress granules (SGs) are among the most studied membraneless organelles that form upon heat stress (HS) to sequester unfolded, misfolded, or aggregated protein, supporting protein quality control (PQC) clearance. The folding states that are primarily associated with SGs, as well as the function of the phase separated environment in adjusting the energy landscapes, remain unknown. Here, we investigate the association of superoxide dismutase 1 (SOD1) proteins with different folding stabilities and aggregation propensities with condensates in cells, in vitro and by simulation. We find that irrespective of aggregation the folding stability determines the association of SOD1 with SGs in cells. In vitro and in silico experiments however suggest that the increased flexibility of the unfolded state constitutes only a minor driving force to associate with the dynamic biomolecular network of the condensate. Specific protein–protein interactions in the cytoplasm in comparison to SGs determine the partitioning of folding states between the respective phases during HS.

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Introduction

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The intracellular liquid–liquid phase separation (LLPS) of proteins and RNA leading to the formation of membraneless organelles (MLOs) is now established as a fundamental process in cell biology, exhibiting functions in cellular signaling, (1,2) transcriptional (3) and translational (4) regulation, enzymatic activity, (5) and stress response. (6,7) In fact, MLOs formed during stress conditions are part of a more complex orchestrated cellular feedback that also encompasses the formation of reversible protein aggregates and filaments, all serving to increase fitness and survival. (6,8,9) Stress granules (SGs) are one of such type of cytoprotective assemblies, formed in response to heat stress (HS), that among under functions are known to recruit and transiently store misfolded proteins. (6,10−13)
HS leads to local or global unfolding of proteins, resulting in the exposure of hydrophobic segments that were otherwise buried in native conditions. (14) The protein quality control (PQC) machinery (chaperones, ubiquitin-proteasome, or autophagy), engages with such unfolded/misfolded states, refolding, shielding, or degrading them, thus precluding toxic aggregation. (12,15−17) These actions are usually rationalized with partition models, where clients can directly bind to PQC as monomers, oligomers, or aggregates, with all these states being further sequestered by SGs and thus optimizing client clearance. (6,11,17−21) Nevertheless, mutations or failure in the PQC promoted by cellular stress or aging leads to deleterious changes in the material properties of the SG components, triggering pathological phase transitions. (18,22−24)
The formation of SGs involves homotypic as well as heterotypic multivalent interactions among proteins with folded and intrinsically disordered regions (IDPRs) and RNA. (25) Simplistic approaches developed to reconstitute in vitro SGs have shown that homotypic interactions within their single components such as Fused in Sarcoma (FUS), (22,26) ribonucleoprotein A1 (hnRNPA1), (27) Ras GTPase-activating protein-binding protein (G3BP), (28) or RNA binding protein TDP-43 (29) are sufficient to induce LLPS. While lower multivalence and structural rigidity of globular proteins are thought to inherently reduce the propensity for LLPS, (30) their unfolded forms were recently shown to drive aggregation-mediated phase separation via homotypic interactions. (31) Nevertheless, it remains unknown if unfolded states constitute an omnipotent driving force when considering heterotypic interactions in functional condensates (32,33) such as SGs. This is important as homotypic-driven aberrant phase transitions are bypassed inside MLOs by “heterotypic buffering”, which refers to the ability of heterotypic interactions to suppress the excess of homotypic interactions within the condensates. (34) Considering the intrinsic stickiness of unfolded states and their well-known tendency to form (toxic) aggregates, (35) it is crucial to investigate how these states engage in the condensate network and how they modify homotypic/heterotypic contacts preserving functional as opposed to pathological phase transitions.
In this study, we investigate the function of LLPS as a cytoprotective mechanism, sequestering unfolded, misfolded, or aggregated protein under cellular stress condition. Considering the in-cell folding and aggregation landscape of superoxide dismutase 1 (SOD1), (36−39) involved in the disease progression of amyotrophic lateral sclerosis (ALS), (40) we identify the folding states that are sequestered by SGs. In this context, we test the hypothesis if the protein unfolding process constitutes an omnipotent driving force for the association with condensates.

Results and Discussion

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The Load of Unfolded Protein and Its Effect on Condensate Association under HS in HeLa Cells and In Vitro

ALS-related full-length (SOD1FL) mutants were previously shown to accumulate and aggregate inside SGs; (18,41,42) however, the folding states that associate with these condensates remain unknown. To investigate the role of unfolded SOD1 in SG association independently of aggregation, we study SOD1bar in reference to SOD1FL. SOD1bar is a monomeric β-barrel protein, where loops IV and VII from SOD1FL were swapped by Gly-Ala-Gly linkers, leading to breakage of the dimer interface, catalytic inactivation and higher thermodynamic stability (43) (Figure 1A). Importantly, removal of Zn2+ and Cu2+ coordination sites as well as replacement of Cys-6 and Cys-111,146 by Ala and Ser diminishes the metal-loss-induced misfolding (44) and the in-cell aggregation propensities at both physiological (45) and HS conditions (Figure S1). On the basis of our prior knowledge of the in-cell modified standard state folding free energies at 37 °C (ΔGf°′), (39) we examined the SG association of a set of specifically destabilized SOD1bar mutants (G85R, A4V, G41D, G93V, and I35A) (Figure 1A). The choice of mutants with distinct ΔGf°′, combined with fast and reversible 2-state folding and lack of aggregation (except A4V, Figure S2), (39) allowed us to vary the amount of unfolded protein upon HS in the respective experiments. Stability measurements to determine Tm and ΔGf°′ under different conditions were conducted by Fast Relaxation Imaging (FReI) (46) (Figure 1B and S3); aggregation and SG association were monitored by temperature-controlled confocal laser scanning techniques (see the Supporting Information, “Materials and Methods” section).

Figure 1

Figure 1. SOD1bar as a sensor to measure protein unfolding and SG association in-cell and in vitro. (A) SOD1FL (Protein Data Bank (PDB) ID: IHL5) and SOD1bar (PDB ID: 4BCZ), with mutations A4V, I35A, G41D, G85R, and G93V highlighted. Images were assembled using UCSF Chimera. (52) (B) Schematic representation of the FReI technique with two cameras (CCD1 and CCD2). DM and BS represent dichroic mirror and beam splitter, respectively. (C) Exemplary images of HeLaFUS-mCh cells showing different partitioning of the SOD1bar-AcGFP1 mutants G93V and A4V. Enrichment of SOD1bar-AcGFP1 was defined by a local apparent partition coefficient (PC) > 1, determined as the ratio of the mean fluorescence intensity inside the SG (region 1) and the mean fluorescence intensity at 0.6 μm (region 2) from the SG (right zoom-in). (D) PC of SOD1bar after 120 min at 43 °C (N = 437 to 523 SGs). PCs of mutants A4V, G93V, G41D, and G85R are significantly higher than Wt. (E) PCs as a function of ΔGf°′ at 37 °C. The values are significantly different between each of the three defined clusters: Wt and I35A (light gray), G93V, G41D, and G85R (light brown) and A4V (light red). (F) PCs of SOD1bar-Wt and SOD1FL-Wt and the respective A4V mutants. No statistical significance is found between the PCs of SOD1bar and SOD1FL for both Wt and A4V. (G) Correlation between SOD1bar PCs and the scaled sum [norm. ΔfU37–43°C + norm. Hphob]. We normalized ΔfU37–43°C values for the different constructs (1 represents the highest ΔfU37–43°C, while 0 represents the lowest one). Similarly, Hphob was normalized from 1 (highest) to the 0 (lowest) within the different SOD1bar sequences. Finally, we sum up the normalized values, [norm. ΔfU37–43°C + norm. Hphob] and scaled between 0 and 1 (lowest to highest). (H) Correlation between mobile fractions (MFs) inside SGs and PCs. The statistical analyses reported in panels D–F were carried out using one-way ANOVA, followed by a post hoc Tukey test for multiple comparisons, computed with a confidence interval of 95%. The values in panels E, G, and H are presented as mean ± SEM.

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Sequestration of SOD1 in SGs under HS Conditions

We first investigated if partitioning of SOD1bar into SGs depends on the in-cell ΔGf°′. HeLaFUS-mCh cells coexpressing AcGFP1-SOD1bar-Wt and mutants were exposed to HS at 43 °C for 120 min following a previously established protocol. (18) We quantified the partitioning of SOD1bar by determining the local apparent partition coefficient (PC) as the ratio between the mean fluorescence intensity inside SGs (FImeanSG) and the mean fluorescence intensity in the immediate surroundings (FImeanOut) using confocal microscopy. Exemplary images and corresponding PCs of SOD1bar-Wt and mutants G93V and A4V are depicted in Figure 1C. PC values of >1 are specific indicators of SOD1bar accumulation inside SGs, as defined previously by colocalization analysis carried out for SOD1FL and FUS or G3BP1. (18) Importantly, PCs are concentration-independent as long as binding-site occupancy does not reach its maximum. (47) Indeed, plotting PCs against the different SOD1bar expression levels (based on the average fluorescence intensity in the cytoplasm) for each construct shows no dependency on the intracellular concentrations (Figure S4), suggesting that such levels are below saturation.
We observed a significant increase in the PCs for SOD1bar mutants (A4V, G93V, G41D, and G85R) in comparison to the Wt protein, while no difference was found for I35A (Figure 1D). We further plotted PC values as a function of ΔGf°′ at 37 °C for each construct and classified them into three clusters, with each cluster containing PC values which are statistically not distinguishable between them (Figure 1E). Both the most and least stable constructs (Wt and I35A) display no preferential partitioning inside SGs, while constructs of intermediate stabilities (G93V, G41D, and G85R) lead to SOD1bar enrichment, being distinctively high for A4V. This mutational effect is consistent with earlier reports of the accumulation of the corresponding SOD1FL mutants inside SGs, (18,41,42) suggesting that the destabilization of the β-barrel structure may constitute the main driving force for SGs accumulation. We further verified this conclusion by comparing the PCs of SOD1bar-Wt and SOD1bar-A4V with the more aggregation-prone SOD1FL-Wt and SOD1FL-A4V. (45) As expected, we found no significant change between SOD1bar and SOD1FL PCs for both Wt and A4V (Figure 1F), while this same construct (SOD1FL-A4V) shows higher aggregation in the cytoplasm when compared to its barrel counterpart (Figure S1). Comparable levels of aggregation were found for all SOD1bar mutations (Figure S2), except A4V, suggesting that SGs-mediated sequestration is independent of the intracellular aggregation propensity of the protein during HS.
We next explored the idea that the extra load of unfolded protein upon HS (14) could reinforce partitioning of SOD1bar mutants into SGs, as pre-existing unfolded proteins remain bound to PQC (17,48) and are thus unavailable to be recruited. We determined the unfolded fractions for each construct at 37 and 43 °C from ΔGf°′ at these same temperatures and plotted the change, ΔfU37–43°C against the different PCs (Figure S5A). We found no apparent correlation (Pearson r = 0.74) indicating that HS induced unfolding is not the only requisite for SG association. We then tested if accounting for the increase in hydrophobicity upon unfolding increases this correlation, as previous studies suggested that recruitment of mutant SOD1FL into SGs was mediated by hydrophobic interactions. (41) We analyzed if mutations resulting in increased hydrophobicity could promote partitioning of HS-induced unfolded SOD1bar in SGs. As such, we first computed the hydrophobicity (Hphob) based on solvent accessible surface areas (SASAs) and overall transfer free energies from water to cyclohexane resulting from adding up the individual values obtained for each residue in the unfolded SOD1bar (Table S1, see the Supporting Information, “Materials and Methods” section). To consider the hydrophobicity change Figure S5B upon unfolding in the ΔfU37 to 43 °C (no correlation of PCs was found with the hydrophobicity alone, Figure S5B), we plotted the scaled sum [norm. ΔfU37–43°C + norm. Hphob.] (1 to 0, highest to lowest), following the strategy proposed previously. (49,50) We found a significant positive correlation with the PCs (Pearson r = 0.95, Figure 1G). We also plotted PCs as a function of ΔfU37–43°C and Hphob in a 3D plot (see Figure S5C). Both the approaches suggest that the increased amount of unfolded protein by HS, paired with an increase in hydrophobicity of the unfolded state, contributes to SOD1bar partitioning into SGs. Specifically, this effect likely relies on interactions of hydrophobic residues accessible in unfolded SOD1bar. This interpretation would be in line with prior assignments of homotypic intermolecular hydrophobic contacts as determinants for LLPS of unfolded barnase (31) and elastin. (51)
Finally, we investigated diffusion properties of SOD1bar in SGs to probe for a reduced mobility of the protein in comparison to the cytoplasm, suggesting stronger interactions and association. (18) We therefore carried out fluorescence recovery after photo bleaching (FRAP) experiments in SGs and in the cytosol (Figure S6A). In the SG environment, we found lower mobile fractions for A4V and G41D mutations when compared to the Wt and higher cytosolic mobility of A4V in contrast to that associated with the condensates (Figure S6B), suggestive of stronger interactions in SGs versus the cytoplasm. Consistently, we observed a significant negative correlation between the PCs and MFs of all SOD1bar proteins, with larger partitioning of mutant SOD1bar leading to lower mobility and thus stronger association with SGs (Figure 1H).
In summary, ALS-linked mutants of SOD1bar, as well as their SOD1FL counterparts associated with SGs under HS conditions, suggesting that such partitioning is independent of metal binding, dimerization sites, and aggregation propensity. The sequestration of proteins in SGs rather relies on the in-cell folding stability suggesting that conformers belonging to the unfolded state ensemble participate in the homotypic and heterotypic protein–protein interactions that compose these cytoprotective assemblies.

SOD1bar Partitioning into FUS Droplets In Vitro under HS Conditions

We next investigate the sequestration of unfolded SOD1 proteins in a cell-free environment by using a simplistic three-component system encompassing FUS condensates and SOD1 protein in dilute solution. The rationale is to probe if SOD1bar condensate association observed in the cell can be solely attributed to protein unfolding as a driving force. We followed a pre-established protocol to purify (see the Supporting Information, “Materials and Methods” section, Figure S7) and induce droplet formation of full-length FUS (Figure 2A), (18,22,26,53−55) together with SOD1bar. We studied the association of SOD1bar with FUS droplets before and after the HS, using confocal microscopy, where the transmitted signal (for the bright field image, Figure S8A) allowed the detection of FUS condensates and mCherry fluorescence emission was used to identify SOD1bar. While at 23 °C the FUS droplets were either visually depleted or enriched in the peripheral region with SOD1bar-Wt and mutants, at the HS temperature (43 °C) we found a significant enhancement of SOD1bar accumulation for all the constructs (Figure 2B). We further determined the PC (FlmeanDroplet/FlmeanoutDroplets) of SOD1bar to quantify its enrichment in the FUS compartments at the HS condition. Similar to the in-cell measurements, where partitioning of SOD1 inside SGs reaches a steady-state condition after 120 min, (18) we conducted HS over 60 min, until such equilibrium conditions were equally attained (Figure S9). As depicted in Figure 2C, the PC values for all the SOD1bar constructs increase after HS to values of >1, suggesting a general preferential partitioning into FUS condensates. This HS induced SOD1bar partitioning is corroborating the observed SG sequestration in cells. In contrast, the PC values do not show a correlation between ΔGf°′ of the SOD1bar and condensate association, as SOD1bar-Wt showed higher PCs than all mutants (Figure 2D). Thus, the in vitro experiments suggest that unfolding of SOD1bar, and the acquired flexibility and hydrophobicity of the unfolded state, may not constitute the main driving force for condensate association upon HS.

Figure 2

Figure 2. Association of SOD1bar with FUS droplets in vitro. (A) Schematic representation of the different FUS domains with predicted (dis)ordered domains by Prediction of Natural Disorder Regions (PONDR) Score bioinformatics tool. (56) (B) Exemplary images (mCherry channel) of SOD1bar added to FUS droplets (in buffer, pH 7.5) after 60 min of incubation at 23 or 43 °C. (C) PCs after 60 min of incubation shown as whisker box plots (N = 41–203 droplets for 23 °C, N = 422–895 droplets for 43 °C). The values are significantly different (except G85R vs A4V) among each other. Significant tests were carried out by one-way ANOVA analysis, followed by post hoc Tukey test for multiple comparisons, computed with a confidence interval of 95%. (D) Mean values of PCs (at 43 °C) plotted against ΔGf°′ at 43 °C.

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The results rather suggest that a delicate balance of protein–protein interactions in the two phases governs the partitioning. The SOD1bar point mutations appear to be decisive in changing this balance, in line with our previous studies showing that the stability of SOD1bar in the cell compared to in vitro changes significantly with specific point mutations. (39) Correspondingly, we here observed that single-point mutations can even lead to the formation of multiphase condensates. (57,58) While we found that SOD1bar-G93V and SOD1bar-G41D accumulate homogeneously in the droplets, SOD1bar-Wt, SOD1bar-A4V, and SOD1bar-G85R accumulate predominantly in the periphery regions (Figure S8B).
Changes in protein–protein interactions may also be due to altered FUS–FUS interactions upon heat shock. (59) Indeed, we found that FUS droplets in the absence and presence of SOD1bar, show a noticeably modified size and morphology (see Figure S8A and S10) after HS, indicative that the HS condition modifies the FUS–FUS interactions as well as the FUS–SOD1bar network. (60,61) We speculate that part of this change may be attributed to the unfolding of the RBD of FUS (residue numbers 285–371, Figure 2A), exposing additional residues accessible for preferential interactions with SOD1bar. (62,63)
In summary, the HS conditions enhance SOD1bar partitioning into FUS condensates depending on single-point mutations, but in contrast to in-cell experiments, independent of folding stability.

SOD1bar Partitioning in Crowded Solutions

The in vitro experiments suggest that the protein–protein interactions that govern partitioning of SOD1bar are sensitive to environmental changes. This may be of particular relevance in cellular health and disease conditions. (64,65) Concerning effects in the crowded cell arising from volume-exclusion, quinary interactions, or chaperone engagement, it is evident that these factors can modulate both the folding equilibrium (17,39,66−71) as well as phase separation. (18,72−75) We deciphered those in cytomimetic media by using Ficoll 70 (15% w/v), known to exert stabilizing excluded-volume effects (76) (ΔΔGf°′(37 °C) < 0), and BSA (20% w/v), known to mimic destabilizing quinary interactions (39,76) (ΔΔGf°′(37 °C) > 0) (Figure S11). We therefore investigated whether these cosolutes that are known to change ΔGf°′ have an influence on SOD1bar association with FUS condensates. As depicted in Figure 3A,B, Ficoll 70 solution significantly promotes the SOD1bar partitioning to the FUS condensates, both at 23 and 43 °C, when compared to dilute solution. In contrast, in BSA solution, we observed a considerable decrease of the PCs for SOD1bar even after 60 min of HS (Figure 3C,D).

Figure 3

Figure 3. Effect of crowding (in vitro) on SOD1bar association with FUS droplets at different temperatures. Exemplary images (mCherry channel) of SOD1bar enrichment/depletion in the presence of (A) 15% (w/v) Ficoll 70 and (C) 20% (w/v) BSA, after 60 min of incubation at 23 and 43 °C. (B and D) PCs measured after 60 min at 23 and 43 °C in the presence of Ficoll and BSA. PCs in buffer (data from Figure 2) are shown as gray box plots for comparison. (N = 96–304 droplets for 23 °C, N = 263–706 droplets for 43 °C). The values are significantly different among each other [except for (B) G85R (in buffer) vs G85R (in Ficoll), Wt (in Ficoll) vs I35A (in Ficoll), and (D) Wt (in BSA) vs I35A (in BSA)]. Significance tests were carried out by one-way ANOVA analysis, followed by post hoc Tukey test for multiple comparisons, computed with a confidence interval of 95%.

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Both observations suggest that shifting the folding equilibrium toward the folded or unfolded states does not determine SOD1bar partitioning (Figure S12). The results can be rather attributed to the cosolute effects on the LLPS (74,77,78) driving the separation of both proteins in the system, FUS and SOD1bar. Thereby, Ficoll 70 induced phase separation can be explained by increased depletion attraction (72,75,77) arising from its exclusion from the FUS–SOD1bar condensate. Likewise, BSA suppresses phase separation presumably by protein–protein interactions weakening the condensate. (74,77) In fact, BSA was previously shown to suppress phase separation of FUS IDR (amino acids 1–237) due to favorable interactions with FUS, (75) whose interpretation with the mass action model (79) suggests few free binding sites in the FUS droplet network, impeding the accommodation of SOD1bar. In other words, BSA colocalizes in FUS droplets as FUS–BSA interactions compete with FUS–SOD1bar association and thus suppress SOD1bar partitioning into the condensates. Alternatively, SOD1bar preferentially binds to BSA in the dilute phase and thus reduces its availability to interact in the FUS droplet phase.
Remarkably, the PCs in BSA are similar to those measured in cellular SGs (Figure 1D), suggesting that BSA acts as a good cytomimetic cosolute for LLPS, in accordance with our previous studies on SOD1bar folding stabilty. (39) Overall, tweaking the folding equilibrium does not modulate SOD1bar association in FUS condensates accordingly, but rather it changes the protein–protein interactions in the respective phases to determine SOD1bar partitioning.

Lattice Boltzmann Molecular Dynamics (LBMD) Simulation to Investigate Interactions of SOD1bar with FUS Low-Complexity Domain (LCD) and BSA

To quantify how the energetics of SOD1bar partitioning into a condensate depends on the folding state of the barrel, we carried out coarse-grained LBMD simulations (80,81) of folded and unfolded SOD1bar molecules embedded in a high-concentration FUS solution composed of 70 chains of the FUS LCD, mimicking a droplet solution concentration of 150 g/L (Figure S13A). Evaluation of the SOD1bar–FUS LCD interaction energies showed that from the enthalpic point of view the unfolded state of SOD1bar does not interact more favorably with the FUS LCD domain in comparison to the folded form, with the energy difference for one SOD1bar protein being less than 1 kJ mol–1 (Table 1). In fact, unfolding leads to a change in the specific contacts between SOD1bar and FUS LCD (Figure S13E,F), disfavoring the association of both proteins. These findings agree with the experimental in vitro results showing that partitioning of SOD1bar is independent of its folding stability.
Table 1. Interaction Energies of 10 SOD1bar with BSA and FUS LCD in the Two Solutionsa
 FUS LCD–SOD1bar(f)BSA–SOD1bar(f)FUS LCD–SOD1bar(u)BSA–SOD1bar(u)
energy [kJ mol–1]–35.2 ± 1.7–41.4 ± 6.3–29.3 ± 2.5–46.8 ± 1.7
a

Solutions were obtained at 27 °C and considering the folded (f) and unfolded (u) SOD1bar. Error bars were estimated via block analysis. The 100 g/L BSA concentration was used in this comparison, and the SOD1bar unfolded state was modeled as a floppy elastic network (see the “Materials and Methods” section in the Supporting Information). The observed trends are also maintained at a higher temperature; see Figure S13C.

A comparison with the interaction energies of SOD1bar embedded in BSA solutions (Table 1) yielded similar results for folded SOD1bar in BSA (100 g/L) (Figure S13B) as in FUS (150 g/L), with SOD1bar experiencing numerous favorable charge–charge contacts in BSA (Figure S13G,H). Moreover, a slight preference for BSA arises when SOD1bar unfolds. However, the magnitude of these interaction differences is very small, on the order of a few kJ mol–1 (Table 1). The comparable energetics in BSA and FUS that we quantified in the simulations rationalizes the suppressing effect of BSA on the SOD1bar partitioning into the FUS phase by proposing that SOD1bar–BSA interactions are at least as favorable as SOD1bar–FUS LCD interactions. Finally, we observed a strong dependence of the SOD1bar–BSA interaction on the concentration of the BSA solution, with the interaction energies increasing more than by a factor of 5 at 300 g/L (see Figure S13D). This points to the delicate role of local protein concentration inside and outside the condensate in determining the partitioning. Estimating the density distribution of proteins across the two-phase boundary is a current challenge and might help to rationalize the differences observed between in-cell and in vitro experiments.

Conclusion

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In this study, we showed that destabilized SOD1bar mutants, independent of the aggregation processes, associate with SGs under HS conditions, suggesting that the unfolded states engage in the homotypic and heterotypic interactions that drive their formation. In vitro studies in the reconstituted FUS environment, however, revealed that the intrinsic flexibility of the SOD1bar unfolded state, despite altering the protein’s interactions with the condensate, does not constitute an omnipotent driving force for condensate association. Studies in cytomimetic media paired with LBMD simulations suggest that specific protein–protein interactions, subject to single-point mutations, determine the partitioning between the two phases. Further investigations in this direction will require the inclusion of different interaction partners of SOD1 in the SG environment using in vitro reconstitutions with other well-known condensate components, such as G3BP1, (41) or PQC components such as chaperones. (18) In fact, the small but significant changes in the in-cell PC values of the SOD1bar/FL mutants in comparison to those of Wt observed here support the need to integrate the PQC mechanisms (21,24) when generally establishing the main driving forces behind SGs-mediated sequestration of destabilized proteins (including disease-related mutations). In addition to SGs, unfolded and misfolded states also bind to chaperones, (17,31,82,83) are targeted for degradation, or are accumulated in aggresomes and further cleared by autophagy, (16,18,84,85) thus distributing the overall concentration of free unfolded species among these PQC centers. (31) As such previous studies revealed, chaperones can act as suppressors of phase separation of unfolded states by binding to them preferentially in the diluted phase (31) or can be recruited inside SGs to prevent accumulation of misfolded proteins. (18)
Recent studies showed that many proteins involved in functional LLPS are expressed close to their solubility limits; thus, they are more prone to become less soluble with aging, leading to aggregation. (86) In this context, our current results built a foundation for achieving a global understanding concerning the involvement of unfolded states in LLPS, giving the intricate relation between dysfunctional unfolding, toxic aggregation, and aberrant liquid-to-solid phase transitions. (24,87,88)

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.1c09589.

  • Description of materials and methods, visual and plot of agregates of Wt and A4V mutants in HeLaFUS-mCh cells; laser heating and FRET data; kinetic traces and amplitudes as a function of temperature; PCs of SOD1bar-Wt and the various mutants inside SGs as a function of protein expression levels; correlation plot for SOD1bar PCs; mobility of SOD1bar inside SGs and cytosol after 120 minutes of HS; SDS PAGE gel for purified FUS-MBP and SOD1bar constructs; DIC images of FUS droplets in the presence and absence of SOD1bar; line-ROI intensity plots; images of SOD1bar G41D-enriched into or depleted from FUS-Wt droplets; change of the cross section of FUS droplets after HS at 43 °C with time; difference in ΔGf°′ at 37 °C; PCs as a function of ΔGf°′; LBMD simulation snapshots; interaction energies and contacts; fractions of protein surface areas occupied by the different residue types; mean number of contacts between different categories of residues; Tables S1 and S2 (including all the data points shown in the graphics) (PDF)

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  • Corresponding Authors
    • Stepan Timr - CNRS Laboratoire de Biochimie Théorique, Institut de Biologie Physico-Chimique, Université Paris Denis Diderot, Sorbonne Paris Cité, PSL Research University, 13 rue Pierre et Marie Curie, Paris 75005, France;  J. Heyrovský Institute of Physical Chemistry, Czech Academy of Sciences, Dolejskova 2155/3, Prague 8 182 23, Czech Republic;  Orcidhttps://orcid.org/0000-0002-5824-4476;  Email: [email protected]
    • Fabio Sterpone - CNRS Laboratoire de Biochimie Théorique, Institut de Biologie Physico-Chimique, Université Paris Denis Diderot, Sorbonne Paris Cité, PSL Research University, 13 rue Pierre et Marie Curie, Paris 75005, France;  Orcidhttps://orcid.org/0000-0003-0894-8069;  Email: [email protected]
    • Simon Ebbinghaus - Institute of Physical and Theoretical Chemistry, TU Braunschweig, Rebenring 56, D-38106 Braunschweig, Germany;  Orcidhttps://orcid.org/0000-0001-9309-1279;  Email: [email protected]
  • Authors
    • Nirnay Samanta - Institute of Physical and Theoretical Chemistry, TU Braunschweig, Rebenring 56, D-38106 Braunschweig, Germany;  Orcidhttps://orcid.org/0000-0002-5746-6907
    • Sara S. Ribeiro - Institute of Physical and Theoretical Chemistry, TU Braunschweig, Rebenring 56, D-38106 Braunschweig, Germany;  Orcidhttps://orcid.org/0000-0001-6033-8853
    • Mailin Becker - Institute of Physical and Theoretical Chemistry, TU Braunschweig, Rebenring 56, D-38106 Braunschweig, Germany
    • Emeline Laborie - CNRS Laboratoire de Biochimie Théorique, Institut de Biologie Physico-Chimique, Université Paris Denis Diderot, Sorbonne Paris Cité, PSL Research University, 13 rue Pierre et Marie Curie, Paris 75005, France
    • Roland Pollak - Institute of Physical and Theoretical Chemistry, TU Braunschweig, Rebenring 56, D-38106 Braunschweig, Germany
  • Author Contributions

    N.S. and S.S.R. contributed equally to this paper.

  • Funding

    N.S., S.S.R. and SE acknowledge support by the German Research Foundation DFG-SPP 2191 “Molecular Mechanisms of Functional Phase Separation” (Project number 402723784). F.S., S.T., and E.L. acknowledge support from the “Initiative d’Excellence” program from the French State (Grant “DYNAMO”, ANR-11-LABX-0011–01). S.T. acknowledges funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 840395. Part of this work was carried out using HPC resources from GENCI and LBT.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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We thank Simon Alberti, Titus Franzmann, Danny Hatters, Jonas Ahlers and David Gnutt for helpful discussions and Simon Alberti and Dorothee Dormann for providing cells and FUS plasmids, respectively.

References

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This article references 88 other publications.

  1. 1
    Case, L. B.; Zhang, X.; Ditlev, J. A.; Rosen, M. K. Stoichiometry Controls Activity of Phase-Separated Clusters of Actin Signaling Proteins. Science 2019, 363 (6431), 1093– 1097,  DOI: 10.1126/science.aau6313
    Google Scholar
    1
    Stoichiometry controls activity of phase-separated clusters of actin signaling proteins
    Case, Lindsay B.; Zhang, Xu; Ditlev, Jonathon A.; Rosen, Michael K.
    Science (Washington, DC, United States) (2019), 363 (6431), 1093-1097CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Biomol. condensates conc. macromols. into foci without a surrounding membrane. Many condensates appear to form through multivalent interactions that drive liq.-liq. phase sepn. (LLPS). LLPS increases the specific activity of actin regulatory proteins toward actin assembly by the Arp2/3 complex. We show that this increase occurs because LLPS of the Nephrin-Nck-N-WASP signaling pathway on lipid bilayers increases membrane dwell time of N-WASP and Arp2/3 complex, consequently increasing actin assembly. Dwell time varies with relative stoichiometry of the signaling proteins in the phase-sepd. clusters, rendering N-WASP and Arp2/3 activity stoichiometry dependent. This mechanism of controlling protein activity is enabled by the stoichiometrically undefined nature of biomol. condensates. Such regulation should be a general feature of signaling systems that assemble through multivalent interactions and drive nonequil. outputs.
  2. 2
    Huang, W. Y. C.; Alvarez, S.; Kondo, Y.; Lee, Y. K.; Chung, J. K.; Lam, H. Y. M.; Biswas, K. H.; Kuriyan, J.; Groves, J. T. A Molecular Assembly Phase Transition and Kinetic Proofreading Modulate Ras Activation by SOS. Science 2019, 363 (6431), 1098– 1103,  DOI: 10.1126/science.aau5721
    Google Scholar
    2
    A molecular assembly phase transition and kinetic proofreading modulate Ras activation by SOS
    Huang, William Y. C.; Alvarez, Steven; Kondo, Yasushi; Lee, Young Kwang; Chung, Jean K.; Lam, Hiu Yue Monatrice; Biswas, Kabir H.; Kuriyan, John; Groves, Jay T.
    Science (Washington, DC, United States) (2019), 363 (6431), 1098-1103CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    The guanine nucleotide exchange factor (GEF) Son of Sevenless (SOS) is a key Ras activator that is autoinhibited in the cytosol and activates upon membrane recruitment. Autoinhibition release involves structural rearrangements of the protein at the membrane and thus introduces a delay between initial recruitment and activation. In this study, we designed a single-mol. assay to resolve the time between initial receptor-mediated membrane recruitment and the initiation of GEF activity of individual SOS mols. on microarrays of Ras-functionalized supported membranes. The rise-and-fall shape of the measured SOS activation time distribution and the long mean time scale to activation (∼50 s) establish a basis for kinetic proofreading in the receptor-mediated activation of Ras. We further demonstrate that this kinetic proofreading is modulated by the LAT (linker for activation of T cells)-Grb2-SOS phosphotyrosine-driven phase transition at the membrane.
  3. 3
    Lu, Y.; Wu, T.; Gutman, O.; Lu, H.; Zhou, Q.; Henis, Y. I.; Luo, K. Phase Separation of TAZ Compartmentalizes the Transcription Machinery to Promote Gene Expression. Nat. Cell Biol. 2020, 22 (4), 453– 464,  DOI: 10.1038/s41556-020-0485-0
    Google Scholar
    3
    Phase separation of TAZ compartmentalizes the transcription machinery to promote gene expression
    Lu, Yi; Wu, Tiantian; Gutman, Orit; Lu, Huasong; Zhou, Qiang; Henis, Yoav I.; Luo, Kunxin
    Nature Cell Biology (2020), 22 (4), 453-464CODEN: NCBIFN; ISSN:1465-7392. (Nature Research)
    Abstr.: TAZ promotes growth, development and tumorigenesis by regulating the expression of target genes. However, the manner in which TAZ orchestrates the transcriptional responses is poorly defined. Here we demonstrate that TAZ forms nuclear condensates through liq.-liq. phase sepn. to compartmentalize its DNA-binding cofactor TEAD4, coactivators BRD4 and MED1, and the transcription elongation factor CDK9 for transcription. TAZ forms phase-sepd. droplets in vitro and liq.-like nuclear condensates in vivo, and this ability is neg. regulated by Hippo signaling through LATS-mediated phosphorylation and is mediated by the coiled-coil (CC) domain. Deletion of the TAZ CC domain or substitution with the YAP CC domain prevents the phase sepn. of TAZ and its ability to induce the expression of TAZ-specific target genes. Thus, we identify a mechanism of transcriptional activation by TAZ and demonstrate that pathway-specific transcription factors also engage the phase-sepn. mechanism for efficient and specific transcriptional activation.
  4. 4
    Iserman, C.; Desroches Altamirano, C.; Jegers, C.; Friedrich, U.; Zarin, T.; Fritsch, A. W.; Mittasch, M.; Domingues, A.; Hersemann, L.; Jahnel, M. Condensation of Ded1p Promotes a Translational Switch from Housekeeping to Stress Protein Production. Cell 2020, 181, 818,  DOI: 10.1016/j.cell.2020.04.009
    Google Scholar
    4
    Condensation of Ded1p Promotes a Translational Switch from Housekeeping to Stress Protein Production
    Iserman, Christiane; Desroches Altamirano, Christine; Jegers, Ceciel; Friedrich, Ulrike; Zarin, Taraneh; Fritsch, Anatol W.; Mittasch, Matthaeus; Domingues, Antonio; Hersemann, Lena; Jahnel, Marcus; Richter, Doris; Guenther, Ulf-Peter; Hentze, Matthias W.; Moses, Alan M.; Hyman, Anthony A.; Kramer, Guenter; Kreysing, Moritz; Franzmann, Titus M.; Alberti, Simon
    Cell (Cambridge, MA, United States) (2020), 181 (4), 818-831.e19CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    Cells sense elevated temps. and mount an adaptive heat shock response that involves changes in gene expression, but the underlying mechanisms, particularly on the level of translation, remain unknown. Here we report that, in budding yeast, the essential translation initiation factor Ded1p undergoes heat-induced phase sepn. into gel-like condensates. Using ribosome profiling and an in vitro translation assay, we reveal that condensate formation inactivates Ded1p and represses translation of housekeeping mRNAs while promoting translation of stress mRNAs. Testing a variant of Ded1p with altered phase behavior as well as Ded1p homologs from diverse species, we demonstrate that Ded1p condensation is adaptive and fine-tuned to the max. growth temp. of the resp. organism. We conclude that Ded1p condensation is an integral part of an extended heat shock response that selectively represses translation of housekeeping mRNAs to promote survival under conditions of severe heat stress.
  5. 5
    Peeples, W.; Rosen, M. K. Mechanistic Dissection of Increased Enzymatic Rate in a Phase-Separated Compartment. Nat. Chem. Biol. 2021, 17 (6), 693– 702,  DOI: 10.1038/s41589-021-00801-x
    Google Scholar
    5
    Mechanistic dissection of increased enzymatic rate in a phase-separated compartment
    Peeples, William; Rosen, Michael K.
    Nature Chemical Biology (2021), 17 (6), 693-702CODEN: NCBABT; ISSN:1552-4450. (Nature Portfolio)
    Biomol. condensates conc. macromols. into discrete cellular foci without an encapsulating membrane. Condensates are often presumed to increase enzymic reaction rates through increased concns. of enzymes and substrates (mass action), although this idea has not been widely tested and other mechanisms of modulation are possible. Here we describe a synthetic system where the SUMOylation enzyme cascade is recruited into engineered condensates generated by liq.-liq. phase sepn. of multidomain scaffolding proteins. SUMOylation rates can be increased up to 36-fold in these droplets compared to the surrounding bulk, depending on substrate KM. This dependency produces substantial specificity among different substrates. Analyses of reactions above and below the phase-sepn. threshold lead to a quant. model in which reactions in condensates are accelerated by mass action and changes in substrate KM, probaby due to scaffold-induced mol. organization. Thus, condensates can modulate reaction rates both by concg. mols. and phys. organizing them.
  6. 6
    Riback, J. A.; Katanski, C. D.; Kear-Scott, J. L.; Pilipenko, E. V.; Rojek, A. E.; Sosnick, T. R.; Drummond, D. A. Stress-Triggered Phase Separation Is an Adaptive, Evolutionarily Tuned Response. Cell 2017, 168 (6), 1028– 1040,  DOI: 10.1016/j.cell.2017.02.027
    Google Scholar
    6
    Stress-triggered phase separation is an adaptive, evolutionarily tuned response
    Riback, Joshua A.; Katanski, Christopher D.; Kear-Scott, Jamie L.; Pilipenko, Evgeny V.; Rojek, Alexandra E.; Sosnick, Tobin R.; Drummond, D. Allan
    Cell (Cambridge, MA, United States) (2017), 168 (6), 1028-1040.e19CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    In eukaryotic cells, diverse stresses trigger coalescence of RNA-binding proteins into stress granules. In vitro, stress granule-assocd. proteins can demix to form liqs., hydrogels, and other assemblies lacking fixed stoichiometry. Observing these phenomena has generally required conditions far removed from physiol. stresses. Here, we show that poly(A)-binding protein (Pab1 in yeast), a defining marker of stress granules, phase separates and forms hydrogels in vitro upon exposure to physiol. stress conditions. Other RNA-binding proteins depend upon low-complexity regions (LCRs) or RNA for phase sepn., whereas Pab1's LCR is not required for demixing, and RNA inhibits it. Based on unique evolutionary patterns, we created LCR mutations, which systematically tuned its biophys. properties and Pab1 phase sepn. in vitro and in vivo. Mutations that impeded phase sepn. reduced organism fitness during prolonged stress. Poly(A)-binding protein thus acts as a physiol. stress sensor, exploiting phase sepn. to precisely mark stress onset, a broadly generalizable mechanism.
  7. 7
    Franzmann, T. M.; Jahnel, M.; Pozniakovsky, A.; Mahamid, J.; Holehouse, A. S.; Nüske, E.; Richter, D.; Baumeister, W.; Grill, S. W.; Pappu, R. V. Phase Separation of a Yeast Prion Protein Promotes Cellular Fitness. Science 2018, 359 (6371), eaao5654,  DOI: 10.1126/science.aao5654
    Google Scholar
    There is no corresponding record for this reference.
  8. 8
    Petrovska, I.; Nüske, E.; Munder, M. C.; Kulasegaran, G.; Malinovska, L.; Kroschwald, S.; Richter, D.; Fahmy, K.; Gibson, K.; Verbavatz, J.-M.; Alberti, S. Filament Formation by Metabolic Enzymes Is a Specific Adaptation to an Advanced State of Cellular Starvation. eLife 2014, 3, e02409  DOI: 10.7554/eLife.02409
    Google Scholar
    There is no corresponding record for this reference.
  9. 9
    Munder, M. C.; Midtvedt, D.; Franzmann, T.; Nüske, E.; Otto, O.; Herbig, M.; Ulbricht, E.; Müller, P.; Taubenberger, A.; Maharana, S.; Malinovska, L.; Richter, D.; Guck, J.; Zaburdaev, V.; Alberti, S. A PH-Driven Transition of the Cytoplasm from a Fluid- to a Solid-like State Promotes Entry into Dormancy. eLife 2016, 5, e09347  DOI: 10.7554/eLife.09347
    Google Scholar
    9
    A pH-driven transition of the cytoplasm from a fluid- to a solid-like state promotes entry into dormancy
    Munder, Matthias Christoph; Midtvedt, Daniel; Franzmann, Titus; Nueske, Elisabeth; Otto, Oliver; Herbig, Maik; Ulbricht, Elke; Mueller, Paul; Taubenberger, Anna; Maharana, Shovamayee; Malinovska, Liliana; Richter, Doris; Guck, Jochen; Zaburdaev, Vasily; Alberti, Simon
    eLife (2016), 5 (), e09347/1-e09347/30CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)
    Cells can enter into a dormant state when faced with unfavorable conditions. However, how cells enter into and recover from this state is still poorly understood. Here, we study dormancy in different eukaryotic organisms and find it to be assocd. with a significant decrease in the mobility of organelles and foreign tracer particles. We show that this reduced mobility is caused by an influx of protons and a marked acidification of the cytoplasm, which leads to widespread macromol. assembly of proteins and triggers a transition of the cytoplasm to a solid-like state with increased mech. stability. We further demonstrate that this transition is required for cellular survival under conditions of starvation. Our findings have broad implications for understanding alternative physiol. states, such as quiescence and dormancy, and create a new view of the cytoplasm as an adaptable fluid that can reversibly transition into a protective solid-like state.
  10. 10
    Wallace, E. W. J.; Kear-Scott, J. L.; Pilipenko, E. V.; Schwartz, M. H.; Laskowski, P. R.; Rojek, A. E.; Katanski, C. D.; Riback, J. A.; Dion, M. F.; Franks, A. M.; Airoldi, E. M.; Pan, T.; Budnik, B. A.; Drummond, D. A. Reversible, Specific, Active Aggregates of Endogenous Proteins Assemble upon Heat Stress. Cell 2015, 162 (6), 1286– 1298,  DOI: 10.1016/j.cell.2015.08.041
    Google Scholar
    10
    Reversible, Specific, Active Aggregates of Endogenous Proteins Assemble upon Heat Stress
    Wallace, Edward W. J.; Kear-Scott, Jamie L.; Pilipenko, Evgeny V.; Schwartz, Michael H.; Laskowski, Pawel R.; Rojek, Alexandra E.; Katanski, Christopher D.; Riback, Joshua A.; Dion, Michael F.; Franks, Alexander M.; Airoldi, Edoardo M.; Pan, Tao; Budnik, Bogdan A.; Drummond, D. Allan
    Cell (Cambridge, MA, United States) (2015), 162 (6), 1286-1298CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    Heat causes protein misfolding and aggregation and, in eukaryotic cells, triggers aggregation of proteins and RNA into stress granules. We have carried out extensive proteomic studies to quantify heat-triggered aggregation and subsequent disaggregation in budding yeast, identifying >170 endogenous proteins aggregating within minutes of heat shock in multiple subcellular compartments. We demonstrate that these aggregated proteins are not misfolded and destined for degrdn. Stable-isotope labeling reveals that even severely aggregated endogenous proteins are disaggregated without degrdn. during recovery from shock, contrasting with the rapid degrdn. obsd. for many exogenous thermolabile proteins. Although aggregation likely inactivates many cellular proteins, in the case of a heterotrimeric aminoacyl-tRNA synthetase complex, the aggregated proteins remain active with unaltered fidelity. We propose that most heat-induced aggregation of mature proteins reflects the operation of an adaptive, autoregulatory process of functionally significant aggregate assembly and disassembly that aids cellular adaptation to thermal stress.
  11. 11
    Cabrera, M.; Boronat, S.; Marte, L.; Vega, M.; Pérez, P.; Ayté, J.; Hidalgo, E. Chaperone-Facilitated Aggregation of Thermo-Sensitive Proteins Shields Them from Degradation during Heat Stress. Cell Rep. 2020, 30 (7), 2430– 2443,  DOI: 10.1016/j.celrep.2020.01.077
    Google Scholar
    11
    Chaperone-Facilitated Aggregation of Thermo-Sensitive Proteins Shields Them from Degradation during Heat Stress
    Cabrera, Margarita; Boronat, Susanna; Marte, Luis; Vega, Montserrat; Perez, Pilar; Ayte, Jose; Hidalgo, Elena
    Cell Reports (2020), 30 (7), 2430-2443.e4CODEN: CREED8; ISSN:2211-1247. (Cell Press)
    Cells have developed protein quality-control strategies to manage the accumulation of misfolded substrates during heat stress. Using a sol. reporter of misfolding in fission yeast, Rho1. C17R-GFP, we demonstrate that upon mild heat shock, the reporter collapses in protein aggregate centers (PACs). They contain and/or require several chaperones, such as Hsp104, Hsp16, and the Hsp40/70 couple Mas5/Ssa2. Stress granules do not assemble at mild temps. and, therefore, are not required for PAC formation; on the contrary, PACs may serve as nucleation centers for the assembly of stress granules. In contrast to the general belief, the dominant fate of these PACs is not degrdn., and the aggregated reporter can be disassembled by chaperones and recovers native structure and activity. Using mass spectrometry, we show that thermo-unstable endogenous proteins form PACs as well. In conclusion, formation of PACs during heat shock is a chaperone-mediated adaptation strategy.
  12. 12
    Gallardo, P.; Salas-Pino, S.; Daga, R. R. Reversible Protein Aggregation as Cytoprotective Mechanism against Heat Stress. Curr. Genet. 2021, 67, 849– 855,  DOI: 10.1007/s00294-021-01191-2
    Google Scholar
    12
    Reversible protein aggregation as cytoprotective mechanism against heat stress
    Gallardo, Paola; Salas-Pino, Silvia; Daga, Rafael R.
    Current Genetics (2021), 67 (6), 849-855CODEN: CUGED5; ISSN:0172-8083. (Springer)
    A review. Temp. fluctuation is one of the most frequent threats to which organisms are exposed in nature. The activation of gene expression programs that trigger the transcription of heat stress-protective genes is the main cellular response to resist high temps. In addn., reversible accumulation and compartmentalization of thermosensitive proteins in high-order mol. assemblies are emerging as crit. mechanisms to ensure cellular protection upon heat stress. Here, we summarize representative examples of membrane-less intracellular bodies formed upon heat stress in yeasts and human cells and highlight how protein aggregation can be turned into a cytoprotective mechanism.
  13. 13
    Franzmann, T.; Alberti, S. Ubiquitin Protein Helps Cells to Recover from Stress. Nature 2021, 597, 183– 184,  DOI: 10.1038/d41586-021-02197-z
    Google Scholar
    13
    Ubiquitin protein helps cells to recover from stress
    Franzmann, Titus; Alberti, Simon
    Nature (London, United Kingdom) (2021), 597 (7875), 183-184CODEN: NATUAS; ISSN:0028-0836. (Nature Portfolio)
    In stressed cells, proteins and RNA mols. cluster together to form stress granules. It emerges that the small protein modifier ubiquitin is needed to disassemble stress granules in recovering cells.
  14. 14
    Zhao, L.; Vecchi, G.; Vendruscolo, M.; Körner, R.; Hayer-Hartl, M.; Hartl, F. U. The Hsp70 Chaperone System Stabilizes a Thermo-Sensitive Subproteome in E. Coli. Cell Rep. 2019, 28 (5), 1335– 1345,  DOI: 10.1016/j.celrep.2019.06.081
    Google Scholar
    14
    The Hsp70 Chaperone System Stabilizes a Thermo-sensitive Subproteome in E. coli
    Zhao, Liang; Vecchi, Giulia; Vendruscolo, Michele; Koerner, Roman; Hayer-Hartl, Manajit; Hartl, F. Ulrich
    Cell Reports (2019), 28 (5), 1335-1345.e6CODEN: CREED8; ISSN:2211-1247. (Cell Press)
    Stress-inducible mol. chaperones have essential roles in maintaining protein homeostasis, but the extent to which they affect overall proteome stability remains unclear. Here, we analyze the effects of the DnaK (Hsp70) system on protein stability in Escherichia coli using pulse proteolysis combined with quant. proteomics. We quantify ∼1,500 sol. proteins and find ∼500 of these to be protease sensitive under normal growth conditions, indicating a high prevalence of conformationally dynamic proteins, forming a metastable subproteome. Acute heat stress results in the unfolding of an addnl. ∼200 proteins, reflected in the exposure of otherwise buried hydrophobic regions. Overexpression of the DnaK chaperone system markedly stabilizes numerous thermo-sensitive proteins, including multiple ribosomal proteins and large, hetero-oligomeric proteins contg. the evolutionarily ancient c.37 fold (P loop nucleoside triphosphate hydrolases). Thus, the Hsp70 system, in addn. to its known chaperone functions, has a remarkable capacity to stabilize proteins in their folded states under denaturing stress conditions.
  15. 15
    Vabulas, R. M.; Raychaudhuri, S.; Hayer-Hartl, M.; Hartl, F. U. Protein Folding in the Cytoplasm and the Heat Shock Response. Cold Spring Harbor Perspect. Biol. 2010, 2 (12), a004390,  DOI: 10.1101/cshperspect.a004390
    Google Scholar
    15
    Protein folding in the cytoplasm and the heat shock response
    Vabulas, R. Martin; Raychaudhuri, Swasti; Hayer-Hartl, Manajit; Hartl, F. Ulrich
    Cold Spring Harbor Perspectives in Biology (2010), 2 (12), a004390CODEN: CSHPEU; ISSN:1943-0264. (Cold Spring Harbor Laboratory Press)
    A review. Proteins generally must fold into precise three-dimensional conformations to fulfill their biol. functions. In the cell, this fundamental process is aided by mol. chaperones, which act in preventing protein misfolding and aggregation. How this machinery assists newly synthesized polypeptide chains in navigating the complex folding energy landscape is now being understood in considerable detail. The mechanisms that ensure the maintenance of a functional proteome under normal and stress conditions are also of great medical relevance, as the aggregation of proteins that escape the cellular quality control underlies a range of debilitating diseases, including many age-of-onset neurodegenerative disorders.
  16. 16
    Ganesan, S.; Rohde, G.; Eckermann, K.; Sroka, K.; Schaefer, M. K. E.; Dohm, C. P.; Kermer, P.; Haase, G.; Wouters, F.; Bähr, M.; Weishaupt, J. H. Mutant SOD1 Detoxification Mechanisms in Intact Single Cells. Cell Death Differ. 2008, 15 (2), 312– 321,  DOI: 10.1038/sj.cdd.4402262
    Google Scholar
    16
    Mutant SOD1 detoxification mechanisms in intact single cells
    Ganesan, S.; Rohde, G.; Eckermann, K.; Sroka, K.; Schaefer, M. K. E.; Dohm, C. P.; Kermer, P.; Haase, G.; Wouters, F.; Baehr, M.; Weishaupt, J. H.
    Cell Death and Differentiation (2008), 15 (2), 312-321CODEN: CDDIEK; ISSN:1350-9047. (Nature Publishing Group)
    Mutant superoxide dismutase 1 (mtSOD1) causes dominantly inherited amyotrophic lateral sclerosis (ALS). The mechanism for mtSOD1 toxicity remains unknown. Two main hypotheses are the impairment of proteasomal function and chaperone depletion by misfolded mtSOD1. Here, we employed FRET/FLIM and biosensor imaging to quant. localize ubiquitination, as well as chaperone binding of mtSOD1, and to assess their effect on proteasomal and protein folding activities. We found large differences in ubiquitination and chaperone interaction levels for wild-type (wt) SOD1 vs. mtSOD1 in intact single cells. Moreover, SOD1 ubiquitination levels differ between proteasomal structures and cytoplasmic material. Hsp70 binding and ubiquitination of wt and mtSOD1 species are highly correlated, demonstrating the coupled upregulation of both cellular detoxification mechanisms upon mtSOD1 expression. Biosensor imaging in single cells revealed that mtSOD1 expression alters cellular protein folding activity but not proteasomal function in the neuronal cell line examd. Our results provide the first cell-by-cell-anal. of SOD1 ubiquitination and chaperone interaction. Moreover, our study opens new methodol. avenues for cell biol. research on ALS. Cell Death and Differentiation (2008) 15, 312-321; doi:10.1038/sj.cdd.4402262; published online 9 Nov. 2007.
  17. 17
    Wood, R. J.; Ormsby, A. R.; Radwan, M.; Cox, D.; Sharma, A.; Vöpel, T.; Ebbinghaus, S.; Oliveberg, M.; Reid, G. E.; Dickson, A.; Hatters, D. M. A Biosensor-Based Framework to Measure Latent Proteostasis Capacity. Nat. Commun. 2018, 9 (1), 287,  DOI: 10.1038/s41467-017-02562-5
    Google Scholar
    17
    A biosensor-based framework to measure latent proteostasis capacity
    Wood Rebecca J; Ormsby Angelique R; Radwan Mona; Cox Dezerae; Reid Gavin E; Hatters Danny M; Sharma Abhishek; Vopel Tobias; Ebbinghaus Simon; Oliveberg Mikael; Reid Gavin E; Dickson Alex; Dickson Alex
    Nature communications (2018), 9 (1), 287 ISSN:.
    The pool of quality control proteins (QC) that maintains protein-folding homeostasis (proteostasis) is dynamic but can become depleted in human disease. A challenge has been in quantitatively defining the depth of the QC pool. With a new biosensor, flow cytometry-based methods and mathematical modeling we measure the QC capacity to act as holdases and suppress biosensor aggregation. The biosensor system comprises a series of barnase kernels with differing folding stability that engage primarily with HSP70 and HSP90 family proteins. Conditions of proteostasis stimulation and stress alter QC holdase activity and aggregation rates. The method reveals the HSP70 chaperone cycle to be rate limited by HSP70 holdase activity under normal conditions, but this is overcome by increasing levels of the BAG1 nucleotide exchange factor to HSPA1A or activation of the heat shock gene cluster by HSF1 overexpression. This scheme opens new paths for biosensors of disease and proteostasis systems.
  18. 18
    Mateju, D.; Franzmann, T. M.; Patel, A.; Kopach, A.; Boczek, E. E.; Maharana, S.; Lee, H. O.; Carra, S.; Hyman, A. A.; Alberti, S. An Aberrant Phase Transition of Stress Granules Triggered by Misfolded Protein and Prevented by Chaperone Function. EMBO J. 2017, 36 (12), 1669– 1687,  DOI: 10.15252/embj.201695957
    Google Scholar
    18
    An aberrant phase transition of stress granules triggered by misfolded protein and prevented by chaperone function
    Mateju, Daniel; Franzmann, Titus M.; Patel, Avinash; Kopach, Andrii; Boczek, Edgar E.; Maharana, Shovamayee; Lee, Hyun O.; Carra, Serena; Hyman, Anthony A.; Alberti, Simon
    EMBO Journal (2017), 36 (12), 1669-1687CODEN: EMJODG; ISSN:0261-4189. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Stress granules (SG) are membrane-less compartments involved in regulating mRNAs during stress. Aberrant forms of SGs have been implicated in age-related diseases, such as amyotrophic lateral sclerosis (ALS), but the mol. events triggering their formation are still unknown. Here, we found that misfolded proteins, such as ALS-linked variants of SOD1, specifically accumulated and aggregated within SGs in human cells. This decreased the dynamics of SGs, changed SG compn., and triggered an aberrant liq.-to-solid transition of in vitro reconstituted compartments. We showed that chaperone recruitment prevented the formation of aberrant SGs and promoted SG disassembly when the stress subsided. Moreover, we identified a backup system for SG clearance, which involved transport of aberrant SGs to the aggresome and their degrdn. by autophagy. Thus, cells employ a system of SG quality control to prevent accumulation of misfolded proteins and maintain the dynamic state of SGs, which may have relevance for ALS and related diseases.
  19. 19
    Boronat, S.; Cabrera, M.; Hidalgo, E. Spatial Sequestration of Misfolded Proteins as an Active Chaperone-Mediated Process during Heat Stress. Curr. Genet. 2021, 67 (2), 237– 243,  DOI: 10.1007/s00294-020-01135-2
    Google Scholar
    19
    Spatial sequestration of misfolded proteins as an active chaperone-mediated process during heat stress
    Boronat, Susanna; Cabrera, Margarita; Hidalgo, Elena
    Current Genetics (2021), 67 (2), 237-243CODEN: CUGED5; ISSN:0172-8083. (Springer)
    A review. Under thermal stress, different protein quality control (PQC) strategies are activated to maintain an intact proteome, which may vary from one model system to another. Hence thermo-sensitive proteins that lose their active conformation might be refolded with the aid of chaperones or removed by the ubiquitin-proteasome system or the process of autophagy. We have recently developed thermo-sensitive reporters to study PQC in fission yeast and shown the relevance of a third adaptation strategy: the sequestration of misfolded proteins into inclusions which will prevent a rapid degrdn. and allow the refolding once stress ends. These protein inclusions, protein aggregate centers (PACs), contain a broad spectrum of misfolding/aggregation-prone proteins and chaperones involved in their assembly or dissoln. The chaperone couple Mas5/Ssa2 plays a crucial role in PAC formation, whereas the Hsp104 chaperone promotes their disassembly. The absence of aggregates obsd. in cells lacking Mas5 could be also explained by the activation of the transcription factor Hsf1 and the induction of chaperone genes, we have excluded this possibility here demonstrating that increased Hsf1 activity and the subsequent overexpression of chaperones do not prevent the assembly of protein aggregates. Protein deposition at certain locations also constitutes a tactic to inactivate proteins temporally. This is the case of Pyp1, the main phosphatase of the stress response kinase Sty1. Upon stress imposition, misfolded Pyp1 is sequestered into cytosolic protein foci while active Sty1 at the nucleus switches on the transcriptional response. In conclusion, we propose that the assembly of aggregation-like foci, PACs in fission yeast, is a crucial PQC strategy during heat stress, and that the Hsp40 chaperone Mas5 is required for PAC assembly and connects physiol. and heat-shock triggered PQC.
  20. 20
    Cherkasov, V.; Hofmann, S.; Druffel-Augustin, S.; Mogk, A.; Tyedmers, J.; Stoecklin, G.; Bukau, B. Coordination of Translational Control and Protein Homeostasis during Severe Heat Stress. Curr. Biol. 2013, 23 (24), 2452– 2462,  DOI: 10.1016/j.cub.2013.09.058
    Google Scholar
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    Coordination of Translational Control and Protein Homeostasis during Severe Heat Stress
    Cherkasov, Valeria; Hofmann, Sarah; Druffel-Augustin, Silke; Mogk, Axel; Tyedmers, Jens; Stoecklin, Georg; Bukau, Bernd
    Current Biology (2013), 23 (24), 2452-2462CODEN: CUBLE2; ISSN:0960-9822. (Cell Press)
    Exposure of cells to severe heat stress causes not only misfolding and aggregation of proteins but also inhibition of translation and storage of mRNA in cytosolic heat stress granules (heat-SGs), limiting newly synthesized protein influx into overloaded proteome repair systems. How these two heat stress responses connect is unclear. Here, we show that both S. cerevisiae and D. melanogaster heat-SGs contain mRNA, translation machinery components (excluding ribosomes), and mol. chaperones and that heat-SGs coassemble with aggregates of misfolded, heat-labile proteins. Components in these mixed assemblies exhibit distinct mol. motilities reflecting differential trapping. We demonstrate that heat-SG disassembly and restoration of translation activity during heat stress recovery is intimately linked to disaggregation of damaged proteins present in the mixed assemblies and requires Hsp104 and Hsp70 activity. Chaperone-driven protein disaggregation directly coordinates timing of translation reinitiation with protein folding capacity during cellular protein quality surveillance, enabling efficient protein homeostasis.
  21. 21
    Hipp, M. S.; Kasturi, P.; Hartl, F. U. The Proteostasis Network and Its Decline in Ageing. Nat. Rev. Mol. Cell Biol. 2019, 20 (7), 421,  DOI: 10.1038/s41580-019-0101-y
    Google Scholar
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    The proteostasis network and its decline in ageing
    Hipp, Mark S.; Kasturi, Prasad; Hartl, F. Ulrich
    Nature Reviews Molecular Cell Biology (2019), 20 (7), 421-435CODEN: NRMCBP; ISSN:1471-0072. (Nature Research)
    Ageing is a major risk factor for the development of many diseases, prominently including neurodegenerative disorders such as Alzheimer disease and Parkinson disease. A hallmark of many age-related diseases is the dysfunction in protein homeostasis (proteostasis), leading to the accumulation of protein aggregates. In healthy cells, a complex proteostasis network, comprising mol. chaperones and proteolytic machineries and their regulators, operates to ensure the maintenance of proteostasis. These factors coordinate protein synthesis with polypeptide folding, the conservation of protein conformation and protein degrdn. However, sustaining proteome balance is a challenging task in the face of various external and endogenous stresses that accumulate during ageing. These stresses lead to the decline of proteostasis network capacity and proteome integrity. The resulting accumulation of misfolded and aggregated proteins affects, in particular, postmitotic cell types such as neurons, manifesting in disease. Recent analyses of proteome-wide changes that occur during ageing inform strategies to improve proteostasis. The possibilities of pharmacol. augmentation of the capacity of proteostasis networks hold great promise for delaying the onset of age-related pathologies assocd. with proteome deterioration and for extending healthspan.
  22. 22
    Patel, A.; Lee, H. O.; Jawerth, L.; Maharana, S.; Jahnel, M.; Hein, M. Y.; Stoynov, S.; Mahamid, J.; Saha, S.; Franzmann, T. M.; Pozniakovski, A.; Poser, I.; Maghelli, N.; Royer, L. A.; Weigert, M.; Myers, E. W.; Grill, S.; Drechsel, D.; Hyman, A. A.; Alberti, S. A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation. Cell 2015, 162 (5), 1066– 1077,  DOI: 10.1016/j.cell.2015.07.047
    Google Scholar
    22
    A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation
    Patel, Avinash; Lee, Hyun O.; Jawerth, Louise; Maharana, Shovamayee; Jahnel, Marcus; Hein, Marco Y.; Stoynov, Stoyno; Mahamid, Julia; Saha, Shambaditya; Franzmann, Titus M.; Pozniakovski, Andrej; Poser, Ina; Maghelli, Nicola; Royer, Loic A.; Weigert, Martin; Myers, Eugene W.; Grill, Stephan; Drechsel, David; Hyman, Anthony A.; Alberti, Simon
    Cell (Cambridge, MA, United States) (2015), 162 (5), 1066-1077CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    Many proteins contain disordered regions of low-sequence complexity, which cause aging-assocd. diseases because they are prone to aggregate. Here, we study FUS, a prion-like protein contg. intrinsically disordered domains assocd. with the neurodegenerative disease ALS. We show that, in cells, FUS forms liq. compartments at sites of DNA damage and in the cytoplasm upon stress. We confirm this by reconstituting liq. FUS compartments in vitro. Using an in vitro "aging" expt., we demonstrate that liq. droplets of FUS protein convert with time from a liq. to an aggregated state, and this conversion is accelerated by patient-derived mutations. We conclude that the physiol. role of FUS requires forming dynamic liq.-like compartments. We propose that liq.-like compartments carry the trade-off between functionality and risk of aggregation and that aberrant phase transitions within liq.-like compartments lie at the heart of ALS and, presumably, other age-related diseases.
  23. 23
    Marrone, L.; Drexler, H. C. A.; Wang, J.; Tripathi, P.; Distler, T.; Heisterkamp, P.; Anderson, E. N.; Kour, S.; Moraiti, A.; Maharana, S.; Bhatnagar, R.; Belgard, T. G.; Tripathy, V.; Kalmbach, N.; Hosseinzadeh, Z.; Crippa, V.; Abo-Rady, M.; Wegner, F.; Poletti, A.; Troost, D.; Aronica, E.; Busskamp, V.; Weis, J.; Pandey, U. B.; Hyman, A. A.; Alberti, S.; Goswami, A.; Sterneckert, J. FUS Pathology in ALS Is Linked to Alterations in Multiple ALS-Associated Proteins and Rescued by Drugs Stimulating Autophagy. Acta Neuropathol. 2019, 138 (1), 67– 84,  DOI: 10.1007/s00401-019-01998-x
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    FUS pathology in ALS is linked to alterations in multiple ALS-associated proteins and rescued by drugs stimulating autophagy
    Marrone, Lara; Drexler, Hannes C. A.; Wang, Jie; Tripathi, Priyanka; Distler, Tania; Heisterkamp, Patrick; Anderson, Eric Nathaniel; Kour, Sukhleen; Moraiti, Anastasia; Maharana, Shovamayee; Bhatnagar, Rajat; Belgard, T. Grant; Tripathy, Vadreenath; Kalmbach, Norman; Hosseinzadeh, Zohreh; Crippa, Valeria; Abo-Rady, Masin; Wegner, Florian; Poletti, Angelo; Troost, Dirk; Aronica, Eleonora; Busskamp, Volker; Weis, Joachim; Pandey, Udai Bhan; Hyman, Anthony A.; Alberti, Simon; Goswami, Anand; Sterneckert, Jared
    Acta Neuropathologica (2019), 138 (1), 67-84CODEN: ANPTAL; ISSN:0001-6322. (Springer)
    Amyotrophic lateral sclerosis (ALS) is a lethal disease characterized by motor neuron degeneration and assocd. with aggregation of nuclear RNA-binding proteins (RBPs), including FUS. How FUS aggregation and neurodegeneration are prevented in healthy motor neurons remain critically unanswered questions. Here, we use a combination of ALS patient autopsy tissue and induced pluripotent stem cell-derived neurons to study the effects of FUS mutations on RBP homeostasis. We show that FUS' tendency to aggregate is normally buffered by interacting RBPs, but this buffering is lost when FUS mislocalizes to the cytoplasm due to ALS mutations. The presence of aggregation-prone FUS in the cytoplasm causes imbalances in RBP homeostasis that exacerbate neurodegeneration. However, enhancing autophagy using small mols. reduces cytoplasmic FUS, restores RBP homeostasis and rescues motor function in vivo. We conclude that disruption of RBP homeostasis plays a crit. role in FUS-ALS and can be treated by stimulating autophagy.
  24. 24
    Alberti, S.; Hyman, A. A. Biomolecular Condensates at the Nexus of Cellular Stress, Protein Aggregation Disease and Ageing. Nat. Rev. Mol. Cell Biol. 2021, 22 (3), 196– 213,  DOI: 10.1038/s41580-020-00326-6
    Google Scholar
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    Biomolecular condensates at the nexus of cellular stress, protein aggregation disease and ageing
    Alberti, Simon; Hyman, Anthony A.
    Nature Reviews Molecular Cell Biology (2021), 22 (3), 196-213CODEN: NRMCBP; ISSN:1471-0072. (Nature Research)
    A review. Biomol. condensates are membraneless intracellular assemblies that often form via liq.-liq. phase sepn. and have the ability to conc. biopolymers. Research over the past 10 years has revealed that condensates play fundamental roles in cellular organization and physiol., and our understanding of the mol. principles, components and forces underlying their formation has substantially increased. Condensate assembly is tightly regulated in the intracellular environment, and failure to control condensate properties, formation and dissoln. can lead to protein misfolding and aggregation, which are often the cause of ageing-assocd. diseases. In this Review, we describe the mechanisms and regulation of condensate assembly and dissoln., highlight recent advances in understanding the role of biomol. condensates in ageing and disease, and discuss how cellular stress, ageing-related loss of homeostasis and a decline in protein quality control may contribute to the formation of aberrant, disease-causing condensates. Our improved understanding of condensate pathol. provides a promising path for the treatment of protein aggregation diseases.
  25. 25
    Hofmann, S.; Kedersha, N.; Anderson, P.; Ivanov, P. Molecular Mechanisms of Stress Granule Assembly and Disassembly. Biochim. Biophys. Acta, Mol. Cell Res. 2021, 1868 (1), 118876,  DOI: 10.1016/j.bbamcr.2020.118876
    Google Scholar
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    Molecular mechanisms of stress granule assembly and disassembly
    Hofmann, Sarah; Kedersha, Nancy; Anderson, Paul; Ivanov, Pavel
    Biochimica et Biophysica Acta, Molecular Cell Research (2021), 1868 (1), 118876CODEN: BBAMCO; ISSN:0167-4889. (Elsevier B.V.)
    A review. Stress granules (SGs) are membrane-less ribonucleoprotein (RNP)-based cellular compartments that form in the cytoplasm of a cell upon exposure to various environmental stressors. SGs contain a large set of proteins, as well as mRNAs that have been stalled in translation as a result of stress-induced polysome disassembly. Despite the fact that SGs have been extensively studied for many years, their function is still not clear. They presumably help the cell to cope with the encountered stress, and facilitate the recovery process after stress removal upon which SGs disassemble. Aberrant formation of SGs and impaired SG disassembly majorly contribute to various pathol. phenomena in cancer, viral infections, and neurodegeneration. The assembly of SGs is largely driven by liq.-liq. phase sepn. (LLPS), however, the mol. mechanisms behind that are not fully understood. Recent studies have proposed a novel mechanism for SG formation that involves the interplay of a large interaction network of mRNAs and proteins. Here, we review this novel concept of SG assembly, and discuss the current insights into SG disassembly.
  26. 26
    Burke, K. A.; Janke, A. M.; Rhine, C. L.; Fawzi, N. L. Residue-by-Residue View of In Vitro FUS Granules That Bind the C-Terminal Domain of RNA Polymerase II. Mol. Cell 2015, 60 (2), 231– 241,  DOI: 10.1016/j.molcel.2015.09.006
    Google Scholar
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    Residue-by-Residue View of In Vitro FUS Granules that Bind the C-Terminal Domain of RNA Polymerase II
    Burke, Kathleen A.; Janke, Abigail M.; Rhine, Christy L.; Fawzi, Nicolas L.
    Molecular Cell (2015), 60 (2), 231-241CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)
    Phase-sepd. states of proteins underlie ribonucleoprotein (RNP) granules and nuclear RNA-binding protein assemblies that may nucleate protein inclusions assocd. with neurodegenerative diseases. We report that the N-terminal low-complexity domain of the RNA-binding protein Fused in Sarcoma (FUS LC) is structurally disordered and forms a liq.-like phase-sepd. state resembling RNP granules. This state directly binds the C-terminal domain of RNA polymerase II. Phase-sepd. FUS lacks static structures as probed by fluorescence microscopy, indicating they are distinct from both protein inclusions and hydrogels. We use soln. NMR spectroscopy to directly probe the dynamic architecture within FUS liq. phase-sepd. assemblies. Importantly, we find that FUS LC retains disordered secondary structure even in the liq. phase-sepd. state. Therefore, we propose that disordered protein granules, even those made of aggregation-prone prion-like domains, are dynamic and disordered mol. assemblies with transiently formed protein-protein contacts.
  27. 27
    Molliex, A.; Temirov, J.; Lee, J.; Coughlin, M.; Kanagaraj, A. P.; Kim, H. J.; Mittag, T.; Taylor, J. P. Phase Separation by Low Complexity Domains Promotes Stress Granule Assembly and Drives Pathological Fibrillization. Cell 2015, 163 (1), 123– 133,  DOI: 10.1016/j.cell.2015.09.015
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    Phase Separation by Low Complexity Domains Promotes Stress Granule Assembly and Drives Pathological Fibrillization
    Molliex, Amandine; Temirov, Jamshid; Lee, Jihun; Coughlin, Maura; Kanagaraj, Anderson P.; Kim, Hong Joo; Mittag, Tanja; Taylor, J. Paul
    Cell (Cambridge, MA, United States) (2015), 163 (1), 123-133CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    Stress granules are membrane-less organelles composed of RNA-binding proteins (RBPs) and RNA. Functional impairment of stress granules has been implicated in amyotrophic lateral sclerosis, frontotemporal dementia, and multisystem proteinopathy-diseases that are characterized by fibrillar inclusions of RBPs. Genetic evidence suggests a link between persistent stress granules and the accumulation of pathol. inclusions. Here, we demonstrate that the disease-related RBP hnRNPA1 undergoes liq.-liq. phase sepn. (LLPS) into protein-rich droplets mediated by a low complexity sequence domain (LCD). While the LCD of hnRNPA1 is sufficient to mediate LLPS, the RNA recognition motifs (RRMs) contribute to LLPS in the presence of RNA, giving rise to several mechanisms for regulating assembly. Importantly, while not required for LLPS, fibrillization is enhanced in protein-rich droplets. We suggest that LCD-mediated LLPS contributes to the assembly of stress granules and their liq. properties and provides a mechanistic link between persistent stress granules and fibrillar protein pathol. in disease.
  28. 28
    Guillén-Boixet, J.; Kopach, A.; Holehouse, A. S.; Wittmann, S.; Jahnel, M.; Schlüßler, R.; Kim, K.; Trussina, I. R. E. A.; Wang, J.; Mateju, D.; Poser, I.; Maharana, S.; Ruer-Gruß, M.; Richter, D.; Zhang, X.; Chang, Y.-T.; Guck, J.; Honigmann, A.; Mahamid, J.; Hyman, A. A.; Pappu, R. V.; Alberti, S.; Franzmann, T. M. RNA-Induced Conformational Switching and Clustering of G3BP Drive Stress Granule Assembly by Condensation. Cell 2020, 181 (2), 346– 361,  DOI: 10.1016/j.cell.2020.03.049
    Google Scholar
    28
    RNA-Induced Conformational Switching and Clustering of G3BP Drive Stress Granule Assembly by Condensation
    Guillen-Boixet, Jordina; Kopach, Andrii; Holehouse, Alex S.; Wittmann, Sina; Jahnel, Marcus; Schluessler, Raimund; Kim, Kyoohyun; Trussina, Irmela R. E. A.; Wang, Jie; Mateju, Daniel; Poser, Ina; Maharana, Shovamayee; Ruer-Gruss, Martine; Richter, Doris; Zhang, Xiaojie; Chang, Young-Tae; Guck, Jochen; Honigmann, Alf; Mahamid, Julia; Hyman, Anthony A.; Pappu, Rohit V.; Alberti, Simon; Franzmann, Titus M.
    Cell (Cambridge, MA, United States) (2020), 181 (2), 346-361.e17CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    Stressed cells shut down translation, release mRNA mols. from polysomes, and form stress granules (SGs) via a network of interactions that involve G3BP. Here we focus on the mechanistic underpinnings of SG assembly. We show that, under non-stress conditions, G3BP adopts a compact auto-inhibited state stabilized by electrostatic intramol. interactions between the intrinsically disordered acidic tracts and the pos. charged arginine-rich region. Upon release from polysomes, unfolded mRNAs outcompete G3BP auto-inhibitory interactions, engendering a conformational transition that facilitates clustering of G3BP through protein-RNA interactions. Subsequent phys. crosslinking of G3BP clusters drives RNA mols. into networked RNA/protein condensates. We show that G3BP condensates impede RNA entanglement and recruit addnl. client proteins that promote SG maturation or induce a liq.-to-solid transition that may underlie disease. We propose that condensation coupled to conformational rearrangements and heterotypic multivalent interactions may be a general principle underlying RNP granule assembly.
  29. 29
    Conicella, A. E.; Zerze, G. H.; Mittal, J.; Fawzi, N. L. ALS Mutations Disrupt Phase Separation Mediated by α-Helical Structure in the TDP-43 Low-Complexity C-Terminal Domain. Structure 2016, 24 (9), 1537– 1549,  DOI: 10.1016/j.str.2016.07.007
    Google Scholar
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    ALS Mutations Disrupt Phase Separation Mediated by α-Helical Structure in the TDP-43 Low-Complexity C-Terminal Domain
    Conicella, Alexander E.; Zerze, Gul H.; Mittal, Jeetain; Fawzi, Nicolas L.
    Structure (Oxford, United Kingdom) (2016), 24 (9), 1537-1549CODEN: STRUE6; ISSN:0969-2126. (Elsevier Ltd.)
    RNA-binding protein TDP-43 mediates essential RNA processing but forms cytoplasmic neuronal inclusions via its C-terminal domain (CTD) in amyotrophic lateral sclerosis (ALS). It remains unclear if aggregated TDP-43 is neurotoxic and if ∼50 ALS-assocd. missense mutations in TDP-43 CTD promote aggregation, or if loss of normal function plays a role in disease. Recent work points to the ability of related proteins to assemble into functional phase-sepd. ribonucleoprotein granules via their structurally disordered prion-like domains. Here, we provide at. details on the structure and assembly of the low-complexity CTD of TDP-43 into liq.-liq. phase-sepd. in vitro granules and demonstrate that ALS-assocd. variants disrupt interactions within granules. Using NMR spectroscopy, simulation, and microscopy, we find that a subregion cooperatively but transiently folds into a helix that mediates TDP-43 phase sepn. ALS-assocd. mutations disrupt phase sepn. by inhibiting interaction and helical stabilization. Therefore, ALS-assocd. mutations can disrupt TDP-43 interactions, affecting function beyond encouraging aggregation.
  30. 30
    Zhou, H.-X.; Nguemaha, V.; Mazarakos, K.; Qin, S. Why Do Disordered and Structured Proteins Behave Differently in Phase Separation?. Trends Biochem. Sci. 2018, 43 (7), 499– 516,  DOI: 10.1016/j.tibs.2018.03.007
    Google Scholar
    30
    Why Do Disordered and Structured Proteins Behave Differently in Phase Separation?
    Zhou, Huan-Xiang; Nguemaha, Valery; Mazarakos, Konstantinos; Qin, Sanbo
    Trends in Biochemical Sciences (2018), 43 (7), 499-516CODEN: TBSCDB; ISSN:0968-0004. (Elsevier Ltd.)
    A review. Intracellular membraneless organelles and their myriad cellular functions have garnered tremendous recent interest. It is becoming well accepted that they form via liq.-liq. phase sepn. (LLPS) of protein mixts. (often including RNA), where the organelles correspond to a protein-rich droplet phase coexisting with a protein-poor bulk phase. The major protein components contain disordered regions and often also RNA-binding domains, and the disordered fragments on their own easily undergo LLPS. By contrast, LLPS for structured proteins has been obsd. infrequently. The contrasting phase behaviors can be explained by modeling disordered and structured proteins, resp., as polymers and colloids. These phys. models also provide a better understanding of the regulation of droplet formation by cellular signals and its dysregulation leading to diseases.
  31. 31
    Ruff, K. M.; Choi, Y. H.; Cox, D.; Ormsby, A. R.; Myung, Y.; Ascher, D. B.; Radford, S. E.; Pappu, R. V.; Hatters, D. M. Sequence Grammar Underlying Unfolding and Phase Separation of Globular Proteins. bioRxiv (Biophysics) , August 20, 2021, 2021.08.20.457073. DOI: 10.1101/2021.08.20.457073 (accessed 2021–09–08).
    Google Scholar
    There is no corresponding record for this reference.
  32. 32
    Ribeiro, S.; Ebbinghaus, S.; Marcos, J. C. Protein Folding and Quinary Interactions: Creating Cellular Organisation through Functional Disorder. FEBS Lett. 2018, 592 (18), 3040– 3053,  DOI: 10.1002/1873-3468.13211
    Google Scholar
    32
    Protein folding and quinary interactions: creating cellular organisation through functional disorder
    Ribeiro, Sara; Ebbinghaus, Simon; Marcos, Joao C.
    FEBS Letters (2018), 592 (18), 3040-3053CODEN: FEBLAL; ISSN:0014-5793. (Wiley-Blackwell)
    The marginal stability of globular proteins in the cell is detd. by the balance between excluded vol. effect and soft interactions. Quinary interactions are a type of soft interactions involved in intracellular organization and known to have stabilizing or destabilizing effects on globular proteins. Recent studies suggest that globular proteins have structural flexibility, exhibiting more than one functional state. Here, we propose that the quinary-induced destabilization can be sufficient to produce functional partially unfolded states of globular proteins. The biol. relevance of this mechanism is explored, involving intracellular phase sepn. and regulatory stress response mechanisms.
  33. 33
    Ribeiro, S. S.; Samanta, N.; Ebbinghaus, S.; Marcos, J. C. The Synergic Effect of Water and Biomolecules in Intracellular Phase Separation. Nat. Rev. Chem. 2019, 3 (9), 552– 561,  DOI: 10.1038/s41570-019-0120-4
    Google Scholar
    33
    The synergic effect of water and biomolecules in intracellular phase separation
    Ribeiro, Sara S.; Samanta, Nirnay; Ebbinghaus, Simon; Marcos, Joao C.
    Nature Reviews Chemistry (2019), 3 (9), 552-561CODEN: NRCAF7; ISSN:2397-3358. (Nature Research)
    Phase sepn. has long been obsd. within aq. mixts. of two or more different compds., such as proteins, salts, polysaccharides and synthetic polymers. A growing body of exptl. evidence indicates that phase sepn. also takes place inside living cells, where intrinsically disordered proteins and other mols. such as RNA are thought to assemble into membraneless organelles. These structures represent a new paradigm of intracellular organization and compartmentalization, in which biochem. processes can be coordinated in space and time. Two thermodn. driving forces have been proposed for phase sepn.: the strengths of macromol.-macromol. and macromol.-H2O interactions, and the perturbation of H2O structure about different macromols. In this Perspective, we propose that both driving forces act in a concerted manner to promote phase sepn., which we describe in the context of the well-known structural dynamics of intrinsically disordered proteins in the cellular milieu. We further suggest that this effect can be extended to explain how the partial unfolding of globular proteins can lead to intracellular phase sepn.
  34. 34
    Mathieu, C.; Pappu, R. V.; Taylor, J. P. Beyond Aggregation: Pathological Phase Transitions in Neurodegenerative Disease. Science 2020, 370 (6512), 56– 60,  DOI: 10.1126/science.abb8032
    Google Scholar
    34
    Beyond aggregation: Pathological phase transitions in neurodegenerative disease
    Mathieu, Cecile; Pappu, Rohit V.; Taylor, J. Paul
    Science (Washington, DC, United States) (2020), 370 (6512), 56-60CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
    Over the past decade, phase transitions have emerged as a fundamental mechanism of cellular organization. In parallel, a wealth of evidence has accrued indicating that aberrations in phase transitions are early events in the pathogenesis of several neurodegenerative diseases. We review the key evidence of defects at multiple levels, from phase transition of individual proteins to the dynamic behavior of complex, multicomponent condensates in neurodegeneration. We also highlight two concepts, dynamical arrest and heterotypic buffering, that are key to understanding how pathol. phase transitions relate to pleiotropic defects in cellular functions and the accrual of proteinaceous deposits at end-stage disease. These insights not only illuminate disease etiol. but also are likely to guide the development of therapeutic interventions to restore homeostasis.
  35. 35
    Jahn, T. R.; Radford, S. E. Folding versus Aggregation: Polypeptide Conformations on Competing Pathways. Arch. Biochem. Biophys. 2008, 469 (1), 100– 117,  DOI: 10.1016/j.abb.2007.05.015
    Google Scholar
    35
    Folding versus aggregation: Polypeptide conformations on competing pathways
    Jahn, Thomas R.; Radford, Sheena E.
    Archives of Biochemistry and Biophysics (2008), 469 (1), 100-117CODEN: ABBIA4; ISSN:0003-9861. (Elsevier)
    A review. Protein aggregation has now become recognized as an important and generic aspect of protein energy landscapes. Since the discovery that numerous human diseases are caused by protein aggregation, the biophys. characterization of misfolded states and their aggregation mechanisms has received increased attention. Utilizing exptl. techniques and computational approaches established for the anal. of protein folding reactions has ensured rapid advances in the study of pathways leading to amyloid fibrils and amyloid-related aggregates. Here, the authors describe recent exptl. and theor. advances in the elucidation of the conformational properties of dynamic, heterogeneous and/or insol. protein ensembles populated on complex, multidimensional protein energy landscapes. The authors discuss the current understanding of aggregation mechanisms in this context and describe how the synergy between biochem., biophys., and cell-biol. expts. are beginning to provide detailed insights into the partitioning of non-native species between protein folding and aggregation pathways.
  36. 36
    Grad, L. I.; Yerbury, J. J.; Turner, B. J.; Guest, W. C.; Pokrishevsky, E.; O’Neill, M. A.; Yanai, A.; Silverman, J. M.; Zeineddine, R.; Corcoran, L.; Kumita, J. R.; Luheshi, L. M.; Yousefi, M.; Coleman, B. M.; Hill, A. F.; Plotkin, S. S.; Mackenzie, I. R.; Cashman, N. R. Intercellular Propagated Misfolding of Wild-Type Cu/Zn Superoxide Dismutase Occurs via Exosome-Dependent and -Independent Mechanisms. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (9), 3620– 3625,  DOI: 10.1073/pnas.1312245111
    Google Scholar
    36
    Intercellular propagated misfolding of wild-type Cu/Zn superoxide dismutase occurs via exosome-dependent and -independent mechanisms
    Grad, Leslie I.; Yerbury, Justin J.; Turner, Bradley J.; Guest, William C.; Pokrishevsky, Edward; O'Neill, Megan A.; Yanai, Anat; Silverman, Judith M.; Zeineddine, Rafaa; Corcoran, Lisa; Kumita, Janet R.; Luheshi, Leila M.; Yousefi, Masoud; Coleman, Bradley M.; Hill, Andrew F.; Plotkin, Steven S.; MacKenzie, Ian R.; Cashman, Neil R.
    Proceedings of the National Academy of Sciences of the United States of America (2014), 111 (9), 3620-3625CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Amyotrophic lateral sclerosis (ALS) is predominantly sporadic, but assocd. with heritable genetic mutations in 5-10% of cases, including those in Cu/Zn superoxide dismutase (SOD1). We previously showed that misfolding of SOD1 can be transmitted to endogenous human wild-type SOD1 (HuWtSOD1) in an intracellular compartment. Using NSC-34 motor neuron-like cells, we now demonstrate that misfolded mutant and HuWtSOD1 can traverse between cells via two nonexclusive mechanisms: protein aggregates released from dying cells and taken up by macropinocytosis, and exosomes secreted from living cells. Furthermore, once HuWtSOD1 propagation has been established, misfolding of HuWtSOD1 can be efficiently and repeatedly propagated between HEK293 cell cultures via conditioned media over multiple passages, and to cultured mouse primary spinal cord cells transgenically expressing HuWtSOD1, but not to cells derived from nontransgenic littermates. Conditioned media transmission of HuWtSOD1 misfolding in HEK293 cells is blocked by HuWtSOD1 siRNA knockdown, consistent with human SOD1 being a substrate for conversion, and attenuated by ultracentrifugation or incubation with SOD1 misfolding-specific antibodies, indicating a relatively massive transmission particle which possesses antibody-accessible SOD1. Finally, misfolded and protease-sensitive HuWtSOD1 comprises up to 4% of total SOD1 in spinal cords of patients with sporadic ALS (SALS). Propagation of HuWtSOD1 misfolding, and its subsequent cell-to-cell transmission, is thus a candidate process for the mol. pathogenesis of SALS, which may provide novel treatment and biomarker targets for this devastating disease.
  37. 37
    Sen Mojumdar, S.; Scholl, Z. N.; Dee, D. R.; Rouleau, L.; Anand, U.; Garen, C.; Woodside, M. T. Partially Native Intermediates Mediate Misfolding of SOD1 in Single-Molecule Folding Trajectories. Nat. Commun. 2017, 8 (1), 1881,  DOI: 10.1038/s41467-017-01996-1
    Google Scholar
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    Partially native intermediates mediate misfolding of SOD1 in single-molecule folding trajectories
    Sen Mojumdar Supratik; N Scholl Zackary; Dee Derek R; Rouleau Logan; Anand Uttam; Garen Craig; Woodside Michael T; Woodside Michael T
    Nature communications (2017), 8 (1), 1881 ISSN:.
    Prion-like misfolding of superoxide dismutase 1 (SOD1) is associated with the disease ALS, but the mechanism of misfolding remains unclear, partly because misfolding is difficult to observe directly. Here we study the most misfolding-prone form of SOD1, reduced un-metallated monomers, using optical tweezers to measure unfolding and refolding of single molecules. We find that the folding is more complex than suspected, resolving numerous previously undetected intermediate states consistent with the formation of individual β-strands in the native structure. We identify a stable core of the protein that unfolds last and refolds first, and directly observe several distinct misfolded states that branch off from the native folding pathways at specific points after the formation of the stable core. Partially folded intermediates thus play a crucial role mediating between native and non-native folding. These results suggest an explanation for SOD1's propensity for prion-like misfolding and point to possible targets for therapeutic intervention.
  38. 38
    Lang, L.; Zetterström, P.; Brännström, T.; Marklund, S. L.; Danielsson, J.; Oliveberg, M. SOD1 Aggregation in ALS Mice Shows Simplistic Test Tube Behavior. Proc. Natl. Acad. Sci. U. S. A. 2015, 112 (32), 9878– 9883,  DOI: 10.1073/pnas.1503328112
    Google Scholar
    38
    SOD1 aggregation in ALS mice shows simplistic test tube behavior
    Lang, Lisa; Zetterstroem, Per; Braennstroem, Thomas; Marklund, Stefan L.; Danielsson, Jens; Oliveberg, Mikael
    Proceedings of the National Academy of Sciences of the United States of America (2015), 112 (32), 9878-9883CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    A longstanding challenge in studies of neurodegenerative disease has been that the pathol. protein aggregates in live tissue are not amenable to structural and kinetic anal. by conventional methods. The situation is put in focus by current progress in demarcating protein aggregation in vitro, exposing new mechanistic details that are now calling for quant. in vivo comparison. Here, the authors bridge this gap by presenting a direct comparison of the aggregation kinetics of amyotrophic lateral sclerosis (ALS)-assocd. superoxide dismutase 1 (SOD1) in vitro and in transgenic mice. The results based on tissue sampling by quant. antibody assays showed that the SOD1 fibrillation kinetics in vitro mirrored with remarkable accuracy the spinal cord aggregate buildup and disease progression in transgenic mice. This similarity between in vitro and in vivo data suggested that, despite the complexity of live tissue, SOD1 aggregation follows robust and simplistic rules, providing new mechanistic insights into the ALS pathol. and organism-level manifestation of protein aggregation phenomena in general.
  39. 39
    Gnutt, D.; Timr, S.; Ahlers, J.; König, B.; Manderfeld, E.; Heyden, M.; Sterpone, F.; Ebbinghaus, S. Stability Effect of Quinary Interactions Reversed by Single Point Mutations. J. Am. Chem. Soc. 2019, 141 (11), 4660– 4669,  DOI: 10.1021/jacs.8b13025
    Google Scholar
    39
    Stability Effect of Quinary Interactions Reversed by Single Point Mutations
    Gnutt, David; Timr, Stepan; Ahlers, Jonas; Koenig, Benedikt; Manderfeld, Emily; Heyden, Matthias; Sterpone, Fabio; Ebbinghaus, Simon
    Journal of the American Chemical Society (2019), 141 (11), 4660-4669CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    In cells, proteins are embedded in a crowded environment that controls their properties via manifold avenues including weak protein-macromol. interactions. A mol. level understanding of these quinary interactions and their contribution to protein stability, function, and localization in the cell is central to modern structural biol. Using a mutational anal. to quantify the energetic contributions of single amino acids to the stability of the ALS related protein superoxide dismutase I (SOD1) in mammalian cells, we show that quinary interactions destabilize SOD1 by a similar energetic offset for most of the mutants, but there are notable exceptions: Mutants that alter its surface properties can even lead to a stabilization of the protein in the cell as compared to the test tube. In conclusion, quinary interactions can amplify and even reverse the mutational response of proteins, being a key aspect in pathogenic protein misfolding and aggregation.
  40. 40
    Wright, G. S. A.; Antonyuk, S. V.; Hasnain, S. S. The Biophysics of Superoxide Dismutase-1 and Amyotrophic Lateral Sclerosis. Q. Rev. Biophys. 2019, 52, e12,  DOI: 10.1017/S003358351900012X
    Google Scholar
    40
    The biophysics of superoxide dismutase-1 and amyotrophic lateral sclerosis
    Wright, Gareth S. A.; Antonyuk, Svetlana V.; Hasnain, S. Samar
    Quarterly Reviews of Biophysics (2019), 52 (), e12CODEN: QURBAW; ISSN:0033-5835. (Cambridge University Press)
    The possibility of reduced structural integrity was suggested by the first crystal structures of human SOD1 even before deleterious mutations in the sod1 gene were linked to the ALS. This concept evolved in the intervening years as an impressive array of biophys. studies examd. the characteristics of mutant SOD1 in great detail. We now recognize how ALS-related mutations perturb the SOD1 maturation processes, reduce its ability to fold and reduce its thermal stability and half-life. Mutant SOD1 is therefore predisposed to monomerisation, non-canonical self-interactions, the formation of small misfolded oligomers and ultimately accumulation in the tell-tale insol. inclusions found within the neurons of ALS patients. We have also seen that several post-translational modifications could push wildtype SOD1 down this toxic pathway. Recently we have come to view ALS as a prion-like disease where both the symptoms, and indeed SOD1 misfolding itself, are transmitted to neighboring cells. This raises the possibility of intervention after the initial disease presentation. Several small-mol. and biol.-based strategies have been devised which directly target the SOD1 mol. to change the behavior thought to be responsible for ALS. Here we provide a comprehensive review of the many biophys. advances that sculpted our view of SOD1 biol. and the recent work that aims to apply this knowledge for therapeutic outcomes in ALS.
  41. 41
    Gal, J.; Kuang, L.; Barnett, K. R.; Zhu, B. Z.; Shissler, S. C.; Korotkov, K. V.; Hayward, L. J.; Kasarskis, E. J.; Zhu, H. ALS Mutant SOD1 Interacts with G3BP1 and Affects Stress Granule Dynamics. Acta Neuropathol. 2016, 132 (4), 563– 576,  DOI: 10.1007/s00401-016-1601-x
    Google Scholar
    41
    ALS mutant SOD1 interacts with G3BP1 and affects stress granule dynamics
    Gal, Jozsef; Kuang, Lisha; Barnett, Kelly R.; Zhu, Brian Z.; Shissler, Susannah C.; Korotkov, Konstantin V.; Hayward, Lawrence J.; Kasarskis, Edward J.; Zhu, Haining
    Acta Neuropathologica (2016), 132 (4), 563-576CODEN: ANPTAL; ISSN:0001-6322. (Springer)
    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. Mutations in Cu/Zn superoxide dismutase (SOD1) are responsible for approx. 20 % of the familial ALS cases. ALS-causing SOD1 mutants display a gain-of-toxicity phenotype, but the nature of this toxicity is still not fully understood. The Ras GTPase-activating protein-binding protein G3BP1 plays a crit. role in stress granule dynamics. Alterations in the dynamics of stress granules have been reported in several other forms of ALS unrelated to SOD1. To our surprise, the mutant G93A SOD1 transgenic mice exhibited pathol. cytoplasmic inclusions that co-localized with G3BP1-pos. granules in spinal cord motor neurons. The co-localization was also obsd. in fibroblast cells derived from familial ALS patient carrying SOD1 mutation L144F. Mutant SOD1, unlike wild-type SOD1, interacted with G3BP1 in an RNA-independent manner. Moreover, the interaction is specific for G3BP1 since mutant SOD1 showed little interaction with four other RNA-binding proteins implicated in ALS. The RNA-binding RRM domain of G3BP1 and two particular phenylalanine residues (F380 and F382) are crit. for this interaction. Mutant SOD1 delayed the formation of G3BP1- and TIA1-pos. stress granules in response to hyperosmolar shock and arsenite treatment in N2A cells. In summary, the aberrant mutant SOD1-G3BP1 interaction affects stress granule dynamics, suggesting a potential link between pathogenic SOD1 mutations and RNA metab. alterations in ALS.
  42. 42
    Da Ros, M.; Deol, H. K.; Savard, A.; Guo, H.; Meiering, E. M.; Gibbings, D. Wild-Type and Mutant SOD1 Localizes to RNA-Rich Structures in Cells and Mice but Does Not Bind RNA. J. Neurochem. 2021, 156 (4), 524– 538,  DOI: 10.1111/jnc.15126
    Google Scholar
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    Wild-type and mutant SOD1 localizes to RNA-rich structures in cells and mice but does not bind RNA
    Da Ros, Matteo; Deol, Harmeen K.; Savard, Alexandre; Guo, Huishan; Meiering, Elizabeth M.; Gibbings, Derrick
    Journal of Neurochemistry (2021), 156 (4), 524-538CODEN: JONRA9; ISSN:0022-3042. (Wiley-Blackwell)
    Many of the genes whose mutation causes Amyotrophic Lateral Sclerosis (ALS) are RNA-binding proteins which localize to stress granules, while others impact the assembly, stability, and elimination of stress granules. This has led to the hypothesis that alterations in the dynamics of stress granules and RNA biol. cause ALS. Genetic mutations in Superoxide Dismutase 1 (SOD1) also cause ALS. Evidence demonstrates that SOD1 harboring ALS-linked mutations is recruited to stress granules, induces changes in alternative splicing, and could be an RNA-binding protein. Whether SOD1 inclusions contain RNA in disease models and whether SOD1 directly binds RNA remains uncertain. We applied methods including crosslinking immunopptn. and in vitro gel shift assays to detect binding of SOD1 to RNA in vitro, in cells with and without stress granules, and in mice expressing human SOD1 G93A. We find that SOD1 localizes to RNA-rich structures including stress granules, and SOD1 inclusions in mice contain mRNA. However, we find no evidence that SOD1 directly binds RNA. This suggests that SOD1 may impact stress granules, alternative splicing and RNA biol. without binding directly to RNA.
  43. 43
    Danielsson, J.; Kurnik, M.; Lang, L.; Oliveberg, M. Cutting Off Functional Loops from Homodimeric Enzyme Superoxide Dismutase 1 (SOD1) Leaves Monomeric β-Barrels. J. Biol. Chem. 2011, 286 (38), 33070– 33083,  DOI: 10.1074/jbc.M111.251223
    Google Scholar
    43
    Cutting Off Functional Loops from Homodimeric Enzyme Superoxide Dismutase 1 (SOD1) Leaves Monomeric β-Barrels
    Danielsson, Jens; Kurnik, Martin; Lang, Lisa; Oliveberg, Mikael
    Journal of Biological Chemistry (2011), 286 (38), 33070-33083, S33070/1-S33070/18CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Demetallation of the homodimeric enzyme Cu/Zn-superoxide dismutase (SOD1) is known to unleash pronounced dynamic motions in the long active-site loops that comprise almost a third of the folded structure. The resulting apo species, which shows increased propensity to aggregate, stands out as the prime disease precursor in amyotrophic lateral sclerosis (ALS). Even so, the detailed structural properties of the apoSOD1 framework have remained elusive and controversial. In this study, we examine the structural interplay between the central apoSOD1 barrel and the active-site loops by simply cutting them off; loops IV and VII were substituted with short Gly-Ala-Gly linkers. The results show that loop removal breaks the dimer interface and leads to sol., monomeric β-barrels with high structural integrity. NMR-detected nuclear Overhauser effects are found between all of the constituent β-strands, confirming ordered interactions across the whole barrel. Moreover, the breathing motions of the SOD1 barrel are overall insensitive to loop removal and yield hydrogen/deuterium protection factors typical for cooperatively folded proteins (i.e. the active-site loops act as a "bolt-on" domain with little dynamic influence on its structural foundation). The sole exceptions are the relatively low protection factors in β-strand 5 and the turn around Gly-93, a hot spot for ALS-provoking mutations, which decrease even further upon loop removal. Taken together, these data suggest that the cytotoxic function of apoSOD1 does not emerge from its folded ground state but from a high energy intermediate or even from the denatured ensemble.
  44. 44
    Nordlund, A.; Leinartaitė, L.; Saraboji, K.; Aisenbrey, C.; Gröbner, G.; Zetterström, P.; Danielsson, J.; Logan, D. T.; Oliveberg, M. Functional Features Cause Misfolding of the ALS-Provoking Enzyme SOD1. Proc. Natl. Acad. Sci. U. S. A. 2009, 106 (24), 9667– 9672,  DOI: 10.1073/pnas.0812046106
    Google Scholar
    44
    Functional features cause misfolding of the ALS-provoking enzyme SOD1
    Nordlund, Anna; Leinartaite, Lina; Saraboji, Kahhirvel; Aisenbrey, Christopher; Grobner, Gerhard; Zetterstrom, Per; Danielsson, Jens; Logan, Derek T.; Oliveberg, Mikael
    Proceedings of the National Academy of Sciences of the United States of America (2009), 106 (24), 9667-9672, S9667/1-S9667/12CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The structural integrity of the ubiquitous enzyme, Cu,Zn-superoxide dismutase (SOD1), relies critically on the correct coordination of Cu and Zn. The loss of these cofactors not only promotes SOD1 aggregation in vitro but also seems to be a key prerequisite for pathogenic misfolding in the neurodegenerative disease, amyotrophic lateral sclerosis (ALS). Here, the authors examd. the consequences of Zn2+ loss by selectively removing the Zn site, which had previously been implicated as the main modulator of SOD1 stability and disease competence. After Zn-site removal, the remaining Cu ligands could coordinate a non-native Zn2+ ion with micromolar affinity in the denatured state, and then retain this ion throughout the folding reaction. Without the restriction of a metalated Zn site, however, the Cu ligands failed to correctly coordinate the non-native Zn2+ ion; trapping of a water mol. caused the His-48 residue to change rotamer and swing outward. The misligation was sterically incompatible with the native structure. As a consequence, SOD1 unfolded locally and interacted with neighboring mols. in the crystal lattice. Thus, the findings point to a crit. role for the native Zn site in controlling SOD1 misfolding, and show that even subtle changes of the metal-loading sequence can render the wild-type protein the same structural properties as ALS-provoking mutations. This frustrated character of the SOD1 mol. seems to arise from a compromise between optimization of functional and structural features.
  45. 45
    Niwa, J.; Yamada, S.; Ishigaki, S.; Sone, J.; Takahashi, M.; Katsuno, M.; Tanaka, F.; Doyu, M.; Sobue, G. Disulfide Bond Mediates Aggregation, Toxicity, and Ubiquitylation of Familial Amyotrophic Lateral Sclerosis-Linked Mutant SOD1 *. J. Biol. Chem. 2007, 282 (38), 28087– 28095,  DOI: 10.1074/jbc.M704465200
    Google Scholar
    45
    Disulfide Bond Mediates Aggregation, Toxicity, and Ubiquitylation of Familial Amyotrophic Lateral Sclerosis-linked Mutant SOD1
    Niwa, Jun-ichi; Yamada, Shin-ichi; Ishigaki, Shinsuke; Sone, Jun; Takahashi, Miho; Katsuno, Masahisa; Tanaka, Fumiaki; Doyu, Manabu; Sobue, Gen
    Journal of Biological Chemistry (2007), 282 (38), 28087-28095CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Mutations in the Cu/Zn-superoxide dismutase (SOD1) gene cause familial amyotrophic lateral sclerosis (ALS) through the gain of a toxic function; however, the nature of this toxic function remains largely unknown. Ubiquitylated aggregates of mutant SOD1 proteins in affected brain lesions are pathol. hallmarks of the disease and are suggested to be involved in several proposed mechanisms of motor neuron death. Recent studies suggest that mutant SOD1 readily forms an incorrect disulfide bond upon mild oxidative stress in vitro, and the insol. SOD1 aggregates in spinal cord of ALS model mice contain multimers cross-linked via intermol. disulfide bonds. Here we show that a non-physiol. intermol. disulfide bond between cysteines at positions 6 and 111 of mutant SOD1 is important for high mol. wt. aggregate formation, ubiquitylation, and neurotoxicity, all of which were dramatically reduced when the pertinent cysteines were replaced in mutant SOD1 expressed in Neuro-2a cells. Dorfin is a ubiquityl ligase that specifically binds familial ALS-linked mutant SOD1 and ubiquitylates it, thereby promoting its degrdn. We found that Dorfin ubiquitylated mutant SOD1 by recognizing the Cys6- and Cys111-disulfide cross-linked form and targeted it for proteasomal degrdn.
  46. 46
    Ebbinghaus, S.; Dhar, A.; McDonald, J. D.; Gruebele, M. Protein Folding Stability and Dynamics Imaged in a Living Cell. Nat. Methods 2010, 7 (4), 319– 323,  DOI: 10.1038/nmeth.1435
    Google Scholar
    46
    Protein folding stability and dynamics imaged in a living cell
    Ebbinghaus, Simon; Dhar, Apratim; McDonald, J. Douglas; Gruebele, Martin
    Nature Methods (2010), 7 (4), 319-323CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    Biomol. dynamics and stability are predominantly investigated in vitro and extrapolated to explain function in the living cell. Here, the authors present fast relaxation imaging (FreI), which combines fluorescence microscopy and temp. jumps to probe biomol. dynamics and stability inside a single living cell with high spatiotemporal resoln. Here, the authors demonstrated the method by measuring the reversible fast folding kinetics as well as folding thermodn. of a FRET probe-labeled phosphoglycerate kinase construct in 2 human cell lines. Comparison with in vitro expts. at 23-49° showed that the cell environment influenced the protein stability and folding rate. FReI should also be applicable to the study of protein-protein interactions and heat-shock responses as well as to comparative studies of cell populations or whole organisms.
  47. 47
    van Lente, J. J.; Claessens, M. M. A. E.; Lindhoud, S. Charge-Based Separation of Proteins Using Polyelectrolyte Complexes as Models for Membraneless Organelles. Biomacromolecules 2019, 20 (10), 3696– 3703,  DOI: 10.1021/acs.biomac.9b00701
    Google Scholar
    47
    Charge-Based Separation of Proteins Using Polyelectrolyte Complexes as Models for Membraneless Organelles
    van Lente, Jere J.; Claessens, Mireille M. A. E.; Lindhoud, Saskia
    Biomacromolecules (2019), 20 (10), 3696-3703CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
    Membraneless organelles are liq. compartments within cells with different solvent properties than the surrounding environment. This difference in solvent properties is thought to result in function-related selective partitioning of proteins. Proteins have also been shown to accumulate in polyelectrolyte complexes, but whether the uptake in these complexes is selective has not been ascertained yet. Here, we show the selective partitioning of two structurally similar but oppositely charged proteins into polyelectrolyte complexes. We demonstrate that these proteins can be sepd. from a mixt. by altering the polyelectrolyte complex compn. and released from the complex by lowering the pH. Combined, we demonstrate that polyelectrolyte complexes can sep. proteins from a mixt. based on protein charge. Besides providing deeper insight into the selective partitioning in membraneless organelles, potential applications for selective biomol. partitioning in polyelectrolyte complexes include drug delivery or extn. processes.
  48. 48
    Raeburn, C. B.; Ormsby, A.; Moily, N. S.; Cox, D.; Ebbinghaus, S.; Dickson, A.; McColl, G.; Hatters, D. M. A Biosensor to Gauge Protein Homeostasis Resilience Differences in the Nucleus Compared to Cytosol of Mammalian Cells. bioRxiv (Biochemistry) , April 19, 2021, 2021.04.19.440383. DOI: 10.1101/2021.04.19.440383 (accessed 2021–09–08).
    Google Scholar
    There is no corresponding record for this reference.
  49. 49
    Vassall, K. A.; Stubbs, H. R.; Primmer, H. A.; Tong, M. S.; Sullivan, S. M.; Sobering, R.; Srinivasan, S.; Briere, L.-A. K.; Dunn, S. D.; Colón, W.; Meiering, E. M. Decreased Stability and Increased Formation of Soluble Aggregates by Immature Superoxide Dismutase Do Not Account for Disease Severity in ALS. Proc. Natl. Acad. Sci. U. S. A. 2011, 108 (6), 2210– 2215,  DOI: 10.1073/pnas.0913021108
    Google Scholar
    49
    Decreased stability and increased formation of soluble aggregates by immature superoxide dismutase do not account for disease severity in ALS
    Vassall, Kenrick A.; Stubbs, Helen R.; Primmer, Heather A.; Tong, Ming Sze; Sullivan, Sarah M.; Sobering, Ryan; Srinivasan, Saipraveen; Briere, Lee-Ann K.; Dunn, Stanley D.; Colon, Wilfredo; Meiering, Elizabeth M.
    Proceedings of the National Academy of Sciences of the United States of America (2011), 108 (6), 2210-2215, S2210/1-S2210/11CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Protein aggregation is a hallmark of many diseases, including amyotrophic lateral sclerosis (ALS), where aggregation of Cu/Zn superoxide dismutase (SOD1) is implicated in causing neurodegeneration. Recent studies have suggested that destabilization and aggregation of the most immature form of SOD1, the disulfidereduced, unmetallated (apo) protein is particularly important in causing ALS. We report herein in depth analyses of the effects of chem. and structurally diverse ALS-assocd. mutations on the stability and aggregation of reduced apo SOD1. In contrast with previous studies, we find that various reduced apo SOD1 mutants undergo highly reversible thermal denaturation with little aggregation, enabling quant. thermodn. stability analyses. In the absence of ALS-assocd. mutations, reduced apo SOD1 is marginally stable but predominantly folded. Mutations generally result in slight decreases to substantial increases in the fraction of unfolded protein. Calorimetry, ultracentrifugation, and light scattering show that all mutations enhance aggregation propensity, with the effects varying widely, from subtle increases in most cases, to pronounced formation of 40-100 nm sol. aggregates by A4V, a mutation that is assocd. with particularly short disease duration. Interestingly, although there is a correlation between obsd. aggregation and stability, there is minimal to no correlation between obsd. aggregation, predicted aggregation propensity, and disease characteristics. These findings suggest that reduced apo SOD1 does not play a dominant role in modulating disease. Rather, addnl. and/or multiple forms of SOD1 and addnl. biophys. and biol. factors are needed to account for the toxicity of mutant SOD1 in ALS.
  50. 50
    Wang, Q.; Johnson, J. L.; Agar, N. Y. R.; Agar, J. N. Protein Aggregation and Protein Instability Govern Familial Amyotrophic Lateral Sclerosis Patient Survival. PLoS Biol. 2008, 6 (7), e170,  DOI: 10.1371/journal.pbio.0060170
    Google Scholar
    There is no corresponding record for this reference.
  51. 51
    Rauscher, S.; Pomès, R. The Liquid Structure of Elastin. eLife 2017, 6, e26526  DOI: 10.7554/eLife.26526
    Google Scholar
    51
    The liquid structure of elastin
    Rauscher, Sarah; Pomes, Regis
    eLife (2017), 6 (), e26526/1-e26526/21CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)
    The protein elastin imparts extensibility, elastic recoil, and resilience to tissues including arterial walls, skin, lung alveoli, and the uterus. Elastin and elastin-like peptides are hydrophobic, disordered, and undergo liq.-liq. phase sepn. upon self-assembly. Despite extensive study, the structure of elastin remains controversial. We use mol. dynamics simulations on a massive scale to elucidate the structural ensemble of aggregated elastin-like peptides. Consistent with the entropic nature of elastic recoil, the aggregated state is stabilized by the hydrophobic effect. However, self-assembly does not entail formation of a hydrophobic core. The polypeptide backbone forms transient, sparse hydrogen-bonded turns and remains significantly hydrated even as self-assembly triples the extent of non-polar side chain contacts. Individual chains in the assembly approach a maximally-disordered, melt-like state which may be called the liq. state of proteins. These findings resolve long-standing controversies regarding elastin structure and function and afford insight into the phase sepn. of disordered proteins.
  52. 52
    Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF Chimera--a Visualization System for Exploratory Research and Analysis. J. Comput. Chem. 2004, 25 (13), 1605– 1612,  DOI: 10.1002/jcc.20084
    Google Scholar
    52
    UCSF Chimera-A visualization system for exploratory research and analysis
    Pettersen, Eric F.; Goddard, Thomas D.; Huang, Conrad C.; Couch, Gregory S.; Greenblatt, Daniel M.; Meng, Elaine C.; Ferrin, Thomas E.
    Journal of Computational Chemistry (2004), 25 (13), 1605-1612CODEN: JCCHDD; ISSN:0192-8651. (John Wiley & Sons, Inc.)
    The design, implementation, and capabilities of an extensible visualization system, UCSF Chimera, are discussed. Chimera is segmented into a core that provides basic services and visualization, and extensions that provide most higher level functionality. This architecture ensures that the extension mechanism satisfies the demands of outside developers who wish to incorporate new features. Two unusual extensions are presented: Multiscale, which adds the ability to visualize large-scale mol. assemblies such as viral coats, and Collab., which allows researchers to share a Chimera session interactively despite being at sep. locales. Other extensions include Multalign Viewer, for showing multiple sequence alignments and assocd. structures; ViewDock, for screening docked ligand orientations; Movie, for replaying mol. dynamics trajectories; and Vol. Viewer, for display and anal. of volumetric data. A discussion of the usage of Chimera in real-world situations is given, along with anticipated future directions. Chimera includes full user documentation, is free to academic and nonprofit users, and is available for Microsoft Windows, Linux, Apple Mac OS X, SGI IRIX, and HP Tru64 Unix from http://www.cgl.ucsf.edu/chimera/.
  53. 53
    Hofweber, M.; Hutten, S.; Bourgeois, B.; Spreitzer, E.; Niedner-Boblenz, A.; Schifferer, M.; Ruepp, M.-D.; Simons, M.; Niessing, D.; Madl, T.; Dormann, D. Phase Separation of FUS Is Suppressed by Its Nuclear Import Receptor and Arginine Methylation. Cell 2018, 173 (3), 706– 719,  DOI: 10.1016/j.cell.2018.03.004
    Google Scholar
    53
    Phase Separation of FUS Is Suppressed by Its Nuclear Import Receptor and Arginine Methylation
    Hofweber, Mario; Hutten, Saskia; Bourgeois, Benjamin; Spreitzer, Emil; Niedner-Boblenz, Annika; Schifferer, Martina; Ruepp, Marc-David; Simons, Mikael; Niessing, Dierk; Madl, Tobias; Dormann, Dorothee
    Cell (Cambridge, MA, United States) (2018), 173 (3), 706-719.e13CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    Cytoplasmic FUS aggregates are a pathol. hallmark in a subset of patients with frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS). A key step that is disrupted in these patients is nuclear import of FUS mediated by the import receptor Transportin/Karyopherin-β2. In ALS-FUS patients, this is caused by mutations in the nuclear localization signal (NLS) of FUS that weaken Transportin binding. In FTD-FUS patients, Transportin is aggregated, and post-translational arginine methylation, which regulates the FUS-Transportin interaction, is lost. Here, we show that Transportin and arginine methylation have a crucial function beyond nuclear import-namely to suppress RGG/RG-driven phase sepn. and stress granule assocn. of FUS. ALS-assocd. FUS-NLS mutations weaken the chaperone activity of Transportin and loss of FUS arginine methylation, as seen in FTD-FUS, promote phase sepn., and stress granule partitioning of FUS. Our findings reveal two regulatory mechanisms of liq.-phase homeostasis that are disrupted in FUS-assocd. neurodegeneration.
  54. 54
    Alexander, E. J.; Ghanbari Niaki, A.; Zhang, T.; Sarkar, J.; Liu, Y.; Nirujogi, R. S.; Pandey, A.; Myong, S.; Wang, J. Ubiquilin 2 Modulates ALS/FTD-Linked FUS–RNA Complex Dynamics and Stress Granule Formation. Proc. Natl. Acad. Sci. U. S. A. 2018, 115 (49), E11485– E11494,  DOI: 10.1073/pnas.1811997115
    Google Scholar
    54
    Ubiquilin 2 modulates ALS/FTD-linked FUS-RNA complex dynamics and stress granule formation
    Alexander, Elizabeth J.; Niaki, Amirhossein Ghanbari; Zhang, Tao; Sarkar, Jaya; Liu, Yang; Nirujogi, Raja Sekhar; Pandey, Akhilesh; Myong, Sua; Wang, Jiou
    Proceedings of the National Academy of Sciences of the United States of America (2018), 115 (49), E11485-E11494CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The ubiquitin-like protein ubiquilin 2 (UBQLN2) has been genetically and pathol. linked to the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but its normal cellular functions are not well understood. In a search for UBQLN2-interacting proteins, we found an enrichment of stress granule (SG) components, including ALS/FTD-linked heterogeneous ribonucleoprotein fused in sarcoma (FUS). Through the use of an optimized SG detection method, we obsd. UBQLN2 and its interactors at SGs. A low complexity, Sti1-like repeat region in UBQLN2 was sufficient for its localization to SGs. Functionally, UBQLN2 neg. regulated SG formation. UBQLN2 increased the dynamics of FUS-RNA interaction and promoted the fluidity of FUS-RNA complexes at a single-mol. level. This solubilizing effect corresponded to a dispersal of FUS liq. droplets in vitro and a suppression of FUS SG formation in cells. ALS-linked mutations in UBQLN2 reduced its assocn. with FUS and impaired its function in regulating FUS-RNA complex dynamics and SG formation. These results reveal a previously unrecognized role for UBQLN2 in regulating the early stages of liq.-liq. phase sepn. by directly modulating the fluidity of protein-RNA complexes and the dynamics of SG formation.
  55. 55
    Ahlers, J.; Adams, E. M.; Bader, V.; Pezzotti, S.; Winklhofer, K. F.; Tatzelt, J.; Havenith, M. The Key Role of Solvent in Condensation: Mapping Water in Liquid-Liquid Phase-Separated FUS. Biophys. J. 2021, 120, 1266,  DOI: 10.1016/j.bpj.2021.01.019
    Google Scholar
    55
    The key role of solvent in condensation: mapping water in liquid-liquid phase-separated FUS
    Ahlers, Jonas; Adams, Ellen M.; Bader, Verian; Pezzotti, Simone; Winklhofer, Konstanze F.; Tatzelt, Joerg; Havenith, Martina
    Biophysical Journal (2021), 120 (7), 1266-1275CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
    Formation of biomol. condensates through liq.-liq. phase sepn. (LLPS) has emerged as a pervasive principle in cell biol., allowing compartmentalization and spatiotemporal regulation of dynamic cellular processes. Proteins that form condensates under physiol. conditions often contain intrinsically disordered regions with low-complexity domains. Among them, the RNA-binding proteins FUS and TDP-43 have been a focus of intense investigation because aberrant condensation and aggregation of these proteins is linked to neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia. LLPS occurs when protein-rich condensates form surrounded by a dil. aq. soln. LLPS is per se entropically unfavorable. Energetically favorable multivalent protein-protein interactions are one important aspect to offset entropic costs. Another proposed aspect is the release of entropically unfavorable preordered hydration water into the bulk. We used attenuated total reflection spectroscopy in the terahertz frequency range to characterize the changes in the hydrogen bonding network accompanying the FUS enrichment in liq.-liq. phase-sepd. droplets to provide exptl. evidence for the key role of the solvent as a thermodn. driving force. The FUS concn. inside LLPS droplets was detd. to be increased to 2.0 mM independent of the initial protein concn. (5 or 10μM solns.) by fluorescence measurements. With terahertz spectroscopy, we revealed a dewetting of hydrophobic side chains in phase-sepd. FUS. Thus, the release of entropically unfavorable water populations into the bulk goes hand in hand with enthalpically favorable protein-protein interaction. Both changes are energetically favorable, and our study shows that both contribute to the thermodn. driving force in phase sepn.
  56. 56
    Li, X.; Romero, P.; Rani, M.; Dunker, A. K.; Obradovic, Z. Predicting Protein Disorder for N-, C-, and Internal Regions. Genome Inform. Workshop Genome Inform. 1999, 10, 30– 40
    Google Scholar
    56
    Predicting Protein Disorder for N-, C-, and Internal Regions
    Li; Romero; Rani; Dunker; Obradovic
    Genome informatics. Workshop on Genome Informatics (1999), 10 (), 30-40 ISSN:.
    Logistic regression (LR), discriminant analysis (DA), and neural networks (NN) were used to predict ordered and disordered regions in proteins. Training data were from a set of non-redundant X-ray crystal structures, with the data being partitioned into N-terminal, C-terminal and internal (I) regions. The DA and LR methods gave almost identical 5-cross validation accuracies that averaged to the following values: 75.9 +/- 3.1% (N-regions), 70.7 +/- 1.5% (I-regions), and 74.6 +/- 4.4% (C-regions). NN predictions gave slightly higher scores: 78.8 +/- 1.2% (N-regions), 72.5 +/- 1.2% (I-regions), and 75.3 +/- 3.3% (C-regions). Predictions improved with length of the disordered regions. Averaged over the three methods, values ranged from 52% to 78% for length = 9-14 to >/= 21, respectively, for I-regions, from 72% to 81% for length = 5 to 12-15, respectively, for N-regions, and from 70% to 80% for length = 5 to 12-15, respectively, for C-regions. These data support the hypothesis that disorder is encoded by the amino acid sequence.
  57. 57
    Boeynaems, S.; Holehouse, A. S.; Weinhardt, V.; Kovacs, D.; Van Lindt, J.; Larabell, C.; Van Den Bosch, L.; Das, R.; Tompa, P. S.; Pappu, R. V.; Gitler, A. D. Spontaneous Driving Forces Give Rise to Protein–RNA Condensates with Coexisting Phases and Complex Material Properties. Proc. Natl. Acad. Sci. U. S. A. 2019, 116, 7889– 7898,  DOI: 10.1073/pnas.1821038116
    Google Scholar
    57
    Spontaneous driving forces give rise to protein-RNA condensates with coexisting phases and complex material properties
    Boeynaems, Steven; Holehouse, Alex S.; Weinhardt, Venera; Kovacs, Denes; Van Lindt, Joris; Larabell, Carolyn; Van Den Bosch, Ludo; Das, Rhiju; Tompa, Peter S.; Pappu, Rohit V.; Gitler, Aaron D.
    Proceedings of the National Academy of Sciences of the United States of America (2019), 116 (16), 7889-7898CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Phase sepn. of multivalent protein and RNA mols. underlies the biogenesis of biomol. condensates such as membraneless organelles. In vivo, these condensates encompass hundreds of distinct types of mols. that typically organize into multilayered structures supporting the differential partitioning of mols. into distinct regions with distinct material properties. The interplay between driven (active) vs. spontaneous (passive) processes that are required for enabling the formation of condensates with coexisting layers of distinct material properties remains unclear. Here, the authors deploy systematic expts. and simulations based on coarse-grained models to show that the collective interactions among the simplest, biol. relevant proteins and archetypal RNA mols. are sufficient for driving the spontaneous emergence of multilayered condensates with distinct material properties. These studies yield a set of rules regarding homotypic and heterotypic interactions that are likely to be relevant for understanding the interplay between active and passive processes that control the formation of functional biomol. condensates.
  58. 58
    Kaur, T.; Raju, M.; Alshareedah, I.; Davis, R. B.; Potoyan, D. A.; Banerjee, P. R. Sequence-Encoded and Composition-Dependent Protein-RNA Interactions Control Multiphasic Condensate Morphologies. Nat. Commun. 2021, 12 (1), 872,  DOI: 10.1038/s41467-021-21089-4
    Google Scholar
    58
    Sequence-encoded and composition-dependent protein-RNA interactions control multiphasic condensate morphologies
    Kaur, Taranpreet; Raju, Muralikrishna; Alshareedah, Ibraheem; Davis, Richoo B.; Potoyan, Davit A.; Banerjee, Priya R.
    Nature Communications (2021), 12 (1), 872CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    Multivalent protein-protein and protein-RNA interactions are the drivers of biol. phase sepn. Biomol. condensates typically contain a dense network of multiple proteins and RNAs, and their competing mol. interactions play key roles in regulating the condensate compn. and structure. Employing a ternary system comprising of a prion-like polypeptide (PLP), arginine-rich polypeptide (RRP), and RNA, we show that competition between the PLP and RNA for a single shared partner, the RRP, leads to RNA-induced demixing of PLP-RRP condensates into stable coexisting phases-homotypic PLP condensates and heterotypic RRP-RNA condensates. The morphol. of these biphasic condensates (non-engulfing/ partial engulfing/ complete engulfing) is detd. by the RNA-to-RRP stoichiometry and the hierarchy of intermol. interactions, providing a glimpse of the broad range of multiphasic patterns that are accessible to these condensates. Our findings provide a minimal set of phys. rules that govern the compn. and spatial organization of multicomponent and multiphasic biomol. condensates.
  59. 59
    Dignon, G. L.; Zheng, W.; Kim, Y. C.; Mittal, J. Temperature-Controlled Liquid–Liquid Phase Separation of Disordered Proteins. ACS Cent. Sci. 2019, 5 (5), 821– 830,  DOI: 10.1021/acscentsci.9b00102
    Google Scholar
    59
    Temperature-Controlled Liquid-Liquid Phase Separation of Disordered Proteins
    Dignon, Gregory L.; Zheng, Wenwei; Kim, Young C.; Mittal, Jeetain
    ACS Central Science (2019), 5 (5), 821-830CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
    The liq.-liq. phase sepn. (LLPS) of intrinsically disordered proteins (IDPs) is a commonly obsd. phenomenon within the cell, and such condensates are also highly attractive for applications in biomaterials and drug delivery. A better understanding of the sequence-dependent thermoresponsive behavior is of immense interest as it will aid in the design of protein sequences with desirable properties and in the understanding of cellular response to heat stress. In this work, the authors use a transferable coarse-grained model to directly probe the sequence-dependent thermoresponsive phase behavior of IDPs. To achieve this goal, the authors develop a unique knowledge-based amino acid potential that accounts for the temp.-dependent effects on solvent-mediated interactions for different types of amino acids. Remarkably, the authors are able to distinguish between more than 35 IDPs with upper or lower crit. soln. temps. at exptl. conditions, thus providing direct evidence that incorporating the temp.-dependent solvent-mediated interactions to IDP assemblies can capture the difference in the shape of the resulting phase diagrams. Given the success of the model in predicting exptl. behavior, the authors use it as a high-throughput screening framework to scan through millions of disordered sequences to characterize the compn. dependence of protein phase sepn.
  60. 60
    Zwicker, D.; Seyboldt, R.; Weber, C. A.; Hyman, A. A.; Jülicher, F. Growth and Division of Active Droplets Provides a Model for Protocells. Nat. Phys. 2017, 13 (4), 408– 413,  DOI: 10.1038/nphys3984
    Google Scholar
    60
    Growth and division of active droplets provides a model for protocells
    Zwicker, David; Seyboldt, Rabea; Weber, Christoph A.; Hyman, Anthony A.; Juelicher, Frank
    Nature Physics (2017), 13 (4), 408-413CODEN: NPAHAX; ISSN:1745-2473. (Nature Publishing Group)
    It has been proposed that during the early steps in the origin of life, small droplets could have formed via the segregation of mols. from complex mixts. by phase sepn. These droplets could have provided chem. reaction centers. However, whether these droplets could divide and propagate is unclear. Here we examine the behavior of droplets in systems that are maintained away from thermodn. equil. by an external supply of energy. In these systems, droplets grow by the addn. of droplet material generated by chem. reactions. Surprisingly, we find that chem. driven droplet growth can lead to shape instabilities that trigger the division of droplets into two smaller daughters. Therefore, chem. active droplets can exhibit cycles of growth and division that resemble the proliferation of living cells. Dividing active droplets could serve as a model for prebiotic protocells, where chem. reactions in the droplet play the role of a prebiotic metab.
  61. 61
    Wang, J.; Choi, J.-M.; Holehouse, A. S.; Lee, H. O.; Zhang, X.; Jahnel, M.; Maharana, S.; Lemaitre, R.; Pozniakovsky, A.; Drechsel, D.; Poser, I.; Pappu, R. V.; Alberti, S.; Hyman, A. A. A Molecular Grammar Governing the Driving Forces for Phase Separation of Prion-like RNA Binding Proteins. Cell 2018, 174 (3), 688– 699,  DOI: 10.1016/j.cell.2018.06.006
    Google Scholar
    61
    A Molecular Grammar Governing the Driving Forces for Phase Separation of Prion-like RNA Binding Proteins
    Wang, Jie; Choi, Jeong-Mo; Holehouse, Alex S.; Lee, Hyun O.; Zhang, Xiaojie; Jahnel, Marcus; Maharana, Shovamayee; Lemaitre, Regis; Pozniakovsky, Andrei; Drechsel, David; Poser, Ina; Pappu, Rohit V.; Alberti, Simon; Hyman, Anthony A.
    Cell (Cambridge, MA, United States) (2018), 174 (3), 688-699.e16CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    Proteins such as FUS (Fused in Sarcoma) phase sep. to form liq.-like condensates that can harden into less dynamic structures. However, how these properties emerge from the collective interactions of many amino acids remains largely unknown. Here, we use extensive mutagenesis to identify a sequence-encoded mol. grammar underlying the driving forces of phase sepn. of proteins in the FUS family and test aspects of this grammar in cells. Phase sepn. is primarily governed by multivalent interactions among tyrosine residues from prion-like domains (PLDs) and arginine residues from RNA-binding domains (RBDs), which are modulated by neg. charged residues. Glycine residues enhance the fluidity, whereas glutamine and serine residues promote hardening. We develop a model to show that the measured satn. concns. of phase sepn. are inversely proportional to the product of the nos. of arginine and tyrosine residues. These results suggest it is possible to predict phase-sepn. properties based on amino acid sequences.
  62. 62
    Lu, Y.; Lim, L.; Song, J. RRM Domain of ALS/FTD-Causing FUS Characteristic of Irreversible Unfolding Spontaneously Self-Assembles into Amyloid Fibrils. Sci. Rep. 2017, 7 (1), 1– 14,  DOI: 10.1038/s41598-017-01281-7
    Google Scholar
    62
    Transgenic Nicotiana benthamiana plants expressing a hairpin RNAi construct of a nematode Rs-cps gene exhibit enhanced resistance to Radopholus similis
    Li, Yu; Wang, Ke; Lu, Qisen; Du, Juan; Wang, Zhenyue; Wang, Desen; Sun, Bingjian; Li, Honglian
    Scientific Reports (2017), 7 (1), 1-11CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)
    Burrowing nematodes (Radopholus similis) cause severe harm in many agronomic and horticultural crops and are very difficult to manage. Cathepsin S is one of the most important cysteine proteinases and plays key roles in nematodes and many other parasites. To evaluate the effect of in planta RNAi on the control of this nematode, a specific fragment from the protease gene, cathepsin S (Rs-cps), was cloned into the binary vector pFGC5941 in the forward and reverse orientations to construct recombinant plant RNAi vectors. Transgenic Nicotiana benthamiana plants expressing Rs-cps dsRNA were obtained and studied. The transcript abundance of Rs-cps dsRNA appeared to be diverse in the different transgenic lines. Moreover, the bioassay results revealed that Rs-cps transgenic N. benthamiana plants were resistant to R. similis and the transcription level of Rs-cps in R. similis was drastically decreased. In addn., the reprodn. and hatching rate of R. similis isolated from the Rs-cps transgenic plants were also significantly reduced. Our results suggest that Rs-cps is essential for the reprodn. and pathogenicity of R. similis. This is the first study to employ in planta RNAi approach to target the Rs-cps gene for the control of plant parasitic nematodes.
  63. 63
    Li, S.; Yoshizawa, T.; Yamazaki, R.; Fujiwara, A.; Kameda, T.; Kitahara, R. Pressure and Temperature Phase Diagram for Liquid–Liquid Phase Separation of the RNA-Binding Protein Fused in Sarcoma. J. Phys. Chem. B 2021, 125 (25), 6821– 6829,  DOI: 10.1021/acs.jpcb.1c01451
    Google Scholar
    63
    Pressure and Temperature Phase Diagram for Liquid-Liquid Phase Separation of the RNA-Binding Protein Fused in Sarcoma
    Li, Shujie; Yoshizawa, Takuya; Yamazaki, Ryota; Fujiwara, Ayano; Kameda, Tomoshi; Kitahara, Ryo
    Journal of Physical Chemistry B (2021), 125 (25), 6821-6829CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
    Liq.-liq. phase sepn. (LLPS) of proteins and nucleic acids to form membraneless cellular compartments is considered to be involved in various biol. functions. The RNA-binding protein fused in sarcoma (FUS) undergoes LLPS in vivo and in vitro. Here, the authors studied the effects of pressure and temp. on the LLPS of FUS by high-pressure microscopy and high-pressure UV/visible spectroscopy. The phase-sepd. condensate of FUS was obliterated with increasing pressure but was obsd. again at a higher pressure. The authors generated a pressure-temp. phase diagram that describes the phase sepn. of FUS and provides a general understanding of the thermodn. properties of self-assembly and phase sepn. of proteins. FUS has two types of condensed phases, obsd. at low pressure (LP-LLPS) and high pressure (HP-LLPS). The HP-LLPS state was more condensed and exhibited lower susceptibility to dissoln. by 1,6-hexanediol and karyopherin-β2 than the LP-LLPS state. Moreover, mol. dynamic simulations revealed that electrostatic interactions were destabilized, whereas cation-π, π-π, and hydrophobic interactions were stabilized in HP-LLPS. When cation-π, π-π, and hydrophobic interactions were transiently stabilized in the cellular environment, the phase transition to HP-LLPS occurred; this might be correlated to the aberrant enrichment of cytoplasmic ribonucleoprotein granules, leading to amyotrophic lateral sclerosis.
  64. 64
    Gnutt, D.; Brylski, O.; Edengeiser, E.; Havenith, M.; Ebbinghaus, S. Imperfect Crowding Adaptation of Mammalian Cells towards Osmotic Stress and Its Modulation by Osmolytes. Mol. BioSyst. 2017, 13 (11), 2218– 2221,  DOI: 10.1039/C7MB00432J
    Google Scholar
    64
    Imperfect crowding adaptation of mammalian cells towards osmotic stress and its modulation by osmolytes
    Gnutt, David; Brylski, Oliver; Edengeiser, Eugen; Havenith, Martina; Ebbinghaus, Simon
    Molecular BioSystems (2017), 13 (11), 2218-2221CODEN: MBOIBW; ISSN:1742-2051. (Royal Society of Chemistry)
    Changes of the extracellular milieu could affect cellular crowding. To prevent detrimental effects, cells use adaptation mechanisms to react to such conditions. Using fluorescent crowding sensors, we show that the initial response to osmotic stress is fast but imperfect, while the slow response renders cells more tolerant to stress, particularly in the presence of osmolytes.
  65. 65
    Gnutt, D.; Sistemich, L.; Ebbinghaus, S. Protein Folding Modulation in Cells Subject to Differentiation and Stress. Front. Mol. Biosci. 2019, 6, 38,  DOI: 10.3389/fmolb.2019.00038
    Google Scholar
    65
    Protein folding modulation in cells subject to differentiation and stress
    Gnutt, Dav; Sistemich, Linda; Ebbinghaus, Simon
    Frontiers in Molecular Biosciences (2019), 6 (), 38CODEN: FMBRBS; ISSN:2296-889X. (Frontiers Media S.A.)
    Cytomimetic media are used to mimic the physicochem. properties of the cellular milieu in an in vitro expt. Themotivation is that compared to entire cells, they can be used efficiently in combination with a broad range of exptl. techniques. However, the development and use of cytomimetic media is hampered by the lack of in-cell data that could be used as a hallmark to directly evaluate and improve the performance of cytomimetic media in different applications. Such data must include the study of specific biomol. reactions in different cell types, different compartments of a single cells and different cellular conditions. In previous studies, model systems such as cancer cell lines, bacteria or oocytes were used. Here we studied how the environment of cells that undergo neuronal differentiation or proteostasis stress modulates the protein folding equil. We found that NGF induced differentiation leads to a decrease of the melting temp. and a change of the folding mechanism. Proteomic changes that occur upon differentiation could explain this effect, however, we found that the crowding effect remained unchanged. Using MG132, a common proteasome inhibitor and inducer of the unfolded protein response, we show that changes to the quality control machinery modulate the folding equil., leading to protein destabilization at prolonged stress exposure. Our study explores the range of protein folding modulation within cells subject to differentiation or stress that must be encountered in the development of cytomimetic media.
  66. 66
    Senske, M.; Törk, L.; Born, B.; Havenith, M.; Herrmann, C.; Ebbinghaus, S. Protein Stabilization by Macromolecular Crowding through Enthalpy Rather than Entropy. J. Am. Chem. Soc. 2014, 136 (25), 9036– 9041,  DOI: 10.1021/ja503205y
    Google Scholar
    66
    Protein stabilization by macromolecular crowding through enthalpy rather than entropy
    Senske, Michael; Toerk, Lisa; Born, Benjamin; Havenith, Martina; Herrmann, Christian; Ebbinghaus, Simon
    Journal of the American Chemical Society (2014), 136 (25), 9036-9041CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The interior of the cell is a densely crowded environment in which protein stability is affected differently than in dil. soln. Macromol. crowding is commonly understood in terms of an entropic vol. exclusion effect based on hardcore repulsions among the macromols. Here, the authors studied the thermal unfolding of ubiquitin in the presence of different cosolutes (glucose, dextran, poly(ethylene glycol), KCl, and urea). The results showed that for a correct dissection of the cosolute-induced changes of the free energy into its enthalpic and entropic contributions, the temp. dependence of the heat capacity change needs to be explicitly taken into account. In contrast to the prediction by excluded vol. theory, the authors obsd. an enthalpic stabilization and an entropic destabilization for glucose, dextran, and poly(ethylene glycol). The enthalpic stabilization mechanism induced by the macromol. crowder, dextran, was similar to the enthalpic stabilization mechanism of its monomeric building block, glucose. In the case of poly(ethylene glycol), entropy was dominating over enthalpy leading to an overall destabilization. The authors propose a new model to classify cosolute effects in terms of their enthalpic contributions to protein stability.
  67. 67
    Smith, A. E.; Zhou, L. Z.; Gorensek, A. H.; Senske, M.; Pielak, G. J. In-Cell Thermodynamics and a New Role for Protein Surfaces. Proc. Natl. Acad. Sci. U. S. A. 2016, 113 (7), 1725– 1730,  DOI: 10.1073/pnas.1518620113
    Google Scholar
    67
    In-cell thermodynamics and a new role for protein surfaces
    Smith, Austin E.; Zhou, Larry Z.; Gorensek, Annelise H.; Senske, Michael; Pielak, Gary J.
    Proceedings of the National Academy of Sciences of the United States of America (2016), 113 (7), 1725-1730CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    There is abundant, physiol. relevant knowledge about protein cores; they are hydrophobic, exquisitely well-packed, and nearly all H-bonds are satisfied. An equivalent understanding of protein surfaces has remained elusive because proteins are almost exclusively studied in vitro in simple aq. solns. Here, the authors established the essential physiol. roles played by protein surfaces by measuring the equil. thermodn. and kinetics of protein folding in the complex environment of living Escherichia coli cells, and under physiol. relevant in vitro conditions. Fluorine NMR data on the 7-kDa globular N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) showed that charge-charge interactions were fundamental to protein stability and folding kinetics in cells. These results contradicted predictions from accepted theories of macromol. crowding and showed that cosolutes commonly used to mimic the cellular interior did not yield physiol. relevant information. As such, the authors provide the foundation for a complete picture of protein chem. in cells.
  68. 68
    Monteith, W. B.; Cohen, R. D.; Smith, A. E.; Guzman-Cisneros, E.; Pielak, G. J. Quinary Structure Modulates Protein Stability in Cells. Proc. Natl. Acad. Sci. U. S. A. 2015, 112 (6), 1739– 1742,  DOI: 10.1073/pnas.1417415112
    Google Scholar
    68
    Quinary structure modulates protein stability in cells
    Monteith, William B.; Cohen, Rachel D.; Smith, Austin E.; Guzman-Cisneros, Emilio; Pielak, Gary J.
    Proceedings of the National Academy of Sciences of the United States of America (2015), 112 (6), 1739-1742CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Protein quinary interactions organize the cellular interior and its metab. Although the interactions stabilizing secondary, tertiary, and quaternary protein structure are well defined, details about the protein-matrix contacts that comprise quinary structure remain elusive. This gap exists because proteins function in a crowded cellular environment, but are traditionally studied in simple buffered solns. Here, the authors used NMR-detected H/D exchange to quantify quinary interactions between the B1 domain of protein G and the cytosol of Escherichia coli. The authors demonstrated that a surface mutation in this protein was 10-fold more destabilizing in cells than in buffer, a surprising result that firmly establishes the significance of quinary interactions. Remarkably, the energy involved in these interactions could be as large as the energies that stabilize specific protein complexes. These results will drive the crit. task of implementing quinary structure into models for understanding the proteome.
  69. 69
    Cohen, R. D.; Pielak, G. J. Quinary Interactions with an Unfolded State Ensemble: Quinary Interactions & Unfolded State Ensemble. Protein Sci. 2017, 26 (9), 1698– 1703,  DOI: 10.1002/pro.3206
    Google Scholar
    69
    Quinary interactions with an unfolded state ensemble
    Cohen, Rachel D.; Pielak, Gary J.
    Protein Science (2017), 26 (9), 1698-1703CODEN: PRCIEI; ISSN:1469-896X. (Wiley-Blackwell)
    Anfinsen's thermodn. hypothesis states that the native three-dimensional fold of a protein represents the structure with the lowest Gibbs free energy. Changes in the free energy of denaturation can arise from changes to the folded state, the unfolded state, or both. It has been recently recognized that quinary interactions, transient contacts that take place only in cells, can modulate protein stability through interactions involving the folded state. Here we show that the cellular environment can also remodel the unfolded state ensemble.
  70. 70
    Samanta, N.; Mahanta, D. D.; Hazra, S.; Kumar, G. S.; Mitra, R. K. Short Chain Polyethylene Glycols Unusually Assist Thermal Unfolding of Human Serum Albumin. Biochimie 2014, 104, 81– 89,  DOI: 10.1016/j.biochi.2014.05.009
    Google Scholar
    70
    Short chain polyethylene glycols unusually assist thermal unfolding of human serum albumin
    Samanta, Nirnay; Mahanta, Debasish Das; Hazra, Soumitra; Kumar, Gopinatha Suresh; Mitra, Rajib Kumar
    Biochimie (2014), 104 (), 81-89CODEN: BICMBE; ISSN:0300-9084. (Elsevier Masson SAS)
    In the present study we have investigated the thermal stability of the globular transport protein human serum albumin (HSA), in the presence of two small chain polyethylene glycols (namely PEG 200 and PEG 400). Both near- and far-UV CD (CD) study reveal that addn. of PEG moderately increases the α-helical content of the protein without abruptly changing its tertiary structure. The hydration structure at the protein surface experiences a notable change at 30% PEG (vol./vol.) concn. as evidenced from compressibility and dynamic light scattering (DLS) measurements. Thermal denaturation of HSA in the presence of PEG has been studied by CD and fluorescence spectroscopy using the intrinsic fluorophore tryptophan and it has been found that addn. of PEG makes the protein more prone towards unfolding, which is in contrary to what has been obsd. in case of larger mol. wt. polymers. The energetics of the thermal unfolding process has been obtained using differential scanning calorimetry (DSC) measurements. Our study concludes that both the indirect excluded vol. principle as well as interaction of the polymer at the protein surface is responsible for the obsd. change of the unfolding process.
  71. 71
    Sörensen, T.; Leeb, S.; Danielsson, J.; Oliveberg, M. Polyanions Cause Protein Destabilization Similar to That in Live Cells. Biochemistry 2021, 60 (10), 735– 746,  DOI: 10.1021/acs.biochem.0c00889
    Google Scholar
    71
    Polyanions Cause Protein Destabilization Similar to That in Live Cells
    Sorensen Therese; Leeb Sarah; Danielsson Jens; Oliveberg Mikael
    Biochemistry (2021), 60 (10), 735-746 ISSN:.
    The structural stability of proteins is found to markedly change upon their transfer to the crowded interior of live cells. For some proteins, the stability increases, while for others, it decreases, depending on both the sequence composition and the type of host cell. The mechanism seems to be linked to the strength and conformational bias of the diffusive in-cell interactions, where protein charge is found to play a decisive role. Because most proteins, nucleotides, and membranes carry a net-negative charge, the intracellular environment behaves like a polyanionic (Z:1) system with electrostatic interactions different from those of standard 1:1 ion solutes. To determine how such polyanion conditions influence protein stability, we use negatively charged polyacetate ions to mimic the net-negatively charged cellular environment. The results show that, per Na(+) equivalent, polyacetate destabilizes the model protein SOD1(barrel) significantly more than monoacetate or NaCl. At an equivalent of 100 mM Na(+), the polyacetate destabilization of SOD1(barrel) is similar to that observed in live cells. By the combined use of equilibrium thermal denaturation, folding kinetics, and high-resolution nuclear magnetic resonance, this destabilization is primarily assigned to preferential interaction between polyacetate and the globally unfolded protein. This interaction is relatively weak and involves mainly the outermost N-terminal region of unfolded SOD1(barrel). Our findings point thus to a generic influence of polyanions on protein stability, which adds to the sequence-specific contributions and needs to be considered in the evaluation of in vivo data.
  72. 72
    Kaur, T.; Alshareedah, I.; Wang, W.; Ngo, J.; Moosa, M. M.; Banerjee, P. R. Molecular Crowding Tunes Material States of Ribonucleoprotein Condensates. Biomolecules 2019, 9 (2), 71,  DOI: 10.3390/biom9020071
    Google Scholar
    72
    Molecular crowding tunes material states of ribonucleoprotein condensates
    Kaur, Taranpreet; Alshareedah, Ibraheem; Wang, Wei; Ngo, Jason; Moosa, Mahdi Muhammad; Banerjee, Priya R.
    Biomolecules (2019), 9 (2), 71CODEN: BIOMHC; ISSN:2218-273X. (MDPI AG)
    Ribonucleoprotein (RNP) granules are membraneless liq. condensates that dynamically form, dissolve, and mature into a gel-like state in response to a changing cellular environment. RNP condensation is largely governed by promiscuous attractive inter-chain interactions mediated by low-complexity domains (LCDs). Using an archetypal disordered RNP, fused in sarcoma (FUS), here we study how mol. crowding impacts the RNP liq. condensation. We observe that the liq.-liq. coexistence boundary of FUS is lowered by polymer crowders, consistent with an excluded vol. model. With increasing bulk crowder concn., the RNP partition increases and the diffusion rate decreases in the condensed phase. Furthermore, we show that RNP condensates undergo substantial hardening wherein protein-dense droplets transition from viscous fluid to viscoelastic gel-like states in a crowder concn.-dependent manner. Utilizing two distinct LCDs that broadly represent commonly occurring sequence motifs driving RNP phase transitions, we reveal that the impact of crowding is largely independent of LCD charge and sequence patterns. These results are consistent with a thermodn. model of crowder-mediated depletion interaction, which suggests that inter-RNP attraction is enhanced by mol. crowding. The depletion force is likely to play a key role in tuning the phys. properties of RNP condensates within the crowded cellular space.
  73. 73
    Park, S.; Barnes, R.; Lin, Y.; Jeon, B.; Najafi, S.; Delaney, K. T.; Fredrickson, G. H.; Shea, J.-E.; Hwang, D. S.; Han, S. Dehydration Entropy Drives Liquid-Liquid Phase Separation by Molecular Crowding. Commun. Chem. 2020, 3 (1), 1– 12,  DOI: 10.1038/s42004-020-0328-8
    Google Scholar
    There is no corresponding record for this reference.
  74. 74
    Nguemaha, V.; Zhou, H.-X. Liquid-Liquid Phase Separation of Patchy Particles Illuminates Diverse Effects of Regulatory Components on Protein Droplet Formation. Sci. Rep. 2018, 8 (1), 6728,  DOI: 10.1038/s41598-018-25132-1
    Google Scholar
    74
    Liquid-Liquid Phase Separation of Patchy Particles Illuminates Diverse Effects of Regulatory Components on Protein Droplet Formation
    Nguemaha Valery; Zhou Huan-Xiang; Zhou Huan-Xiang
    Scientific reports (2018), 8 (1), 6728 ISSN:.
    Recently many cellular functions have been associated with membraneless organelles, or protein droplets, formed by liquid-liquid phase separation (LLPS). Proteins in these droplets often contain RNA-binding domains, but the effects of RNA on LLPS have been controversial. To gain better understanding on the roles of RNA and other macromolecular regulators, here we used Gibbs-ensemble simulations to determine phase diagrams of two-component patchy particles, as models for mixtures of proteins with regulatory components. Protein-like particles have four patches, with attraction strength εPP; regulatory particles experience mutual steric repulsion but have two attractive patches toward proteins, with the strength εPR tunable. At low εPR, the regulator, due to steric repulsion, preferentially partitions in the dispersed phase, thereby displacing the protein into the droplet phase and promoting LLPS. At moderate εPR, the regulator starts to partition and displace the protein in the droplet phase, but only to weaken bonding networks and thereby suppress LLPS. At εPR > εPP, the enhanced bonding ability of the regulator initially promotes LLPS, but at higher amounts, the resulting displacement of the protein suppresses LLPS. These results illustrate how RNA can have disparate effects on LLPS, thus able to perform diverse functions in different organelles.
  75. 75
    Protter, D. S. W.; Rao, B. S.; Van Treeck, B.; Lin, Y.; Mizoue, L.; Rosen, M. K.; Parker, R. Intrinsically Disordered Regions Can Contribute Promiscuous Interactions to RNP Granule Assembly. Cell Rep. 2018, 22 (6), 1401– 1412,  DOI: 10.1016/j.celrep.2018.01.036
    Google Scholar
    75
    Intrinsically Disordered Regions Can Contribute Promiscuous Interactions to RNP Granule Assembly
    Protter, David S. W.; Rao, Bhalchandra S.; Van Treeck, Briana; Lin, Yuan; Mizoue, Laura; Rosen, Michael K.; Parker, Roy
    Cell Reports (2018), 22 (6), 1401-1412CODEN: CREED8; ISSN:2211-1247. (Cell Press)
    Eukaryotic cells contain large RNA-protein assemblies referred to as RNP granules, whose assembly is promoted by both traditional protein interactions and intrinsically disordered protein domains. Using RNP granules as an example, we provide evidence for an assembly mechanism of large cellular structures wherein specific protein-protein or protein-RNA interactions act together with promiscuous interactions of intrinsically disordered regions (IDRs). This synergistic assembly mechanism illuminates RNP granule assembly and explains why many components of RNP granules, and other large dynamic assemblies, contain IDRs linked to specific protein-protein or protein-RNA interaction modules. We suggest assemblies based on combinations of specific interactions and promiscuous IDRs are common features of eukaryotic cells.
  76. 76
    Danielsson, J.; Mu, X.; Lang, L.; Wang, H.; Binolfi, A.; Theillet, F.-X.; Bekei, B.; Logan, D. T.; Selenko, P.; Wennerström, H.; Oliveberg, M. Thermodynamics of Protein Destabilization in Live Cells. Proc. Natl. Acad. Sci. U. S. A. 2015, 112 (40), 12402– 12407,  DOI: 10.1073/pnas.1511308112
    Google Scholar
    76
    Thermodynamics of protein destabilization in live cells
    Danielsson, Jens; Mu, Xin; Lang, Lisa; Wang, Huabing; Binolfi, Andres; Theillet, Francois-Xavier; Bekei, Beata; Logan, Derek T.; Selenko, Philipp; Wennerstroem, Haakan; Oliveberg, Mikael
    Proceedings of the National Academy of Sciences of the United States of America (2015), 112 (40), 12402-12407CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at at. resoln. the thermal unfolding of a β-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37° but with a conspicuous twist: while the melting temp. goes down the cold unfolding moves into the physiol. regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, detd. by the individual protein's interplay with the functionally optimized "interaction landscape" of the cellular interior.
  77. 77
    Ghosh, A.; Mazarakos, K.; Zhou, H.-X. Three Archetypical Classes of Macromolecular Regulators of Protein Liquid–Liquid Phase Separation. Proc. Natl. Acad. Sci. U. S. A. 2019, 116 (39), 19474– 19483,  DOI: 10.1073/pnas.1907849116
    Google Scholar
    77
    Three archetypical classes of macromolecular regulators of protein liquid-liquid phase separation
    Ghosh, Archishman; Mazarakos, Konstantinos; Zhou, Huan-Xiang
    Proceedings of the National Academy of Sciences of the United States of America (2019), 116 (39), 19474-19483CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Membraneless organelles, corresponding to the droplet phase upon liq.-liq. phase sepn. (LLPS) of protein or protein-RNA mixts., mediate myriad cellular functions. Cells use a variety of biochem. signals such as expression level and posttranslational modification to regulate droplet formation and dissoln., but the phys. basis of the regulatory mechanisms remains ill-defined and quant. assessment of the effects is largely lacking. Our computational study predicted that the strength of attraction by droplet-forming proteins dictates whether and how macromol. regulators promote or suppress LLPS. We exptl. tested this prediction, using the pentamers of SH3 domains and proline-rich motifs (SH35 and PRM5) as droplet-forming proteins. Detn. of the changes in phase boundary and the partition coeffs. in the droplet phase over a wide range of regulator concns. yielded both a quant. measure and a mechanistic understanding of the regulatory effects. Three archetypical classes of regulatory effects were obsd. Ficoll 70 at high concns. indirectly promoted SH35-PRM5 LLPS, by taking up vol. in the bulk phase and thereby displacing SH35 and PRM5 into the droplet phase. Lysozyme had a moderate partition coeff. and suppressed LLPS by substituting weaker attraction with SH35 for the stronger SH35-PRM5 attraction in the droplet phase. By forming even stronger attraction with PRM5, heparin at low concns. partitioned heavily into the droplet phase and promoted LLPS. These characteristics were recapitulated by computational results of patchy particle models, validating the identification of the 3 classes of macromol. regulators as vol.-exclusion promotors, weak-attraction suppressors, and strong-attraction promotors.
  78. 78
    André, A. A. M.; Spruijt, E. Liquid–Liquid Phase Separation in Crowded Environments. Int. J. Mol. Sci. 2020, 21 (16), 5908,  DOI: 10.3390/ijms21165908
    Google Scholar
    78
    Liquid-liquid phase separation in crowded environments
    Andre, Alain A. M.; Spruijt, Evan
    International Journal of Molecular Sciences (2020), 21 (16), 5908CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
    A review. Biomol. condensates play a key role in organizing cellular fluids such as the cytoplasm and nucleoplasm. Most of these non-membranous organelles show liq.-like properties both in cells and when studied in vitro through liq.-liq. phase sepn. (LLPS) of purified proteins. In general, LLPS of proteins is known to be sensitive to variations in pH, temp. and ionic strength, but the role of crowding remains underappreciated. Several decades of research have shown that macromol. crowding can have profound effects on protein interactions, folding and aggregation, and it must, by extension, also impact LLPS. However, the precise role of crowding in LLPS is far from trivial, as most condensate components have a disordered nature and exhibit multiple weak attractive interactions. Here, we discuss which factors det. the scope of LLPS in crowded environments, and we review the evidence for the impact of macromol. crowding on phase boundaries, partitioning behavior and condensate properties. Based on a comparison of both in vivo and in vitro LLPS studies, we propose that phase sepn. in cells does not solely rely on attractive interactions, but shows important similarities to segregative phase sepn.
  79. 79
    Banani, S. F.; Rice, A. M.; Peeples, W. B.; Lin, Y.; Jain, S.; Parker, R.; Rosen, M. K. Compositional Control of Phase-Separated Cellular Bodies. Cell 2016, 166 (3), 651– 663,  DOI: 10.1016/j.cell.2016.06.010
    Google Scholar
    79
    Compositional control of phase-separated cellular bodies
    Banani, Salman F.; Rice, Allyson M.; Peeples, William B.; Lin, Yuan; Jain, Saumya; Parker, Roy; Rosen, Michael K.
    Cell (Cambridge, MA, United States) (2016), 166 (3), 651-663CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    Cellular bodies such as P bodies and PML nuclear bodies (PML NBs) appear to be phase-sepd. liqs. organized by multivalent interactions among proteins and RNA mols. Although many components of various cellular bodies are known, general principles that define body compn. are lacking. Here, the authors modeled cellular bodies using several engineered multivalent proteins and RNA. In vitro and in cells, these scaffold mols. formed phase-sepd. liqs. that concd. low valency client proteins. Clients partitioned differently depending on the ratio of scaffolds, with a sharp switch across the phase diagram diagonal. The compn. could switch rapidly through changes in scaffold concn. or valency. Natural PML NBs and P bodies showed analogous partitioning behavior, suggesting how their compns. could be controlled by levels of PML SUMOylation or cellular mRNA concn., resp. The data suggested a conceptual framework for considering the compn. and control thereof of cellular bodies assembled through heterotypic multivalent interactions.
  80. 80
    Sterpone, F.; Melchionna, S.; Tuffery, P.; Pasquali, S.; Mousseau, N.; Cragnolini, T.; Chebaro, Y.; St-Pierre, J.-F.; Kalimeri, M.; Barducci, A.; Laurin, Y.; Tek, A.; Baaden, M.; Nguyen, P. H.; Derreumaux, P. The OPEP Protein Model: From Single Molecules, Amyloid Formation, Crowding and Hydrodynamics to DNA/RNA Systems. Chem. Soc. Rev. 2014, 43 (13), 4871– 4893,  DOI: 10.1039/C4CS00048J
    Google Scholar
    80
    The OPEP protein model: from single molecules, amyloid formation, crowding and hydrodynamics to DNA/RNA systems
    Sterpone, Fabio; Melchionna, Simone; Tuffery, Pierre; Pasquali, Samuela; Mousseau, Normand; Cragnolini, Tristan; Chebaro, Yassmine; St-Pierre, Jean-Francois; Kalimeri, Maria; Barducci, Alessandro; Laurin, Yoann; Tek, Alex; Baaden, Marc; Nguyen, Phuong Hoang; Derreumaux, Philippe
    Chemical Society Reviews (2014), 43 (13), 4871-4893CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
    A review. The OPEP coarse-grained protein model has been applied to a wide range of applications since its first release 15 years ago. The model, which combines energetic and structural accuracy and chem. specificity, allows the study of single protein properties, DNA-RNA complexes, amyloid fibril formation and protein suspensions in a crowded environment. Here we first review the current state of the model and the most exciting applications using advanced conformational sampling methods. We then present the current limitations and a perspective on the ongoing developments.
  81. 81
    Stirnemann, F.; Derreumaux, P.; Melchionna, S. Protein Simulations in Fluids: Coupling the OPEP Coarse-Grained Force Field with Hydrodynamics. J. Chem. Theory Comput. 2015, 11 (4), 1843– 1853,  DOI: 10.1021/ct501015h
    Google Scholar
    There is no corresponding record for this reference.
  82. 82
    Claes, F.; Rudyak, S.; Laird, A. S.; Louros, N.; Beerten, J.; Debulpaep, M.; Michiels, E.; van der Kant, R.; Van Durme, J.; De Baets, G.; Houben, B.; Ramakers, M.; Yuan, K.; Gwee, S. S. L.; Hernandez, S.; Broersen, K.; Oliveberg, M.; Moahamed, B.; Kirstein, J.; Robberecht, W.; Rousseau, F.; Schymkowitz, J. Exposure of a Cryptic Hsp70 Binding Site Determines the Cytotoxicity of the ALS-Associated SOD1-Mutant A4V. Protein Eng., Des. Sel. 2019, 32 (10), 443– 457,  DOI: 10.1093/protein/gzaa008
    Google Scholar
    82
    Exposure of a cryptic Hsp70 binding site determines the cytotoxicity of the ALS-associated SOD1-mutant A4V
    Claes, Filip; Rudyak, Stanislav; Laird, Angela S.; Louros, Nikolaos; Beerten, Jacinte; Debulpaep, Maja; Michiels, Emiel; van der Kant, Rob; Van Durme, Joost; De Baets, Greet; Houben, Bert; Ramakers, Meine; Yuan, Kristy; Gwee, Serene S. L.; Hernandez, Sara; Broersen, Kerensa; Oliveberg, Mikael; Moahamed, Barbara; Kirstein, Janine; Robberecht, Wim; Rousseau, Frederic; Schymkowitz, Joost
    Protein Engineering, Design & Selection (2019), 32 (10), 443-457CODEN: PEDSBR; ISSN:1741-0134. (Oxford University Press)
    The accumulation of toxic protein aggregates is thought to play a key role in a range of degenerative pathologies, but it remains unclear why aggregation of polypeptides into non-native assemblies is toxic and why cellular clearance pathways offer ineffective protection. We here study the A4V mutant of SOD1, which forms toxic aggregates in motor neurons of patients with familial amyotrophic lateral sclerosis (ALS). A comparison of the location of aggregation prone regions (APRs) and Hsp70 binding sites in the denatured state of SOD1 reveals that ALS-assocd. mutations promote exposure of the APRs more than the strongest Hsc/Hsp70 binding site that we could detect. Mutations designed to increase the exposure of this Hsp70 interaction site in the denatured state promote aggregation but also display an increased interaction with Hsp70 chaperones. Depending on the cell type, in vitro this resulted in cellular inclusion body formation or increased clearance, accompanied with a suppression of cytotoxicity. The latter was also obsd. in a zebrafish model in vivo. Our results suggest that the uncontrolled accumulation of toxic SOD1A4V aggregates results from insufficient detection by the cellular surveillance network.
  83. 83
    Imamoglu, R.; Balchin, D.; Hayer-Hartl, M.; Hartl, F. U. Bacterial Hsp70 Resolves Misfolded States and Accelerates Productive Folding of a Multi-Domain Protein. Nat. Commun. 2020, 11 (1), 1– 13,  DOI: 10.1038/s41467-019-14245-4
    Google Scholar
    There is no corresponding record for this reference.
  84. 84
    Gal, J.; Ström, A.-L.; Kilty, R.; Zhang, F.; Zhu, H. P62 Accumulates and Enhances Aggregate Formation in Model Systems of Familial Amyotrophic Lateral Sclerosis*. J. Biol. Chem. 2007, 282 (15), 11068– 11077,  DOI: 10.1074/jbc.M608787200
    Google Scholar
    84
    p62 Accumulates and Enhances Aggregate Formation in Model Systems of Familial Amyotrophic Lateral Sclerosis
    Gal, Jozsef; Stroem, Anna-Lena; Kilty, Renee; Zhang, Fujian; Zhu, Haining
    Journal of Biological Chemistry (2007), 282 (15), 11068-11077CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by motor neuron death. A hallmark of the disease is the appearance of protein aggregates in the affected motor neurons. We have found that p62, a protein implicated in protein aggregate formation, accumulated progressively in the G93A mouse spinal cord. The accumulation of p62 was in parallel to the increase of polyubiquitinated proteins and mutant SOD1 aggregates. Immunostaining studies showed that p62, ubiquitin, and mutant SOD1 co-localized in the protein aggregates in affected cells in G93A mouse spinal cord. The p62 protein selectively interacted with familial ALS mutants, but not WT SOD1. When p62 was co-expressed with SOD1 in NSC34 cells, it greatly enhanced the formation of aggregates of the ALS-linked SOD1 mutants, but not wild-type SOD1. Cell viability was measured in the presence and absence of overexpressed p62, and the results suggest that the large aggregates facilitated by p62 were not directly toxic to cells under the conditions in this study. Deletion of the ubiquitin-assocn. (UBA) domain of p62 significantly decreased the p62-facilitated aggregate formation, but did not completely inhibit it. Further protein interaction expts. also showed that the truncated p62 with the UBA domain deletion remained capable of interacting with mutant SOD1. The findings of this study show that p62 plays a crit. role in forming protein aggregates in familial ALS, likely by linking misfolded mutant SOD1 mols. and other cellular proteins together.
  85. 85
    Ciechanover, A.; Kwon, Y. T. Degradation of Misfolded Proteins in Neurodegenerative Diseases: Therapeutic Targets and Strategies. Exp. Mol. Med. 2015, 47 (3), e147– e147,  DOI: 10.1038/emm.2014.117
    Google Scholar
    85
    Degradation of misfolded proteins in neurodegenerative diseases: therapeutic targets and strategies
    Ciechanover, Aaron; Kwon, Yong Tae
    Experimental & Molecular Medicine (2015), 47 (3), e147CODEN: EMMEF3; ISSN:2092-6413. (NPG Nature Asia-Pacific)
    A review. Mammalian cells remove misfolded proteins using various proteolytic systems, including the ubiquitin (Ub)-proteasome system (UPS), chaperone mediated autophagy (CMA) and macroautophagy. The majority of misfolded proteins are degraded by the UPS, in which Ub-conjugated substrates are deubiquitinated, unfolded and cleaved into small peptides when passing through the narrow chamber of the proteasome. The substrates that expose a specific degrdn. signal, the KFERQ sequence motif, can be delivered to and degraded in lysosomes via the CMA. Aggregation-prone substrates resistant to both the UPS and the CMA can be degraded by macroautophagy, in which cargoes are segregated into autophagosomes before degrdn. by lysosomal hydrolases. Although most misfolded and aggregated proteins in the human proteome can be degraded by cellular protein quality control, some native and mutant proteins prone to aggregation into β-sheet-enriched oligomers are resistant to all known proteolytic pathways and can thus grow into inclusion bodies or extracellular plaques. The accumulation of protease-resistant misfolded and aggregated proteins is a common mechanism underlying protein misfolding disorders, including neurodegenerative diseases such as Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), prion diseases and Amyotrophic Lateral Sclerosis (ALS). In this review, we provide an overview of the proteolytic pathways in neurons, with an emphasis on the UPS, CMA and macroautophagy, and discuss the role of protein quality control in the degrdn. of pathogenic proteins in neurodegenerative diseases. Addnl., we examine existing putative therapeutic strategies to efficiently remove cytotoxic proteins from degenerating neurons.
  86. 86
    Vecchi, G.; Sormanni, P.; Mannini, B.; Vandelli, A.; Tartaglia, G. G.; Dobson, C. M.; Hartl, F. U.; Vendruscolo, M. Proteome-Wide Observation of the Phenomenon of Life on the Edge of Solubility. Proc. Natl. Acad. Sci. U. S. A. 2020, 117 (2), 1015– 1020,  DOI: 10.1073/pnas.1910444117
    Google Scholar
    86
    Proteome-wide observation of the phenomenon of life on the edge of solubility
    Vecchi, Giulia; Sormanni, Pietro; Mannini, Benedetta; Vandelli, Andrea; Tartaglia, Gian Gaetano; Dobson, Christopher M.; Hartl, F. Ulrich; Vendruscolo, Michele
    Proceedings of the National Academy of Sciences of the United States of America (2020), 117 (2), 1015-1020CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    To function effectively proteins must avoid aberrant aggregation, and hence they are expected to be expressed at concns. safely below their soly. limits. By analyzing proteome-wide mass spectrometry data of Caenorhabditis elegans, however, we show that the levels of about three-quarters of the nearly 4,000 proteins analyzed in adult animals are close to their intrinsic soly. limits, indeed exceeding them by about 10% on av. We next asked how aging and functional self-assembly influence these soly. limits. We found that despite the fact that the total quantity of proteins within the cellular environment remains approx. const. during aging, protein aggregation sharply increases between days 6 and 12 of adulthood, after the worms have reproduced, as individual proteins lose their stoichiometric balances and the cellular machinery that maintains soly. undergoes functional decline. These findings reveal that these proteins are highly prone to undergoing concn.-dependent phase sepn., which on aging is rationalized in a decrease of their effective solubilities, in particular for proteins assocd. with translation, growth, reprodn., and the chaperone system.
  87. 87
    Dobson, C. M. Principles of Protein Folding, Misfolding and Aggregation. Semin. Cell Dev. Biol. 2004, 15 (1), 3– 16,  DOI: 10.1016/j.semcdb.2003.12.008
    Google Scholar
    87
    Principles of protein folding, misfolding and aggregation
    Dobson, Christopher M.
    Seminars in Cell & Developmental Biology (2004), 15 (1), 3-16CODEN: SCDBFX; ISSN:1084-9521. (Elsevier Science B.V.)
    A review. Here, the author summarizes the current understanding of the underlying and universal mechanism by which newly synthesized proteins achieve their biol. functional states. Protein mols., however, all have a finite tendency either to misfold, or to fail to maintain their correctly folded states, under some circumstances. The author describes some of the consequences of such behavior, particularly in the context of the aggregation events that are frequently assocd. with aberrant folding. The authors focuses in particular on the emerging links between protein aggregation and the increasingly prevalent forms of debilitating disease with which it is now known to be assocd.
  88. 88
    Zbinden, A.; Pérez-Berlanga, M.; De Rossi, P.; Polymenidou, M. Phase Separation and Neurodegenerative Diseases: A Disturbance in the Force. Dev. Cell 2020, 55 (1), 45– 68,  DOI: 10.1016/j.devcel.2020.09.014
    Google Scholar
    88
    Phase Separation and Neurodegenerative Diseases: A Disturbance in the Force
    Zbinden, Aurelie; Perez-Berlanga, Manuela; De Rossi, Pierre; Polymenidou, Magdalini
    Developmental Cell (2020), 55 (1), 45-68CODEN: DCEEBE; ISSN:1534-5807. (Cell Press)
    Protein aggregation is the main hallmark of neurodegenerative diseases. Many proteins found in pathol. inclusions are known to undergo liq.-liq. phase sepn., a reversible process of mol. self-assembly. Emerging evidence supports the hypothesis that aberrant phase sepn. behavior may serve as a trigger of protein aggregation in neurodegeneration, and efforts to understand and control the underlying mechanisms are underway. Here, we review similarities and differences among four main proteins, α-synuclein, FUS, tau, and TDP-43, which are found aggregated in different diseases and were independently shown to phase sep. We discuss future directions in the field that will help shed light on the mol. mechanisms of aggregation and neurodegeneration.

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  14. Sibasankar Panigrahy, Rahul Sahu, Sandeep K. Reddy, Divya Nayar. Structure, energetics and dynamics in crowded amino acid solutions: a molecular dynamics study. Physical Chemistry Chemical Physics 2023, 25 (7) , 5430-5442. https://doi.org/10.1039/D2CP04238J
  15. Bidisha Das, Sumangal Roychowdhury, Priyesh Mohanty, Azamat Rizuan, Joy Chakraborty, Jeetain Mittal, Krishnananda Chattopadhyay. A Zn‐dependent structural transition of SOD1 modulates its ability to undergo phase separation. The EMBO Journal 2023, 42 (2) https://doi.org/10.15252/embj.2022111185
  16. Hong-Yu Hu, Ya-Jun Liu. Sequestration of cellular native factors by biomolecular assemblies: Physiological or pathological?. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 2022, 1869 (12) , 119360. https://doi.org/10.1016/j.bbamcr.2022.119360
  17. Nirnay Samanta, Yasser B. Ruiz‐Blanco, Zamira Fetahaj, David Gnutt, Carter Lantz, Joseph A. Loo, Elsa Sanchez‐Garcia, Simon Ebbinghaus. Superoxide Dismutase 1 Folding Stability as a Target for Molecular Tweezers in SOD1‐Related Amyotrophic Lateral Sclerosis. ChemBioChem 2022, 23 (21) https://doi.org/10.1002/cbic.202200396
  18. Emmanuel Amzallag, Eran Hornstein. Crosstalk between Biomolecular Condensates and Proteostasis. Cells 2022, 11 (15) , 2415. https://doi.org/10.3390/cells11152415
  19. Oliver Brylski, Puja Shrestha, Patricia Gnutt, David Gnutt, Jonathan Wolf Mueller, Simon Ebbinghaus. Cellular ATP Levels Determine the Stability of a Nucleotide Kinase. Frontiers in Molecular Biosciences 2021, 8 https://doi.org/10.3389/fmolb.2021.790304
  20. Stepan Timr, Fabio Sterpone. Computational Insights into the Unfolding of a Destabilized Superoxide Dismutase 1 Mutant. Biology 2021, 10 (12) , 1240. https://doi.org/10.3390/biology10121240
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Cite this: J. Am. Chem. Soc. 2021, 143, 47, 19909–19918
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  • Abstract

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    Figure 1

    Figure 1. SOD1bar as a sensor to measure protein unfolding and SG association in-cell and in vitro. (A) SOD1FL (Protein Data Bank (PDB) ID: IHL5) and SOD1bar (PDB ID: 4BCZ), with mutations A4V, I35A, G41D, G85R, and G93V highlighted. Images were assembled using UCSF Chimera. (52) (B) Schematic representation of the FReI technique with two cameras (CCD1 and CCD2). DM and BS represent dichroic mirror and beam splitter, respectively. (C) Exemplary images of HeLaFUS-mCh cells showing different partitioning of the SOD1bar-AcGFP1 mutants G93V and A4V. Enrichment of SOD1bar-AcGFP1 was defined by a local apparent partition coefficient (PC) > 1, determined as the ratio of the mean fluorescence intensity inside the SG (region 1) and the mean fluorescence intensity at 0.6 μm (region 2) from the SG (right zoom-in). (D) PC of SOD1bar after 120 min at 43 °C (N = 437 to 523 SGs). PCs of mutants A4V, G93V, G41D, and G85R are significantly higher than Wt. (E) PCs as a function of ΔGf°′ at 37 °C. The values are significantly different between each of the three defined clusters: Wt and I35A (light gray), G93V, G41D, and G85R (light brown) and A4V (light red). (F) PCs of SOD1bar-Wt and SOD1FL-Wt and the respective A4V mutants. No statistical significance is found between the PCs of SOD1bar and SOD1FL for both Wt and A4V. (G) Correlation between SOD1bar PCs and the scaled sum [norm. ΔfU37–43°C + norm. Hphob]. We normalized ΔfU37–43°C values for the different constructs (1 represents the highest ΔfU37–43°C, while 0 represents the lowest one). Similarly, Hphob was normalized from 1 (highest) to the 0 (lowest) within the different SOD1bar sequences. Finally, we sum up the normalized values, [norm. ΔfU37–43°C + norm. Hphob] and scaled between 0 and 1 (lowest to highest). (H) Correlation between mobile fractions (MFs) inside SGs and PCs. The statistical analyses reported in panels D–F were carried out using one-way ANOVA, followed by a post hoc Tukey test for multiple comparisons, computed with a confidence interval of 95%. The values in panels E, G, and H are presented as mean ± SEM.

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    Figure 2

    Figure 2. Association of SOD1bar with FUS droplets in vitro. (A) Schematic representation of the different FUS domains with predicted (dis)ordered domains by Prediction of Natural Disorder Regions (PONDR) Score bioinformatics tool. (56) (B) Exemplary images (mCherry channel) of SOD1bar added to FUS droplets (in buffer, pH 7.5) after 60 min of incubation at 23 or 43 °C. (C) PCs after 60 min of incubation shown as whisker box plots (N = 41–203 droplets for 23 °C, N = 422–895 droplets for 43 °C). The values are significantly different (except G85R vs A4V) among each other. Significant tests were carried out by one-way ANOVA analysis, followed by post hoc Tukey test for multiple comparisons, computed with a confidence interval of 95%. (D) Mean values of PCs (at 43 °C) plotted against ΔGf°′ at 43 °C.

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    Figure 3

    Figure 3. Effect of crowding (in vitro) on SOD1bar association with FUS droplets at different temperatures. Exemplary images (mCherry channel) of SOD1bar enrichment/depletion in the presence of (A) 15% (w/v) Ficoll 70 and (C) 20% (w/v) BSA, after 60 min of incubation at 23 and 43 °C. (B and D) PCs measured after 60 min at 23 and 43 °C in the presence of Ficoll and BSA. PCs in buffer (data from Figure 2) are shown as gray box plots for comparison. (N = 96–304 droplets for 23 °C, N = 263–706 droplets for 43 °C). The values are significantly different among each other [except for (B) G85R (in buffer) vs G85R (in Ficoll), Wt (in Ficoll) vs I35A (in Ficoll), and (D) Wt (in BSA) vs I35A (in BSA)]. Significance tests were carried out by one-way ANOVA analysis, followed by post hoc Tukey test for multiple comparisons, computed with a confidence interval of 95%.

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  • References

    This article references 88 other publications.

    1. 1
      Case, L. B.; Zhang, X.; Ditlev, J. A.; Rosen, M. K. Stoichiometry Controls Activity of Phase-Separated Clusters of Actin Signaling Proteins. Science 2019, 363 (6431), 1093– 1097,  DOI: 10.1126/science.aau6313
      1
      Stoichiometry controls activity of phase-separated clusters of actin signaling proteins
      Case, Lindsay B.; Zhang, Xu; Ditlev, Jonathon A.; Rosen, Michael K.
      Science (Washington, DC, United States) (2019), 363 (6431), 1093-1097CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Biomol. condensates conc. macromols. into foci without a surrounding membrane. Many condensates appear to form through multivalent interactions that drive liq.-liq. phase sepn. (LLPS). LLPS increases the specific activity of actin regulatory proteins toward actin assembly by the Arp2/3 complex. We show that this increase occurs because LLPS of the Nephrin-Nck-N-WASP signaling pathway on lipid bilayers increases membrane dwell time of N-WASP and Arp2/3 complex, consequently increasing actin assembly. Dwell time varies with relative stoichiometry of the signaling proteins in the phase-sepd. clusters, rendering N-WASP and Arp2/3 activity stoichiometry dependent. This mechanism of controlling protein activity is enabled by the stoichiometrically undefined nature of biomol. condensates. Such regulation should be a general feature of signaling systems that assemble through multivalent interactions and drive nonequil. outputs.
    2. 2
      Huang, W. Y. C.; Alvarez, S.; Kondo, Y.; Lee, Y. K.; Chung, J. K.; Lam, H. Y. M.; Biswas, K. H.; Kuriyan, J.; Groves, J. T. A Molecular Assembly Phase Transition and Kinetic Proofreading Modulate Ras Activation by SOS. Science 2019, 363 (6431), 1098– 1103,  DOI: 10.1126/science.aau5721
      2
      A molecular assembly phase transition and kinetic proofreading modulate Ras activation by SOS
      Huang, William Y. C.; Alvarez, Steven; Kondo, Yasushi; Lee, Young Kwang; Chung, Jean K.; Lam, Hiu Yue Monatrice; Biswas, Kabir H.; Kuriyan, John; Groves, Jay T.
      Science (Washington, DC, United States) (2019), 363 (6431), 1098-1103CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      The guanine nucleotide exchange factor (GEF) Son of Sevenless (SOS) is a key Ras activator that is autoinhibited in the cytosol and activates upon membrane recruitment. Autoinhibition release involves structural rearrangements of the protein at the membrane and thus introduces a delay between initial recruitment and activation. In this study, we designed a single-mol. assay to resolve the time between initial receptor-mediated membrane recruitment and the initiation of GEF activity of individual SOS mols. on microarrays of Ras-functionalized supported membranes. The rise-and-fall shape of the measured SOS activation time distribution and the long mean time scale to activation (∼50 s) establish a basis for kinetic proofreading in the receptor-mediated activation of Ras. We further demonstrate that this kinetic proofreading is modulated by the LAT (linker for activation of T cells)-Grb2-SOS phosphotyrosine-driven phase transition at the membrane.
    3. 3
      Lu, Y.; Wu, T.; Gutman, O.; Lu, H.; Zhou, Q.; Henis, Y. I.; Luo, K. Phase Separation of TAZ Compartmentalizes the Transcription Machinery to Promote Gene Expression. Nat. Cell Biol. 2020, 22 (4), 453– 464,  DOI: 10.1038/s41556-020-0485-0
      3
      Phase separation of TAZ compartmentalizes the transcription machinery to promote gene expression
      Lu, Yi; Wu, Tiantian; Gutman, Orit; Lu, Huasong; Zhou, Qiang; Henis, Yoav I.; Luo, Kunxin
      Nature Cell Biology (2020), 22 (4), 453-464CODEN: NCBIFN; ISSN:1465-7392. (Nature Research)
      Abstr.: TAZ promotes growth, development and tumorigenesis by regulating the expression of target genes. However, the manner in which TAZ orchestrates the transcriptional responses is poorly defined. Here we demonstrate that TAZ forms nuclear condensates through liq.-liq. phase sepn. to compartmentalize its DNA-binding cofactor TEAD4, coactivators BRD4 and MED1, and the transcription elongation factor CDK9 for transcription. TAZ forms phase-sepd. droplets in vitro and liq.-like nuclear condensates in vivo, and this ability is neg. regulated by Hippo signaling through LATS-mediated phosphorylation and is mediated by the coiled-coil (CC) domain. Deletion of the TAZ CC domain or substitution with the YAP CC domain prevents the phase sepn. of TAZ and its ability to induce the expression of TAZ-specific target genes. Thus, we identify a mechanism of transcriptional activation by TAZ and demonstrate that pathway-specific transcription factors also engage the phase-sepn. mechanism for efficient and specific transcriptional activation.
    4. 4
      Iserman, C.; Desroches Altamirano, C.; Jegers, C.; Friedrich, U.; Zarin, T.; Fritsch, A. W.; Mittasch, M.; Domingues, A.; Hersemann, L.; Jahnel, M. Condensation of Ded1p Promotes a Translational Switch from Housekeeping to Stress Protein Production. Cell 2020, 181, 818,  DOI: 10.1016/j.cell.2020.04.009
      4
      Condensation of Ded1p Promotes a Translational Switch from Housekeeping to Stress Protein Production
      Iserman, Christiane; Desroches Altamirano, Christine; Jegers, Ceciel; Friedrich, Ulrike; Zarin, Taraneh; Fritsch, Anatol W.; Mittasch, Matthaeus; Domingues, Antonio; Hersemann, Lena; Jahnel, Marcus; Richter, Doris; Guenther, Ulf-Peter; Hentze, Matthias W.; Moses, Alan M.; Hyman, Anthony A.; Kramer, Guenter; Kreysing, Moritz; Franzmann, Titus M.; Alberti, Simon
      Cell (Cambridge, MA, United States) (2020), 181 (4), 818-831.e19CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      Cells sense elevated temps. and mount an adaptive heat shock response that involves changes in gene expression, but the underlying mechanisms, particularly on the level of translation, remain unknown. Here we report that, in budding yeast, the essential translation initiation factor Ded1p undergoes heat-induced phase sepn. into gel-like condensates. Using ribosome profiling and an in vitro translation assay, we reveal that condensate formation inactivates Ded1p and represses translation of housekeeping mRNAs while promoting translation of stress mRNAs. Testing a variant of Ded1p with altered phase behavior as well as Ded1p homologs from diverse species, we demonstrate that Ded1p condensation is adaptive and fine-tuned to the max. growth temp. of the resp. organism. We conclude that Ded1p condensation is an integral part of an extended heat shock response that selectively represses translation of housekeeping mRNAs to promote survival under conditions of severe heat stress.
    5. 5
      Peeples, W.; Rosen, M. K. Mechanistic Dissection of Increased Enzymatic Rate in a Phase-Separated Compartment. Nat. Chem. Biol. 2021, 17 (6), 693– 702,  DOI: 10.1038/s41589-021-00801-x
      5
      Mechanistic dissection of increased enzymatic rate in a phase-separated compartment
      Peeples, William; Rosen, Michael K.
      Nature Chemical Biology (2021), 17 (6), 693-702CODEN: NCBABT; ISSN:1552-4450. (Nature Portfolio)
      Biomol. condensates conc. macromols. into discrete cellular foci without an encapsulating membrane. Condensates are often presumed to increase enzymic reaction rates through increased concns. of enzymes and substrates (mass action), although this idea has not been widely tested and other mechanisms of modulation are possible. Here we describe a synthetic system where the SUMOylation enzyme cascade is recruited into engineered condensates generated by liq.-liq. phase sepn. of multidomain scaffolding proteins. SUMOylation rates can be increased up to 36-fold in these droplets compared to the surrounding bulk, depending on substrate KM. This dependency produces substantial specificity among different substrates. Analyses of reactions above and below the phase-sepn. threshold lead to a quant. model in which reactions in condensates are accelerated by mass action and changes in substrate KM, probaby due to scaffold-induced mol. organization. Thus, condensates can modulate reaction rates both by concg. mols. and phys. organizing them.
    6. 6
      Riback, J. A.; Katanski, C. D.; Kear-Scott, J. L.; Pilipenko, E. V.; Rojek, A. E.; Sosnick, T. R.; Drummond, D. A. Stress-Triggered Phase Separation Is an Adaptive, Evolutionarily Tuned Response. Cell 2017, 168 (6), 1028– 1040,  DOI: 10.1016/j.cell.2017.02.027
      6
      Stress-triggered phase separation is an adaptive, evolutionarily tuned response
      Riback, Joshua A.; Katanski, Christopher D.; Kear-Scott, Jamie L.; Pilipenko, Evgeny V.; Rojek, Alexandra E.; Sosnick, Tobin R.; Drummond, D. Allan
      Cell (Cambridge, MA, United States) (2017), 168 (6), 1028-1040.e19CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      In eukaryotic cells, diverse stresses trigger coalescence of RNA-binding proteins into stress granules. In vitro, stress granule-assocd. proteins can demix to form liqs., hydrogels, and other assemblies lacking fixed stoichiometry. Observing these phenomena has generally required conditions far removed from physiol. stresses. Here, we show that poly(A)-binding protein (Pab1 in yeast), a defining marker of stress granules, phase separates and forms hydrogels in vitro upon exposure to physiol. stress conditions. Other RNA-binding proteins depend upon low-complexity regions (LCRs) or RNA for phase sepn., whereas Pab1's LCR is not required for demixing, and RNA inhibits it. Based on unique evolutionary patterns, we created LCR mutations, which systematically tuned its biophys. properties and Pab1 phase sepn. in vitro and in vivo. Mutations that impeded phase sepn. reduced organism fitness during prolonged stress. Poly(A)-binding protein thus acts as a physiol. stress sensor, exploiting phase sepn. to precisely mark stress onset, a broadly generalizable mechanism.
    7. 7
      Franzmann, T. M.; Jahnel, M.; Pozniakovsky, A.; Mahamid, J.; Holehouse, A. S.; Nüske, E.; Richter, D.; Baumeister, W.; Grill, S. W.; Pappu, R. V. Phase Separation of a Yeast Prion Protein Promotes Cellular Fitness. Science 2018, 359 (6371), eaao5654,  DOI: 10.1126/science.aao5654
      There is no corresponding record for this reference.
    8. 8
      Petrovska, I.; Nüske, E.; Munder, M. C.; Kulasegaran, G.; Malinovska, L.; Kroschwald, S.; Richter, D.; Fahmy, K.; Gibson, K.; Verbavatz, J.-M.; Alberti, S. Filament Formation by Metabolic Enzymes Is a Specific Adaptation to an Advanced State of Cellular Starvation. eLife 2014, 3, e02409  DOI: 10.7554/eLife.02409
      There is no corresponding record for this reference.
    9. 9
      Munder, M. C.; Midtvedt, D.; Franzmann, T.; Nüske, E.; Otto, O.; Herbig, M.; Ulbricht, E.; Müller, P.; Taubenberger, A.; Maharana, S.; Malinovska, L.; Richter, D.; Guck, J.; Zaburdaev, V.; Alberti, S. A PH-Driven Transition of the Cytoplasm from a Fluid- to a Solid-like State Promotes Entry into Dormancy. eLife 2016, 5, e09347  DOI: 10.7554/eLife.09347
      9
      A pH-driven transition of the cytoplasm from a fluid- to a solid-like state promotes entry into dormancy
      Munder, Matthias Christoph; Midtvedt, Daniel; Franzmann, Titus; Nueske, Elisabeth; Otto, Oliver; Herbig, Maik; Ulbricht, Elke; Mueller, Paul; Taubenberger, Anna; Maharana, Shovamayee; Malinovska, Liliana; Richter, Doris; Guck, Jochen; Zaburdaev, Vasily; Alberti, Simon
      eLife (2016), 5 (), e09347/1-e09347/30CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)
      Cells can enter into a dormant state when faced with unfavorable conditions. However, how cells enter into and recover from this state is still poorly understood. Here, we study dormancy in different eukaryotic organisms and find it to be assocd. with a significant decrease in the mobility of organelles and foreign tracer particles. We show that this reduced mobility is caused by an influx of protons and a marked acidification of the cytoplasm, which leads to widespread macromol. assembly of proteins and triggers a transition of the cytoplasm to a solid-like state with increased mech. stability. We further demonstrate that this transition is required for cellular survival under conditions of starvation. Our findings have broad implications for understanding alternative physiol. states, such as quiescence and dormancy, and create a new view of the cytoplasm as an adaptable fluid that can reversibly transition into a protective solid-like state.
    10. 10
      Wallace, E. W. J.; Kear-Scott, J. L.; Pilipenko, E. V.; Schwartz, M. H.; Laskowski, P. R.; Rojek, A. E.; Katanski, C. D.; Riback, J. A.; Dion, M. F.; Franks, A. M.; Airoldi, E. M.; Pan, T.; Budnik, B. A.; Drummond, D. A. Reversible, Specific, Active Aggregates of Endogenous Proteins Assemble upon Heat Stress. Cell 2015, 162 (6), 1286– 1298,  DOI: 10.1016/j.cell.2015.08.041
      10
      Reversible, Specific, Active Aggregates of Endogenous Proteins Assemble upon Heat Stress
      Wallace, Edward W. J.; Kear-Scott, Jamie L.; Pilipenko, Evgeny V.; Schwartz, Michael H.; Laskowski, Pawel R.; Rojek, Alexandra E.; Katanski, Christopher D.; Riback, Joshua A.; Dion, Michael F.; Franks, Alexander M.; Airoldi, Edoardo M.; Pan, Tao; Budnik, Bogdan A.; Drummond, D. Allan
      Cell (Cambridge, MA, United States) (2015), 162 (6), 1286-1298CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      Heat causes protein misfolding and aggregation and, in eukaryotic cells, triggers aggregation of proteins and RNA into stress granules. We have carried out extensive proteomic studies to quantify heat-triggered aggregation and subsequent disaggregation in budding yeast, identifying >170 endogenous proteins aggregating within minutes of heat shock in multiple subcellular compartments. We demonstrate that these aggregated proteins are not misfolded and destined for degrdn. Stable-isotope labeling reveals that even severely aggregated endogenous proteins are disaggregated without degrdn. during recovery from shock, contrasting with the rapid degrdn. obsd. for many exogenous thermolabile proteins. Although aggregation likely inactivates many cellular proteins, in the case of a heterotrimeric aminoacyl-tRNA synthetase complex, the aggregated proteins remain active with unaltered fidelity. We propose that most heat-induced aggregation of mature proteins reflects the operation of an adaptive, autoregulatory process of functionally significant aggregate assembly and disassembly that aids cellular adaptation to thermal stress.
    11. 11
      Cabrera, M.; Boronat, S.; Marte, L.; Vega, M.; Pérez, P.; Ayté, J.; Hidalgo, E. Chaperone-Facilitated Aggregation of Thermo-Sensitive Proteins Shields Them from Degradation during Heat Stress. Cell Rep. 2020, 30 (7), 2430– 2443,  DOI: 10.1016/j.celrep.2020.01.077
      11
      Chaperone-Facilitated Aggregation of Thermo-Sensitive Proteins Shields Them from Degradation during Heat Stress
      Cabrera, Margarita; Boronat, Susanna; Marte, Luis; Vega, Montserrat; Perez, Pilar; Ayte, Jose; Hidalgo, Elena
      Cell Reports (2020), 30 (7), 2430-2443.e4CODEN: CREED8; ISSN:2211-1247. (Cell Press)
      Cells have developed protein quality-control strategies to manage the accumulation of misfolded substrates during heat stress. Using a sol. reporter of misfolding in fission yeast, Rho1. C17R-GFP, we demonstrate that upon mild heat shock, the reporter collapses in protein aggregate centers (PACs). They contain and/or require several chaperones, such as Hsp104, Hsp16, and the Hsp40/70 couple Mas5/Ssa2. Stress granules do not assemble at mild temps. and, therefore, are not required for PAC formation; on the contrary, PACs may serve as nucleation centers for the assembly of stress granules. In contrast to the general belief, the dominant fate of these PACs is not degrdn., and the aggregated reporter can be disassembled by chaperones and recovers native structure and activity. Using mass spectrometry, we show that thermo-unstable endogenous proteins form PACs as well. In conclusion, formation of PACs during heat shock is a chaperone-mediated adaptation strategy.
    12. 12
      Gallardo, P.; Salas-Pino, S.; Daga, R. R. Reversible Protein Aggregation as Cytoprotective Mechanism against Heat Stress. Curr. Genet. 2021, 67, 849– 855,  DOI: 10.1007/s00294-021-01191-2
      12
      Reversible protein aggregation as cytoprotective mechanism against heat stress
      Gallardo, Paola; Salas-Pino, Silvia; Daga, Rafael R.
      Current Genetics (2021), 67 (6), 849-855CODEN: CUGED5; ISSN:0172-8083. (Springer)
      A review. Temp. fluctuation is one of the most frequent threats to which organisms are exposed in nature. The activation of gene expression programs that trigger the transcription of heat stress-protective genes is the main cellular response to resist high temps. In addn., reversible accumulation and compartmentalization of thermosensitive proteins in high-order mol. assemblies are emerging as crit. mechanisms to ensure cellular protection upon heat stress. Here, we summarize representative examples of membrane-less intracellular bodies formed upon heat stress in yeasts and human cells and highlight how protein aggregation can be turned into a cytoprotective mechanism.
    13. 13
      Franzmann, T.; Alberti, S. Ubiquitin Protein Helps Cells to Recover from Stress. Nature 2021, 597, 183– 184,  DOI: 10.1038/d41586-021-02197-z
      13
      Ubiquitin protein helps cells to recover from stress
      Franzmann, Titus; Alberti, Simon
      Nature (London, United Kingdom) (2021), 597 (7875), 183-184CODEN: NATUAS; ISSN:0028-0836. (Nature Portfolio)
      In stressed cells, proteins and RNA mols. cluster together to form stress granules. It emerges that the small protein modifier ubiquitin is needed to disassemble stress granules in recovering cells.
    14. 14
      Zhao, L.; Vecchi, G.; Vendruscolo, M.; Körner, R.; Hayer-Hartl, M.; Hartl, F. U. The Hsp70 Chaperone System Stabilizes a Thermo-Sensitive Subproteome in E. Coli. Cell Rep. 2019, 28 (5), 1335– 1345,  DOI: 10.1016/j.celrep.2019.06.081
      14
      The Hsp70 Chaperone System Stabilizes a Thermo-sensitive Subproteome in E. coli
      Zhao, Liang; Vecchi, Giulia; Vendruscolo, Michele; Koerner, Roman; Hayer-Hartl, Manajit; Hartl, F. Ulrich
      Cell Reports (2019), 28 (5), 1335-1345.e6CODEN: CREED8; ISSN:2211-1247. (Cell Press)
      Stress-inducible mol. chaperones have essential roles in maintaining protein homeostasis, but the extent to which they affect overall proteome stability remains unclear. Here, we analyze the effects of the DnaK (Hsp70) system on protein stability in Escherichia coli using pulse proteolysis combined with quant. proteomics. We quantify ∼1,500 sol. proteins and find ∼500 of these to be protease sensitive under normal growth conditions, indicating a high prevalence of conformationally dynamic proteins, forming a metastable subproteome. Acute heat stress results in the unfolding of an addnl. ∼200 proteins, reflected in the exposure of otherwise buried hydrophobic regions. Overexpression of the DnaK chaperone system markedly stabilizes numerous thermo-sensitive proteins, including multiple ribosomal proteins and large, hetero-oligomeric proteins contg. the evolutionarily ancient c.37 fold (P loop nucleoside triphosphate hydrolases). Thus, the Hsp70 system, in addn. to its known chaperone functions, has a remarkable capacity to stabilize proteins in their folded states under denaturing stress conditions.
    15. 15
      Vabulas, R. M.; Raychaudhuri, S.; Hayer-Hartl, M.; Hartl, F. U. Protein Folding in the Cytoplasm and the Heat Shock Response. Cold Spring Harbor Perspect. Biol. 2010, 2 (12), a004390,  DOI: 10.1101/cshperspect.a004390
      15
      Protein folding in the cytoplasm and the heat shock response
      Vabulas, R. Martin; Raychaudhuri, Swasti; Hayer-Hartl, Manajit; Hartl, F. Ulrich
      Cold Spring Harbor Perspectives in Biology (2010), 2 (12), a004390CODEN: CSHPEU; ISSN:1943-0264. (Cold Spring Harbor Laboratory Press)
      A review. Proteins generally must fold into precise three-dimensional conformations to fulfill their biol. functions. In the cell, this fundamental process is aided by mol. chaperones, which act in preventing protein misfolding and aggregation. How this machinery assists newly synthesized polypeptide chains in navigating the complex folding energy landscape is now being understood in considerable detail. The mechanisms that ensure the maintenance of a functional proteome under normal and stress conditions are also of great medical relevance, as the aggregation of proteins that escape the cellular quality control underlies a range of debilitating diseases, including many age-of-onset neurodegenerative disorders.
    16. 16
      Ganesan, S.; Rohde, G.; Eckermann, K.; Sroka, K.; Schaefer, M. K. E.; Dohm, C. P.; Kermer, P.; Haase, G.; Wouters, F.; Bähr, M.; Weishaupt, J. H. Mutant SOD1 Detoxification Mechanisms in Intact Single Cells. Cell Death Differ. 2008, 15 (2), 312– 321,  DOI: 10.1038/sj.cdd.4402262
      16
      Mutant SOD1 detoxification mechanisms in intact single cells
      Ganesan, S.; Rohde, G.; Eckermann, K.; Sroka, K.; Schaefer, M. K. E.; Dohm, C. P.; Kermer, P.; Haase, G.; Wouters, F.; Baehr, M.; Weishaupt, J. H.
      Cell Death and Differentiation (2008), 15 (2), 312-321CODEN: CDDIEK; ISSN:1350-9047. (Nature Publishing Group)
      Mutant superoxide dismutase 1 (mtSOD1) causes dominantly inherited amyotrophic lateral sclerosis (ALS). The mechanism for mtSOD1 toxicity remains unknown. Two main hypotheses are the impairment of proteasomal function and chaperone depletion by misfolded mtSOD1. Here, we employed FRET/FLIM and biosensor imaging to quant. localize ubiquitination, as well as chaperone binding of mtSOD1, and to assess their effect on proteasomal and protein folding activities. We found large differences in ubiquitination and chaperone interaction levels for wild-type (wt) SOD1 vs. mtSOD1 in intact single cells. Moreover, SOD1 ubiquitination levels differ between proteasomal structures and cytoplasmic material. Hsp70 binding and ubiquitination of wt and mtSOD1 species are highly correlated, demonstrating the coupled upregulation of both cellular detoxification mechanisms upon mtSOD1 expression. Biosensor imaging in single cells revealed that mtSOD1 expression alters cellular protein folding activity but not proteasomal function in the neuronal cell line examd. Our results provide the first cell-by-cell-anal. of SOD1 ubiquitination and chaperone interaction. Moreover, our study opens new methodol. avenues for cell biol. research on ALS. Cell Death and Differentiation (2008) 15, 312-321; doi:10.1038/sj.cdd.4402262; published online 9 Nov. 2007.
    17. 17
      Wood, R. J.; Ormsby, A. R.; Radwan, M.; Cox, D.; Sharma, A.; Vöpel, T.; Ebbinghaus, S.; Oliveberg, M.; Reid, G. E.; Dickson, A.; Hatters, D. M. A Biosensor-Based Framework to Measure Latent Proteostasis Capacity. Nat. Commun. 2018, 9 (1), 287,  DOI: 10.1038/s41467-017-02562-5
      17
      A biosensor-based framework to measure latent proteostasis capacity
      Wood Rebecca J; Ormsby Angelique R; Radwan Mona; Cox Dezerae; Reid Gavin E; Hatters Danny M; Sharma Abhishek; Vopel Tobias; Ebbinghaus Simon; Oliveberg Mikael; Reid Gavin E; Dickson Alex; Dickson Alex
      Nature communications (2018), 9 (1), 287 ISSN:.
      The pool of quality control proteins (QC) that maintains protein-folding homeostasis (proteostasis) is dynamic but can become depleted in human disease. A challenge has been in quantitatively defining the depth of the QC pool. With a new biosensor, flow cytometry-based methods and mathematical modeling we measure the QC capacity to act as holdases and suppress biosensor aggregation. The biosensor system comprises a series of barnase kernels with differing folding stability that engage primarily with HSP70 and HSP90 family proteins. Conditions of proteostasis stimulation and stress alter QC holdase activity and aggregation rates. The method reveals the HSP70 chaperone cycle to be rate limited by HSP70 holdase activity under normal conditions, but this is overcome by increasing levels of the BAG1 nucleotide exchange factor to HSPA1A or activation of the heat shock gene cluster by HSF1 overexpression. This scheme opens new paths for biosensors of disease and proteostasis systems.
    18. 18
      Mateju, D.; Franzmann, T. M.; Patel, A.; Kopach, A.; Boczek, E. E.; Maharana, S.; Lee, H. O.; Carra, S.; Hyman, A. A.; Alberti, S. An Aberrant Phase Transition of Stress Granules Triggered by Misfolded Protein and Prevented by Chaperone Function. EMBO J. 2017, 36 (12), 1669– 1687,  DOI: 10.15252/embj.201695957
      18
      An aberrant phase transition of stress granules triggered by misfolded protein and prevented by chaperone function
      Mateju, Daniel; Franzmann, Titus M.; Patel, Avinash; Kopach, Andrii; Boczek, Edgar E.; Maharana, Shovamayee; Lee, Hyun O.; Carra, Serena; Hyman, Anthony A.; Alberti, Simon
      EMBO Journal (2017), 36 (12), 1669-1687CODEN: EMJODG; ISSN:0261-4189. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Stress granules (SG) are membrane-less compartments involved in regulating mRNAs during stress. Aberrant forms of SGs have been implicated in age-related diseases, such as amyotrophic lateral sclerosis (ALS), but the mol. events triggering their formation are still unknown. Here, we found that misfolded proteins, such as ALS-linked variants of SOD1, specifically accumulated and aggregated within SGs in human cells. This decreased the dynamics of SGs, changed SG compn., and triggered an aberrant liq.-to-solid transition of in vitro reconstituted compartments. We showed that chaperone recruitment prevented the formation of aberrant SGs and promoted SG disassembly when the stress subsided. Moreover, we identified a backup system for SG clearance, which involved transport of aberrant SGs to the aggresome and their degrdn. by autophagy. Thus, cells employ a system of SG quality control to prevent accumulation of misfolded proteins and maintain the dynamic state of SGs, which may have relevance for ALS and related diseases.
    19. 19
      Boronat, S.; Cabrera, M.; Hidalgo, E. Spatial Sequestration of Misfolded Proteins as an Active Chaperone-Mediated Process during Heat Stress. Curr. Genet. 2021, 67 (2), 237– 243,  DOI: 10.1007/s00294-020-01135-2
      19
      Spatial sequestration of misfolded proteins as an active chaperone-mediated process during heat stress
      Boronat, Susanna; Cabrera, Margarita; Hidalgo, Elena
      Current Genetics (2021), 67 (2), 237-243CODEN: CUGED5; ISSN:0172-8083. (Springer)
      A review. Under thermal stress, different protein quality control (PQC) strategies are activated to maintain an intact proteome, which may vary from one model system to another. Hence thermo-sensitive proteins that lose their active conformation might be refolded with the aid of chaperones or removed by the ubiquitin-proteasome system or the process of autophagy. We have recently developed thermo-sensitive reporters to study PQC in fission yeast and shown the relevance of a third adaptation strategy: the sequestration of misfolded proteins into inclusions which will prevent a rapid degrdn. and allow the refolding once stress ends. These protein inclusions, protein aggregate centers (PACs), contain a broad spectrum of misfolding/aggregation-prone proteins and chaperones involved in their assembly or dissoln. The chaperone couple Mas5/Ssa2 plays a crucial role in PAC formation, whereas the Hsp104 chaperone promotes their disassembly. The absence of aggregates obsd. in cells lacking Mas5 could be also explained by the activation of the transcription factor Hsf1 and the induction of chaperone genes, we have excluded this possibility here demonstrating that increased Hsf1 activity and the subsequent overexpression of chaperones do not prevent the assembly of protein aggregates. Protein deposition at certain locations also constitutes a tactic to inactivate proteins temporally. This is the case of Pyp1, the main phosphatase of the stress response kinase Sty1. Upon stress imposition, misfolded Pyp1 is sequestered into cytosolic protein foci while active Sty1 at the nucleus switches on the transcriptional response. In conclusion, we propose that the assembly of aggregation-like foci, PACs in fission yeast, is a crucial PQC strategy during heat stress, and that the Hsp40 chaperone Mas5 is required for PAC assembly and connects physiol. and heat-shock triggered PQC.
    20. 20
      Cherkasov, V.; Hofmann, S.; Druffel-Augustin, S.; Mogk, A.; Tyedmers, J.; Stoecklin, G.; Bukau, B. Coordination of Translational Control and Protein Homeostasis during Severe Heat Stress. Curr. Biol. 2013, 23 (24), 2452– 2462,  DOI: 10.1016/j.cub.2013.09.058
      20
      Coordination of Translational Control and Protein Homeostasis during Severe Heat Stress
      Cherkasov, Valeria; Hofmann, Sarah; Druffel-Augustin, Silke; Mogk, Axel; Tyedmers, Jens; Stoecklin, Georg; Bukau, Bernd
      Current Biology (2013), 23 (24), 2452-2462CODEN: CUBLE2; ISSN:0960-9822. (Cell Press)
      Exposure of cells to severe heat stress causes not only misfolding and aggregation of proteins but also inhibition of translation and storage of mRNA in cytosolic heat stress granules (heat-SGs), limiting newly synthesized protein influx into overloaded proteome repair systems. How these two heat stress responses connect is unclear. Here, we show that both S. cerevisiae and D. melanogaster heat-SGs contain mRNA, translation machinery components (excluding ribosomes), and mol. chaperones and that heat-SGs coassemble with aggregates of misfolded, heat-labile proteins. Components in these mixed assemblies exhibit distinct mol. motilities reflecting differential trapping. We demonstrate that heat-SG disassembly and restoration of translation activity during heat stress recovery is intimately linked to disaggregation of damaged proteins present in the mixed assemblies and requires Hsp104 and Hsp70 activity. Chaperone-driven protein disaggregation directly coordinates timing of translation reinitiation with protein folding capacity during cellular protein quality surveillance, enabling efficient protein homeostasis.
    21. 21
      Hipp, M. S.; Kasturi, P.; Hartl, F. U. The Proteostasis Network and Its Decline in Ageing. Nat. Rev. Mol. Cell Biol. 2019, 20 (7), 421,  DOI: 10.1038/s41580-019-0101-y
      21
      The proteostasis network and its decline in ageing
      Hipp, Mark S.; Kasturi, Prasad; Hartl, F. Ulrich
      Nature Reviews Molecular Cell Biology (2019), 20 (7), 421-435CODEN: NRMCBP; ISSN:1471-0072. (Nature Research)
      Ageing is a major risk factor for the development of many diseases, prominently including neurodegenerative disorders such as Alzheimer disease and Parkinson disease. A hallmark of many age-related diseases is the dysfunction in protein homeostasis (proteostasis), leading to the accumulation of protein aggregates. In healthy cells, a complex proteostasis network, comprising mol. chaperones and proteolytic machineries and their regulators, operates to ensure the maintenance of proteostasis. These factors coordinate protein synthesis with polypeptide folding, the conservation of protein conformation and protein degrdn. However, sustaining proteome balance is a challenging task in the face of various external and endogenous stresses that accumulate during ageing. These stresses lead to the decline of proteostasis network capacity and proteome integrity. The resulting accumulation of misfolded and aggregated proteins affects, in particular, postmitotic cell types such as neurons, manifesting in disease. Recent analyses of proteome-wide changes that occur during ageing inform strategies to improve proteostasis. The possibilities of pharmacol. augmentation of the capacity of proteostasis networks hold great promise for delaying the onset of age-related pathologies assocd. with proteome deterioration and for extending healthspan.
    22. 22
      Patel, A.; Lee, H. O.; Jawerth, L.; Maharana, S.; Jahnel, M.; Hein, M. Y.; Stoynov, S.; Mahamid, J.; Saha, S.; Franzmann, T. M.; Pozniakovski, A.; Poser, I.; Maghelli, N.; Royer, L. A.; Weigert, M.; Myers, E. W.; Grill, S.; Drechsel, D.; Hyman, A. A.; Alberti, S. A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation. Cell 2015, 162 (5), 1066– 1077,  DOI: 10.1016/j.cell.2015.07.047
      22
      A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation
      Patel, Avinash; Lee, Hyun O.; Jawerth, Louise; Maharana, Shovamayee; Jahnel, Marcus; Hein, Marco Y.; Stoynov, Stoyno; Mahamid, Julia; Saha, Shambaditya; Franzmann, Titus M.; Pozniakovski, Andrej; Poser, Ina; Maghelli, Nicola; Royer, Loic A.; Weigert, Martin; Myers, Eugene W.; Grill, Stephan; Drechsel, David; Hyman, Anthony A.; Alberti, Simon
      Cell (Cambridge, MA, United States) (2015), 162 (5), 1066-1077CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      Many proteins contain disordered regions of low-sequence complexity, which cause aging-assocd. diseases because they are prone to aggregate. Here, we study FUS, a prion-like protein contg. intrinsically disordered domains assocd. with the neurodegenerative disease ALS. We show that, in cells, FUS forms liq. compartments at sites of DNA damage and in the cytoplasm upon stress. We confirm this by reconstituting liq. FUS compartments in vitro. Using an in vitro "aging" expt., we demonstrate that liq. droplets of FUS protein convert with time from a liq. to an aggregated state, and this conversion is accelerated by patient-derived mutations. We conclude that the physiol. role of FUS requires forming dynamic liq.-like compartments. We propose that liq.-like compartments carry the trade-off between functionality and risk of aggregation and that aberrant phase transitions within liq.-like compartments lie at the heart of ALS and, presumably, other age-related diseases.
    23. 23
      Marrone, L.; Drexler, H. C. A.; Wang, J.; Tripathi, P.; Distler, T.; Heisterkamp, P.; Anderson, E. N.; Kour, S.; Moraiti, A.; Maharana, S.; Bhatnagar, R.; Belgard, T. G.; Tripathy, V.; Kalmbach, N.; Hosseinzadeh, Z.; Crippa, V.; Abo-Rady, M.; Wegner, F.; Poletti, A.; Troost, D.; Aronica, E.; Busskamp, V.; Weis, J.; Pandey, U. B.; Hyman, A. A.; Alberti, S.; Goswami, A.; Sterneckert, J. FUS Pathology in ALS Is Linked to Alterations in Multiple ALS-Associated Proteins and Rescued by Drugs Stimulating Autophagy. Acta Neuropathol. 2019, 138 (1), 67– 84,  DOI: 10.1007/s00401-019-01998-x
      23
      FUS pathology in ALS is linked to alterations in multiple ALS-associated proteins and rescued by drugs stimulating autophagy
      Marrone, Lara; Drexler, Hannes C. A.; Wang, Jie; Tripathi, Priyanka; Distler, Tania; Heisterkamp, Patrick; Anderson, Eric Nathaniel; Kour, Sukhleen; Moraiti, Anastasia; Maharana, Shovamayee; Bhatnagar, Rajat; Belgard, T. Grant; Tripathy, Vadreenath; Kalmbach, Norman; Hosseinzadeh, Zohreh; Crippa, Valeria; Abo-Rady, Masin; Wegner, Florian; Poletti, Angelo; Troost, Dirk; Aronica, Eleonora; Busskamp, Volker; Weis, Joachim; Pandey, Udai Bhan; Hyman, Anthony A.; Alberti, Simon; Goswami, Anand; Sterneckert, Jared
      Acta Neuropathologica (2019), 138 (1), 67-84CODEN: ANPTAL; ISSN:0001-6322. (Springer)
      Amyotrophic lateral sclerosis (ALS) is a lethal disease characterized by motor neuron degeneration and assocd. with aggregation of nuclear RNA-binding proteins (RBPs), including FUS. How FUS aggregation and neurodegeneration are prevented in healthy motor neurons remain critically unanswered questions. Here, we use a combination of ALS patient autopsy tissue and induced pluripotent stem cell-derived neurons to study the effects of FUS mutations on RBP homeostasis. We show that FUS' tendency to aggregate is normally buffered by interacting RBPs, but this buffering is lost when FUS mislocalizes to the cytoplasm due to ALS mutations. The presence of aggregation-prone FUS in the cytoplasm causes imbalances in RBP homeostasis that exacerbate neurodegeneration. However, enhancing autophagy using small mols. reduces cytoplasmic FUS, restores RBP homeostasis and rescues motor function in vivo. We conclude that disruption of RBP homeostasis plays a crit. role in FUS-ALS and can be treated by stimulating autophagy.
    24. 24
      Alberti, S.; Hyman, A. A. Biomolecular Condensates at the Nexus of Cellular Stress, Protein Aggregation Disease and Ageing. Nat. Rev. Mol. Cell Biol. 2021, 22 (3), 196– 213,  DOI: 10.1038/s41580-020-00326-6
      24
      Biomolecular condensates at the nexus of cellular stress, protein aggregation disease and ageing
      Alberti, Simon; Hyman, Anthony A.
      Nature Reviews Molecular Cell Biology (2021), 22 (3), 196-213CODEN: NRMCBP; ISSN:1471-0072. (Nature Research)
      A review. Biomol. condensates are membraneless intracellular assemblies that often form via liq.-liq. phase sepn. and have the ability to conc. biopolymers. Research over the past 10 years has revealed that condensates play fundamental roles in cellular organization and physiol., and our understanding of the mol. principles, components and forces underlying their formation has substantially increased. Condensate assembly is tightly regulated in the intracellular environment, and failure to control condensate properties, formation and dissoln. can lead to protein misfolding and aggregation, which are often the cause of ageing-assocd. diseases. In this Review, we describe the mechanisms and regulation of condensate assembly and dissoln., highlight recent advances in understanding the role of biomol. condensates in ageing and disease, and discuss how cellular stress, ageing-related loss of homeostasis and a decline in protein quality control may contribute to the formation of aberrant, disease-causing condensates. Our improved understanding of condensate pathol. provides a promising path for the treatment of protein aggregation diseases.
    25. 25
      Hofmann, S.; Kedersha, N.; Anderson, P.; Ivanov, P. Molecular Mechanisms of Stress Granule Assembly and Disassembly. Biochim. Biophys. Acta, Mol. Cell Res. 2021, 1868 (1), 118876,  DOI: 10.1016/j.bbamcr.2020.118876
      25
      Molecular mechanisms of stress granule assembly and disassembly
      Hofmann, Sarah; Kedersha, Nancy; Anderson, Paul; Ivanov, Pavel
      Biochimica et Biophysica Acta, Molecular Cell Research (2021), 1868 (1), 118876CODEN: BBAMCO; ISSN:0167-4889. (Elsevier B.V.)
      A review. Stress granules (SGs) are membrane-less ribonucleoprotein (RNP)-based cellular compartments that form in the cytoplasm of a cell upon exposure to various environmental stressors. SGs contain a large set of proteins, as well as mRNAs that have been stalled in translation as a result of stress-induced polysome disassembly. Despite the fact that SGs have been extensively studied for many years, their function is still not clear. They presumably help the cell to cope with the encountered stress, and facilitate the recovery process after stress removal upon which SGs disassemble. Aberrant formation of SGs and impaired SG disassembly majorly contribute to various pathol. phenomena in cancer, viral infections, and neurodegeneration. The assembly of SGs is largely driven by liq.-liq. phase sepn. (LLPS), however, the mol. mechanisms behind that are not fully understood. Recent studies have proposed a novel mechanism for SG formation that involves the interplay of a large interaction network of mRNAs and proteins. Here, we review this novel concept of SG assembly, and discuss the current insights into SG disassembly.
    26. 26
      Burke, K. A.; Janke, A. M.; Rhine, C. L.; Fawzi, N. L. Residue-by-Residue View of In Vitro FUS Granules That Bind the C-Terminal Domain of RNA Polymerase II. Mol. Cell 2015, 60 (2), 231– 241,  DOI: 10.1016/j.molcel.2015.09.006
      26
      Residue-by-Residue View of In Vitro FUS Granules that Bind the C-Terminal Domain of RNA Polymerase II
      Burke, Kathleen A.; Janke, Abigail M.; Rhine, Christy L.; Fawzi, Nicolas L.
      Molecular Cell (2015), 60 (2), 231-241CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)
      Phase-sepd. states of proteins underlie ribonucleoprotein (RNP) granules and nuclear RNA-binding protein assemblies that may nucleate protein inclusions assocd. with neurodegenerative diseases. We report that the N-terminal low-complexity domain of the RNA-binding protein Fused in Sarcoma (FUS LC) is structurally disordered and forms a liq.-like phase-sepd. state resembling RNP granules. This state directly binds the C-terminal domain of RNA polymerase II. Phase-sepd. FUS lacks static structures as probed by fluorescence microscopy, indicating they are distinct from both protein inclusions and hydrogels. We use soln. NMR spectroscopy to directly probe the dynamic architecture within FUS liq. phase-sepd. assemblies. Importantly, we find that FUS LC retains disordered secondary structure even in the liq. phase-sepd. state. Therefore, we propose that disordered protein granules, even those made of aggregation-prone prion-like domains, are dynamic and disordered mol. assemblies with transiently formed protein-protein contacts.
    27. 27
      Molliex, A.; Temirov, J.; Lee, J.; Coughlin, M.; Kanagaraj, A. P.; Kim, H. J.; Mittag, T.; Taylor, J. P. Phase Separation by Low Complexity Domains Promotes Stress Granule Assembly and Drives Pathological Fibrillization. Cell 2015, 163 (1), 123– 133,  DOI: 10.1016/j.cell.2015.09.015
      27
      Phase Separation by Low Complexity Domains Promotes Stress Granule Assembly and Drives Pathological Fibrillization
      Molliex, Amandine; Temirov, Jamshid; Lee, Jihun; Coughlin, Maura; Kanagaraj, Anderson P.; Kim, Hong Joo; Mittag, Tanja; Taylor, J. Paul
      Cell (Cambridge, MA, United States) (2015), 163 (1), 123-133CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      Stress granules are membrane-less organelles composed of RNA-binding proteins (RBPs) and RNA. Functional impairment of stress granules has been implicated in amyotrophic lateral sclerosis, frontotemporal dementia, and multisystem proteinopathy-diseases that are characterized by fibrillar inclusions of RBPs. Genetic evidence suggests a link between persistent stress granules and the accumulation of pathol. inclusions. Here, we demonstrate that the disease-related RBP hnRNPA1 undergoes liq.-liq. phase sepn. (LLPS) into protein-rich droplets mediated by a low complexity sequence domain (LCD). While the LCD of hnRNPA1 is sufficient to mediate LLPS, the RNA recognition motifs (RRMs) contribute to LLPS in the presence of RNA, giving rise to several mechanisms for regulating assembly. Importantly, while not required for LLPS, fibrillization is enhanced in protein-rich droplets. We suggest that LCD-mediated LLPS contributes to the assembly of stress granules and their liq. properties and provides a mechanistic link between persistent stress granules and fibrillar protein pathol. in disease.
    28. 28
      Guillén-Boixet, J.; Kopach, A.; Holehouse, A. S.; Wittmann, S.; Jahnel, M.; Schlüßler, R.; Kim, K.; Trussina, I. R. E. A.; Wang, J.; Mateju, D.; Poser, I.; Maharana, S.; Ruer-Gruß, M.; Richter, D.; Zhang, X.; Chang, Y.-T.; Guck, J.; Honigmann, A.; Mahamid, J.; Hyman, A. A.; Pappu, R. V.; Alberti, S.; Franzmann, T. M. RNA-Induced Conformational Switching and Clustering of G3BP Drive Stress Granule Assembly by Condensation. Cell 2020, 181 (2), 346– 361,  DOI: 10.1016/j.cell.2020.03.049
      28
      RNA-Induced Conformational Switching and Clustering of G3BP Drive Stress Granule Assembly by Condensation
      Guillen-Boixet, Jordina; Kopach, Andrii; Holehouse, Alex S.; Wittmann, Sina; Jahnel, Marcus; Schluessler, Raimund; Kim, Kyoohyun; Trussina, Irmela R. E. A.; Wang, Jie; Mateju, Daniel; Poser, Ina; Maharana, Shovamayee; Ruer-Gruss, Martine; Richter, Doris; Zhang, Xiaojie; Chang, Young-Tae; Guck, Jochen; Honigmann, Alf; Mahamid, Julia; Hyman, Anthony A.; Pappu, Rohit V.; Alberti, Simon; Franzmann, Titus M.
      Cell (Cambridge, MA, United States) (2020), 181 (2), 346-361.e17CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      Stressed cells shut down translation, release mRNA mols. from polysomes, and form stress granules (SGs) via a network of interactions that involve G3BP. Here we focus on the mechanistic underpinnings of SG assembly. We show that, under non-stress conditions, G3BP adopts a compact auto-inhibited state stabilized by electrostatic intramol. interactions between the intrinsically disordered acidic tracts and the pos. charged arginine-rich region. Upon release from polysomes, unfolded mRNAs outcompete G3BP auto-inhibitory interactions, engendering a conformational transition that facilitates clustering of G3BP through protein-RNA interactions. Subsequent phys. crosslinking of G3BP clusters drives RNA mols. into networked RNA/protein condensates. We show that G3BP condensates impede RNA entanglement and recruit addnl. client proteins that promote SG maturation or induce a liq.-to-solid transition that may underlie disease. We propose that condensation coupled to conformational rearrangements and heterotypic multivalent interactions may be a general principle underlying RNP granule assembly.
    29. 29
      Conicella, A. E.; Zerze, G. H.; Mittal, J.; Fawzi, N. L. ALS Mutations Disrupt Phase Separation Mediated by α-Helical Structure in the TDP-43 Low-Complexity C-Terminal Domain. Structure 2016, 24 (9), 1537– 1549,  DOI: 10.1016/j.str.2016.07.007
      29
      ALS Mutations Disrupt Phase Separation Mediated by α-Helical Structure in the TDP-43 Low-Complexity C-Terminal Domain
      Conicella, Alexander E.; Zerze, Gul H.; Mittal, Jeetain; Fawzi, Nicolas L.
      Structure (Oxford, United Kingdom) (2016), 24 (9), 1537-1549CODEN: STRUE6; ISSN:0969-2126. (Elsevier Ltd.)
      RNA-binding protein TDP-43 mediates essential RNA processing but forms cytoplasmic neuronal inclusions via its C-terminal domain (CTD) in amyotrophic lateral sclerosis (ALS). It remains unclear if aggregated TDP-43 is neurotoxic and if ∼50 ALS-assocd. missense mutations in TDP-43 CTD promote aggregation, or if loss of normal function plays a role in disease. Recent work points to the ability of related proteins to assemble into functional phase-sepd. ribonucleoprotein granules via their structurally disordered prion-like domains. Here, we provide at. details on the structure and assembly of the low-complexity CTD of TDP-43 into liq.-liq. phase-sepd. in vitro granules and demonstrate that ALS-assocd. variants disrupt interactions within granules. Using NMR spectroscopy, simulation, and microscopy, we find that a subregion cooperatively but transiently folds into a helix that mediates TDP-43 phase sepn. ALS-assocd. mutations disrupt phase sepn. by inhibiting interaction and helical stabilization. Therefore, ALS-assocd. mutations can disrupt TDP-43 interactions, affecting function beyond encouraging aggregation.
    30. 30
      Zhou, H.-X.; Nguemaha, V.; Mazarakos, K.; Qin, S. Why Do Disordered and Structured Proteins Behave Differently in Phase Separation?. Trends Biochem. Sci. 2018, 43 (7), 499– 516,  DOI: 10.1016/j.tibs.2018.03.007
      30
      Why Do Disordered and Structured Proteins Behave Differently in Phase Separation?
      Zhou, Huan-Xiang; Nguemaha, Valery; Mazarakos, Konstantinos; Qin, Sanbo
      Trends in Biochemical Sciences (2018), 43 (7), 499-516CODEN: TBSCDB; ISSN:0968-0004. (Elsevier Ltd.)
      A review. Intracellular membraneless organelles and their myriad cellular functions have garnered tremendous recent interest. It is becoming well accepted that they form via liq.-liq. phase sepn. (LLPS) of protein mixts. (often including RNA), where the organelles correspond to a protein-rich droplet phase coexisting with a protein-poor bulk phase. The major protein components contain disordered regions and often also RNA-binding domains, and the disordered fragments on their own easily undergo LLPS. By contrast, LLPS for structured proteins has been obsd. infrequently. The contrasting phase behaviors can be explained by modeling disordered and structured proteins, resp., as polymers and colloids. These phys. models also provide a better understanding of the regulation of droplet formation by cellular signals and its dysregulation leading to diseases.
    31. 31
      Ruff, K. M.; Choi, Y. H.; Cox, D.; Ormsby, A. R.; Myung, Y.; Ascher, D. B.; Radford, S. E.; Pappu, R. V.; Hatters, D. M. Sequence Grammar Underlying Unfolding and Phase Separation of Globular Proteins. bioRxiv (Biophysics) , August 20, 2021, 2021.08.20.457073. DOI: 10.1101/2021.08.20.457073 (accessed 2021–09–08).
      There is no corresponding record for this reference.
    32. 32
      Ribeiro, S.; Ebbinghaus, S.; Marcos, J. C. Protein Folding and Quinary Interactions: Creating Cellular Organisation through Functional Disorder. FEBS Lett. 2018, 592 (18), 3040– 3053,  DOI: 10.1002/1873-3468.13211
      32
      Protein folding and quinary interactions: creating cellular organisation through functional disorder
      Ribeiro, Sara; Ebbinghaus, Simon; Marcos, Joao C.
      FEBS Letters (2018), 592 (18), 3040-3053CODEN: FEBLAL; ISSN:0014-5793. (Wiley-Blackwell)
      The marginal stability of globular proteins in the cell is detd. by the balance between excluded vol. effect and soft interactions. Quinary interactions are a type of soft interactions involved in intracellular organization and known to have stabilizing or destabilizing effects on globular proteins. Recent studies suggest that globular proteins have structural flexibility, exhibiting more than one functional state. Here, we propose that the quinary-induced destabilization can be sufficient to produce functional partially unfolded states of globular proteins. The biol. relevance of this mechanism is explored, involving intracellular phase sepn. and regulatory stress response mechanisms.
    33. 33
      Ribeiro, S. S.; Samanta, N.; Ebbinghaus, S.; Marcos, J. C. The Synergic Effect of Water and Biomolecules in Intracellular Phase Separation. Nat. Rev. Chem. 2019, 3 (9), 552– 561,  DOI: 10.1038/s41570-019-0120-4
      33
      The synergic effect of water and biomolecules in intracellular phase separation
      Ribeiro, Sara S.; Samanta, Nirnay; Ebbinghaus, Simon; Marcos, Joao C.
      Nature Reviews Chemistry (2019), 3 (9), 552-561CODEN: NRCAF7; ISSN:2397-3358. (Nature Research)
      Phase sepn. has long been obsd. within aq. mixts. of two or more different compds., such as proteins, salts, polysaccharides and synthetic polymers. A growing body of exptl. evidence indicates that phase sepn. also takes place inside living cells, where intrinsically disordered proteins and other mols. such as RNA are thought to assemble into membraneless organelles. These structures represent a new paradigm of intracellular organization and compartmentalization, in which biochem. processes can be coordinated in space and time. Two thermodn. driving forces have been proposed for phase sepn.: the strengths of macromol.-macromol. and macromol.-H2O interactions, and the perturbation of H2O structure about different macromols. In this Perspective, we propose that both driving forces act in a concerted manner to promote phase sepn., which we describe in the context of the well-known structural dynamics of intrinsically disordered proteins in the cellular milieu. We further suggest that this effect can be extended to explain how the partial unfolding of globular proteins can lead to intracellular phase sepn.
    34. 34
      Mathieu, C.; Pappu, R. V.; Taylor, J. P. Beyond Aggregation: Pathological Phase Transitions in Neurodegenerative Disease. Science 2020, 370 (6512), 56– 60,  DOI: 10.1126/science.abb8032
      34
      Beyond aggregation: Pathological phase transitions in neurodegenerative disease
      Mathieu, Cecile; Pappu, Rohit V.; Taylor, J. Paul
      Science (Washington, DC, United States) (2020), 370 (6512), 56-60CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
      Over the past decade, phase transitions have emerged as a fundamental mechanism of cellular organization. In parallel, a wealth of evidence has accrued indicating that aberrations in phase transitions are early events in the pathogenesis of several neurodegenerative diseases. We review the key evidence of defects at multiple levels, from phase transition of individual proteins to the dynamic behavior of complex, multicomponent condensates in neurodegeneration. We also highlight two concepts, dynamical arrest and heterotypic buffering, that are key to understanding how pathol. phase transitions relate to pleiotropic defects in cellular functions and the accrual of proteinaceous deposits at end-stage disease. These insights not only illuminate disease etiol. but also are likely to guide the development of therapeutic interventions to restore homeostasis.
    35. 35
      Jahn, T. R.; Radford, S. E. Folding versus Aggregation: Polypeptide Conformations on Competing Pathways. Arch. Biochem. Biophys. 2008, 469 (1), 100– 117,  DOI: 10.1016/j.abb.2007.05.015
      35
      Folding versus aggregation: Polypeptide conformations on competing pathways
      Jahn, Thomas R.; Radford, Sheena E.
      Archives of Biochemistry and Biophysics (2008), 469 (1), 100-117CODEN: ABBIA4; ISSN:0003-9861. (Elsevier)
      A review. Protein aggregation has now become recognized as an important and generic aspect of protein energy landscapes. Since the discovery that numerous human diseases are caused by protein aggregation, the biophys. characterization of misfolded states and their aggregation mechanisms has received increased attention. Utilizing exptl. techniques and computational approaches established for the anal. of protein folding reactions has ensured rapid advances in the study of pathways leading to amyloid fibrils and amyloid-related aggregates. Here, the authors describe recent exptl. and theor. advances in the elucidation of the conformational properties of dynamic, heterogeneous and/or insol. protein ensembles populated on complex, multidimensional protein energy landscapes. The authors discuss the current understanding of aggregation mechanisms in this context and describe how the synergy between biochem., biophys., and cell-biol. expts. are beginning to provide detailed insights into the partitioning of non-native species between protein folding and aggregation pathways.
    36. 36
      Grad, L. I.; Yerbury, J. J.; Turner, B. J.; Guest, W. C.; Pokrishevsky, E.; O’Neill, M. A.; Yanai, A.; Silverman, J. M.; Zeineddine, R.; Corcoran, L.; Kumita, J. R.; Luheshi, L. M.; Yousefi, M.; Coleman, B. M.; Hill, A. F.; Plotkin, S. S.; Mackenzie, I. R.; Cashman, N. R. Intercellular Propagated Misfolding of Wild-Type Cu/Zn Superoxide Dismutase Occurs via Exosome-Dependent and -Independent Mechanisms. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (9), 3620– 3625,  DOI: 10.1073/pnas.1312245111
      36
      Intercellular propagated misfolding of wild-type Cu/Zn superoxide dismutase occurs via exosome-dependent and -independent mechanisms
      Grad, Leslie I.; Yerbury, Justin J.; Turner, Bradley J.; Guest, William C.; Pokrishevsky, Edward; O'Neill, Megan A.; Yanai, Anat; Silverman, Judith M.; Zeineddine, Rafaa; Corcoran, Lisa; Kumita, Janet R.; Luheshi, Leila M.; Yousefi, Masoud; Coleman, Bradley M.; Hill, Andrew F.; Plotkin, Steven S.; MacKenzie, Ian R.; Cashman, Neil R.
      Proceedings of the National Academy of Sciences of the United States of America (2014), 111 (9), 3620-3625CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Amyotrophic lateral sclerosis (ALS) is predominantly sporadic, but assocd. with heritable genetic mutations in 5-10% of cases, including those in Cu/Zn superoxide dismutase (SOD1). We previously showed that misfolding of SOD1 can be transmitted to endogenous human wild-type SOD1 (HuWtSOD1) in an intracellular compartment. Using NSC-34 motor neuron-like cells, we now demonstrate that misfolded mutant and HuWtSOD1 can traverse between cells via two nonexclusive mechanisms: protein aggregates released from dying cells and taken up by macropinocytosis, and exosomes secreted from living cells. Furthermore, once HuWtSOD1 propagation has been established, misfolding of HuWtSOD1 can be efficiently and repeatedly propagated between HEK293 cell cultures via conditioned media over multiple passages, and to cultured mouse primary spinal cord cells transgenically expressing HuWtSOD1, but not to cells derived from nontransgenic littermates. Conditioned media transmission of HuWtSOD1 misfolding in HEK293 cells is blocked by HuWtSOD1 siRNA knockdown, consistent with human SOD1 being a substrate for conversion, and attenuated by ultracentrifugation or incubation with SOD1 misfolding-specific antibodies, indicating a relatively massive transmission particle which possesses antibody-accessible SOD1. Finally, misfolded and protease-sensitive HuWtSOD1 comprises up to 4% of total SOD1 in spinal cords of patients with sporadic ALS (SALS). Propagation of HuWtSOD1 misfolding, and its subsequent cell-to-cell transmission, is thus a candidate process for the mol. pathogenesis of SALS, which may provide novel treatment and biomarker targets for this devastating disease.
    37. 37
      Sen Mojumdar, S.; Scholl, Z. N.; Dee, D. R.; Rouleau, L.; Anand, U.; Garen, C.; Woodside, M. T. Partially Native Intermediates Mediate Misfolding of SOD1 in Single-Molecule Folding Trajectories. Nat. Commun. 2017, 8 (1), 1881,  DOI: 10.1038/s41467-017-01996-1
      37
      Partially native intermediates mediate misfolding of SOD1 in single-molecule folding trajectories
      Sen Mojumdar Supratik; N Scholl Zackary; Dee Derek R; Rouleau Logan; Anand Uttam; Garen Craig; Woodside Michael T; Woodside Michael T
      Nature communications (2017), 8 (1), 1881 ISSN:.
      Prion-like misfolding of superoxide dismutase 1 (SOD1) is associated with the disease ALS, but the mechanism of misfolding remains unclear, partly because misfolding is difficult to observe directly. Here we study the most misfolding-prone form of SOD1, reduced un-metallated monomers, using optical tweezers to measure unfolding and refolding of single molecules. We find that the folding is more complex than suspected, resolving numerous previously undetected intermediate states consistent with the formation of individual β-strands in the native structure. We identify a stable core of the protein that unfolds last and refolds first, and directly observe several distinct misfolded states that branch off from the native folding pathways at specific points after the formation of the stable core. Partially folded intermediates thus play a crucial role mediating between native and non-native folding. These results suggest an explanation for SOD1's propensity for prion-like misfolding and point to possible targets for therapeutic intervention.
    38. 38
      Lang, L.; Zetterström, P.; Brännström, T.; Marklund, S. L.; Danielsson, J.; Oliveberg, M. SOD1 Aggregation in ALS Mice Shows Simplistic Test Tube Behavior. Proc. Natl. Acad. Sci. U. S. A. 2015, 112 (32), 9878– 9883,  DOI: 10.1073/pnas.1503328112
      38
      SOD1 aggregation in ALS mice shows simplistic test tube behavior
      Lang, Lisa; Zetterstroem, Per; Braennstroem, Thomas; Marklund, Stefan L.; Danielsson, Jens; Oliveberg, Mikael
      Proceedings of the National Academy of Sciences of the United States of America (2015), 112 (32), 9878-9883CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      A longstanding challenge in studies of neurodegenerative disease has been that the pathol. protein aggregates in live tissue are not amenable to structural and kinetic anal. by conventional methods. The situation is put in focus by current progress in demarcating protein aggregation in vitro, exposing new mechanistic details that are now calling for quant. in vivo comparison. Here, the authors bridge this gap by presenting a direct comparison of the aggregation kinetics of amyotrophic lateral sclerosis (ALS)-assocd. superoxide dismutase 1 (SOD1) in vitro and in transgenic mice. The results based on tissue sampling by quant. antibody assays showed that the SOD1 fibrillation kinetics in vitro mirrored with remarkable accuracy the spinal cord aggregate buildup and disease progression in transgenic mice. This similarity between in vitro and in vivo data suggested that, despite the complexity of live tissue, SOD1 aggregation follows robust and simplistic rules, providing new mechanistic insights into the ALS pathol. and organism-level manifestation of protein aggregation phenomena in general.
    39. 39
      Gnutt, D.; Timr, S.; Ahlers, J.; König, B.; Manderfeld, E.; Heyden, M.; Sterpone, F.; Ebbinghaus, S. Stability Effect of Quinary Interactions Reversed by Single Point Mutations. J. Am. Chem. Soc. 2019, 141 (11), 4660– 4669,  DOI: 10.1021/jacs.8b13025
      39
      Stability Effect of Quinary Interactions Reversed by Single Point Mutations
      Gnutt, David; Timr, Stepan; Ahlers, Jonas; Koenig, Benedikt; Manderfeld, Emily; Heyden, Matthias; Sterpone, Fabio; Ebbinghaus, Simon
      Journal of the American Chemical Society (2019), 141 (11), 4660-4669CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      In cells, proteins are embedded in a crowded environment that controls their properties via manifold avenues including weak protein-macromol. interactions. A mol. level understanding of these quinary interactions and their contribution to protein stability, function, and localization in the cell is central to modern structural biol. Using a mutational anal. to quantify the energetic contributions of single amino acids to the stability of the ALS related protein superoxide dismutase I (SOD1) in mammalian cells, we show that quinary interactions destabilize SOD1 by a similar energetic offset for most of the mutants, but there are notable exceptions: Mutants that alter its surface properties can even lead to a stabilization of the protein in the cell as compared to the test tube. In conclusion, quinary interactions can amplify and even reverse the mutational response of proteins, being a key aspect in pathogenic protein misfolding and aggregation.
    40. 40
      Wright, G. S. A.; Antonyuk, S. V.; Hasnain, S. S. The Biophysics of Superoxide Dismutase-1 and Amyotrophic Lateral Sclerosis. Q. Rev. Biophys. 2019, 52, e12,  DOI: 10.1017/S003358351900012X
      40
      The biophysics of superoxide dismutase-1 and amyotrophic lateral sclerosis
      Wright, Gareth S. A.; Antonyuk, Svetlana V.; Hasnain, S. Samar
      Quarterly Reviews of Biophysics (2019), 52 (), e12CODEN: QURBAW; ISSN:0033-5835. (Cambridge University Press)
      The possibility of reduced structural integrity was suggested by the first crystal structures of human SOD1 even before deleterious mutations in the sod1 gene were linked to the ALS. This concept evolved in the intervening years as an impressive array of biophys. studies examd. the characteristics of mutant SOD1 in great detail. We now recognize how ALS-related mutations perturb the SOD1 maturation processes, reduce its ability to fold and reduce its thermal stability and half-life. Mutant SOD1 is therefore predisposed to monomerisation, non-canonical self-interactions, the formation of small misfolded oligomers and ultimately accumulation in the tell-tale insol. inclusions found within the neurons of ALS patients. We have also seen that several post-translational modifications could push wildtype SOD1 down this toxic pathway. Recently we have come to view ALS as a prion-like disease where both the symptoms, and indeed SOD1 misfolding itself, are transmitted to neighboring cells. This raises the possibility of intervention after the initial disease presentation. Several small-mol. and biol.-based strategies have been devised which directly target the SOD1 mol. to change the behavior thought to be responsible for ALS. Here we provide a comprehensive review of the many biophys. advances that sculpted our view of SOD1 biol. and the recent work that aims to apply this knowledge for therapeutic outcomes in ALS.
    41. 41
      Gal, J.; Kuang, L.; Barnett, K. R.; Zhu, B. Z.; Shissler, S. C.; Korotkov, K. V.; Hayward, L. J.; Kasarskis, E. J.; Zhu, H. ALS Mutant SOD1 Interacts with G3BP1 and Affects Stress Granule Dynamics. Acta Neuropathol. 2016, 132 (4), 563– 576,  DOI: 10.1007/s00401-016-1601-x
      41
      ALS mutant SOD1 interacts with G3BP1 and affects stress granule dynamics
      Gal, Jozsef; Kuang, Lisha; Barnett, Kelly R.; Zhu, Brian Z.; Shissler, Susannah C.; Korotkov, Konstantin V.; Hayward, Lawrence J.; Kasarskis, Edward J.; Zhu, Haining
      Acta Neuropathologica (2016), 132 (4), 563-576CODEN: ANPTAL; ISSN:0001-6322. (Springer)
      Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. Mutations in Cu/Zn superoxide dismutase (SOD1) are responsible for approx. 20 % of the familial ALS cases. ALS-causing SOD1 mutants display a gain-of-toxicity phenotype, but the nature of this toxicity is still not fully understood. The Ras GTPase-activating protein-binding protein G3BP1 plays a crit. role in stress granule dynamics. Alterations in the dynamics of stress granules have been reported in several other forms of ALS unrelated to SOD1. To our surprise, the mutant G93A SOD1 transgenic mice exhibited pathol. cytoplasmic inclusions that co-localized with G3BP1-pos. granules in spinal cord motor neurons. The co-localization was also obsd. in fibroblast cells derived from familial ALS patient carrying SOD1 mutation L144F. Mutant SOD1, unlike wild-type SOD1, interacted with G3BP1 in an RNA-independent manner. Moreover, the interaction is specific for G3BP1 since mutant SOD1 showed little interaction with four other RNA-binding proteins implicated in ALS. The RNA-binding RRM domain of G3BP1 and two particular phenylalanine residues (F380 and F382) are crit. for this interaction. Mutant SOD1 delayed the formation of G3BP1- and TIA1-pos. stress granules in response to hyperosmolar shock and arsenite treatment in N2A cells. In summary, the aberrant mutant SOD1-G3BP1 interaction affects stress granule dynamics, suggesting a potential link between pathogenic SOD1 mutations and RNA metab. alterations in ALS.
    42. 42
      Da Ros, M.; Deol, H. K.; Savard, A.; Guo, H.; Meiering, E. M.; Gibbings, D. Wild-Type and Mutant SOD1 Localizes to RNA-Rich Structures in Cells and Mice but Does Not Bind RNA. J. Neurochem. 2021, 156 (4), 524– 538,  DOI: 10.1111/jnc.15126
      42
      Wild-type and mutant SOD1 localizes to RNA-rich structures in cells and mice but does not bind RNA
      Da Ros, Matteo; Deol, Harmeen K.; Savard, Alexandre; Guo, Huishan; Meiering, Elizabeth M.; Gibbings, Derrick
      Journal of Neurochemistry (2021), 156 (4), 524-538CODEN: JONRA9; ISSN:0022-3042. (Wiley-Blackwell)
      Many of the genes whose mutation causes Amyotrophic Lateral Sclerosis (ALS) are RNA-binding proteins which localize to stress granules, while others impact the assembly, stability, and elimination of stress granules. This has led to the hypothesis that alterations in the dynamics of stress granules and RNA biol. cause ALS. Genetic mutations in Superoxide Dismutase 1 (SOD1) also cause ALS. Evidence demonstrates that SOD1 harboring ALS-linked mutations is recruited to stress granules, induces changes in alternative splicing, and could be an RNA-binding protein. Whether SOD1 inclusions contain RNA in disease models and whether SOD1 directly binds RNA remains uncertain. We applied methods including crosslinking immunopptn. and in vitro gel shift assays to detect binding of SOD1 to RNA in vitro, in cells with and without stress granules, and in mice expressing human SOD1 G93A. We find that SOD1 localizes to RNA-rich structures including stress granules, and SOD1 inclusions in mice contain mRNA. However, we find no evidence that SOD1 directly binds RNA. This suggests that SOD1 may impact stress granules, alternative splicing and RNA biol. without binding directly to RNA.
    43. 43
      Danielsson, J.; Kurnik, M.; Lang, L.; Oliveberg, M. Cutting Off Functional Loops from Homodimeric Enzyme Superoxide Dismutase 1 (SOD1) Leaves Monomeric β-Barrels. J. Biol. Chem. 2011, 286 (38), 33070– 33083,  DOI: 10.1074/jbc.M111.251223
      43
      Cutting Off Functional Loops from Homodimeric Enzyme Superoxide Dismutase 1 (SOD1) Leaves Monomeric β-Barrels
      Danielsson, Jens; Kurnik, Martin; Lang, Lisa; Oliveberg, Mikael
      Journal of Biological Chemistry (2011), 286 (38), 33070-33083, S33070/1-S33070/18CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Demetallation of the homodimeric enzyme Cu/Zn-superoxide dismutase (SOD1) is known to unleash pronounced dynamic motions in the long active-site loops that comprise almost a third of the folded structure. The resulting apo species, which shows increased propensity to aggregate, stands out as the prime disease precursor in amyotrophic lateral sclerosis (ALS). Even so, the detailed structural properties of the apoSOD1 framework have remained elusive and controversial. In this study, we examine the structural interplay between the central apoSOD1 barrel and the active-site loops by simply cutting them off; loops IV and VII were substituted with short Gly-Ala-Gly linkers. The results show that loop removal breaks the dimer interface and leads to sol., monomeric β-barrels with high structural integrity. NMR-detected nuclear Overhauser effects are found between all of the constituent β-strands, confirming ordered interactions across the whole barrel. Moreover, the breathing motions of the SOD1 barrel are overall insensitive to loop removal and yield hydrogen/deuterium protection factors typical for cooperatively folded proteins (i.e. the active-site loops act as a "bolt-on" domain with little dynamic influence on its structural foundation). The sole exceptions are the relatively low protection factors in β-strand 5 and the turn around Gly-93, a hot spot for ALS-provoking mutations, which decrease even further upon loop removal. Taken together, these data suggest that the cytotoxic function of apoSOD1 does not emerge from its folded ground state but from a high energy intermediate or even from the denatured ensemble.
    44. 44
      Nordlund, A.; Leinartaitė, L.; Saraboji, K.; Aisenbrey, C.; Gröbner, G.; Zetterström, P.; Danielsson, J.; Logan, D. T.; Oliveberg, M. Functional Features Cause Misfolding of the ALS-Provoking Enzyme SOD1. Proc. Natl. Acad. Sci. U. S. A. 2009, 106 (24), 9667– 9672,  DOI: 10.1073/pnas.0812046106
      44
      Functional features cause misfolding of the ALS-provoking enzyme SOD1
      Nordlund, Anna; Leinartaite, Lina; Saraboji, Kahhirvel; Aisenbrey, Christopher; Grobner, Gerhard; Zetterstrom, Per; Danielsson, Jens; Logan, Derek T.; Oliveberg, Mikael
      Proceedings of the National Academy of Sciences of the United States of America (2009), 106 (24), 9667-9672, S9667/1-S9667/12CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The structural integrity of the ubiquitous enzyme, Cu,Zn-superoxide dismutase (SOD1), relies critically on the correct coordination of Cu and Zn. The loss of these cofactors not only promotes SOD1 aggregation in vitro but also seems to be a key prerequisite for pathogenic misfolding in the neurodegenerative disease, amyotrophic lateral sclerosis (ALS). Here, the authors examd. the consequences of Zn2+ loss by selectively removing the Zn site, which had previously been implicated as the main modulator of SOD1 stability and disease competence. After Zn-site removal, the remaining Cu ligands could coordinate a non-native Zn2+ ion with micromolar affinity in the denatured state, and then retain this ion throughout the folding reaction. Without the restriction of a metalated Zn site, however, the Cu ligands failed to correctly coordinate the non-native Zn2+ ion; trapping of a water mol. caused the His-48 residue to change rotamer and swing outward. The misligation was sterically incompatible with the native structure. As a consequence, SOD1 unfolded locally and interacted with neighboring mols. in the crystal lattice. Thus, the findings point to a crit. role for the native Zn site in controlling SOD1 misfolding, and show that even subtle changes of the metal-loading sequence can render the wild-type protein the same structural properties as ALS-provoking mutations. This frustrated character of the SOD1 mol. seems to arise from a compromise between optimization of functional and structural features.
    45. 45
      Niwa, J.; Yamada, S.; Ishigaki, S.; Sone, J.; Takahashi, M.; Katsuno, M.; Tanaka, F.; Doyu, M.; Sobue, G. Disulfide Bond Mediates Aggregation, Toxicity, and Ubiquitylation of Familial Amyotrophic Lateral Sclerosis-Linked Mutant SOD1 *. J. Biol. Chem. 2007, 282 (38), 28087– 28095,  DOI: 10.1074/jbc.M704465200
      45
      Disulfide Bond Mediates Aggregation, Toxicity, and Ubiquitylation of Familial Amyotrophic Lateral Sclerosis-linked Mutant SOD1
      Niwa, Jun-ichi; Yamada, Shin-ichi; Ishigaki, Shinsuke; Sone, Jun; Takahashi, Miho; Katsuno, Masahisa; Tanaka, Fumiaki; Doyu, Manabu; Sobue, Gen
      Journal of Biological Chemistry (2007), 282 (38), 28087-28095CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Mutations in the Cu/Zn-superoxide dismutase (SOD1) gene cause familial amyotrophic lateral sclerosis (ALS) through the gain of a toxic function; however, the nature of this toxic function remains largely unknown. Ubiquitylated aggregates of mutant SOD1 proteins in affected brain lesions are pathol. hallmarks of the disease and are suggested to be involved in several proposed mechanisms of motor neuron death. Recent studies suggest that mutant SOD1 readily forms an incorrect disulfide bond upon mild oxidative stress in vitro, and the insol. SOD1 aggregates in spinal cord of ALS model mice contain multimers cross-linked via intermol. disulfide bonds. Here we show that a non-physiol. intermol. disulfide bond between cysteines at positions 6 and 111 of mutant SOD1 is important for high mol. wt. aggregate formation, ubiquitylation, and neurotoxicity, all of which were dramatically reduced when the pertinent cysteines were replaced in mutant SOD1 expressed in Neuro-2a cells. Dorfin is a ubiquityl ligase that specifically binds familial ALS-linked mutant SOD1 and ubiquitylates it, thereby promoting its degrdn. We found that Dorfin ubiquitylated mutant SOD1 by recognizing the Cys6- and Cys111-disulfide cross-linked form and targeted it for proteasomal degrdn.
    46. 46
      Ebbinghaus, S.; Dhar, A.; McDonald, J. D.; Gruebele, M. Protein Folding Stability and Dynamics Imaged in a Living Cell. Nat. Methods 2010, 7 (4), 319– 323,  DOI: 10.1038/nmeth.1435
      46
      Protein folding stability and dynamics imaged in a living cell
      Ebbinghaus, Simon; Dhar, Apratim; McDonald, J. Douglas; Gruebele, Martin
      Nature Methods (2010), 7 (4), 319-323CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      Biomol. dynamics and stability are predominantly investigated in vitro and extrapolated to explain function in the living cell. Here, the authors present fast relaxation imaging (FreI), which combines fluorescence microscopy and temp. jumps to probe biomol. dynamics and stability inside a single living cell with high spatiotemporal resoln. Here, the authors demonstrated the method by measuring the reversible fast folding kinetics as well as folding thermodn. of a FRET probe-labeled phosphoglycerate kinase construct in 2 human cell lines. Comparison with in vitro expts. at 23-49° showed that the cell environment influenced the protein stability and folding rate. FReI should also be applicable to the study of protein-protein interactions and heat-shock responses as well as to comparative studies of cell populations or whole organisms.
    47. 47
      van Lente, J. J.; Claessens, M. M. A. E.; Lindhoud, S. Charge-Based Separation of Proteins Using Polyelectrolyte Complexes as Models for Membraneless Organelles. Biomacromolecules 2019, 20 (10), 3696– 3703,  DOI: 10.1021/acs.biomac.9b00701
      47
      Charge-Based Separation of Proteins Using Polyelectrolyte Complexes as Models for Membraneless Organelles
      van Lente, Jere J.; Claessens, Mireille M. A. E.; Lindhoud, Saskia
      Biomacromolecules (2019), 20 (10), 3696-3703CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
      Membraneless organelles are liq. compartments within cells with different solvent properties than the surrounding environment. This difference in solvent properties is thought to result in function-related selective partitioning of proteins. Proteins have also been shown to accumulate in polyelectrolyte complexes, but whether the uptake in these complexes is selective has not been ascertained yet. Here, we show the selective partitioning of two structurally similar but oppositely charged proteins into polyelectrolyte complexes. We demonstrate that these proteins can be sepd. from a mixt. by altering the polyelectrolyte complex compn. and released from the complex by lowering the pH. Combined, we demonstrate that polyelectrolyte complexes can sep. proteins from a mixt. based on protein charge. Besides providing deeper insight into the selective partitioning in membraneless organelles, potential applications for selective biomol. partitioning in polyelectrolyte complexes include drug delivery or extn. processes.
    48. 48
      Raeburn, C. B.; Ormsby, A.; Moily, N. S.; Cox, D.; Ebbinghaus, S.; Dickson, A.; McColl, G.; Hatters, D. M. A Biosensor to Gauge Protein Homeostasis Resilience Differences in the Nucleus Compared to Cytosol of Mammalian Cells. bioRxiv (Biochemistry) , April 19, 2021, 2021.04.19.440383. DOI: 10.1101/2021.04.19.440383 (accessed 2021–09–08).
      There is no corresponding record for this reference.
    49. 49
      Vassall, K. A.; Stubbs, H. R.; Primmer, H. A.; Tong, M. S.; Sullivan, S. M.; Sobering, R.; Srinivasan, S.; Briere, L.-A. K.; Dunn, S. D.; Colón, W.; Meiering, E. M. Decreased Stability and Increased Formation of Soluble Aggregates by Immature Superoxide Dismutase Do Not Account for Disease Severity in ALS. Proc. Natl. Acad. Sci. U. S. A. 2011, 108 (6), 2210– 2215,  DOI: 10.1073/pnas.0913021108
      49
      Decreased stability and increased formation of soluble aggregates by immature superoxide dismutase do not account for disease severity in ALS
      Vassall, Kenrick A.; Stubbs, Helen R.; Primmer, Heather A.; Tong, Ming Sze; Sullivan, Sarah M.; Sobering, Ryan; Srinivasan, Saipraveen; Briere, Lee-Ann K.; Dunn, Stanley D.; Colon, Wilfredo; Meiering, Elizabeth M.
      Proceedings of the National Academy of Sciences of the United States of America (2011), 108 (6), 2210-2215, S2210/1-S2210/11CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Protein aggregation is a hallmark of many diseases, including amyotrophic lateral sclerosis (ALS), where aggregation of Cu/Zn superoxide dismutase (SOD1) is implicated in causing neurodegeneration. Recent studies have suggested that destabilization and aggregation of the most immature form of SOD1, the disulfidereduced, unmetallated (apo) protein is particularly important in causing ALS. We report herein in depth analyses of the effects of chem. and structurally diverse ALS-assocd. mutations on the stability and aggregation of reduced apo SOD1. In contrast with previous studies, we find that various reduced apo SOD1 mutants undergo highly reversible thermal denaturation with little aggregation, enabling quant. thermodn. stability analyses. In the absence of ALS-assocd. mutations, reduced apo SOD1 is marginally stable but predominantly folded. Mutations generally result in slight decreases to substantial increases in the fraction of unfolded protein. Calorimetry, ultracentrifugation, and light scattering show that all mutations enhance aggregation propensity, with the effects varying widely, from subtle increases in most cases, to pronounced formation of 40-100 nm sol. aggregates by A4V, a mutation that is assocd. with particularly short disease duration. Interestingly, although there is a correlation between obsd. aggregation and stability, there is minimal to no correlation between obsd. aggregation, predicted aggregation propensity, and disease characteristics. These findings suggest that reduced apo SOD1 does not play a dominant role in modulating disease. Rather, addnl. and/or multiple forms of SOD1 and addnl. biophys. and biol. factors are needed to account for the toxicity of mutant SOD1 in ALS.
    50. 50
      Wang, Q.; Johnson, J. L.; Agar, N. Y. R.; Agar, J. N. Protein Aggregation and Protein Instability Govern Familial Amyotrophic Lateral Sclerosis Patient Survival. PLoS Biol. 2008, 6 (7), e170,  DOI: 10.1371/journal.pbio.0060170
      There is no corresponding record for this reference.
    51. 51
      Rauscher, S.; Pomès, R. The Liquid Structure of Elastin. eLife 2017, 6, e26526  DOI: 10.7554/eLife.26526
      51
      The liquid structure of elastin
      Rauscher, Sarah; Pomes, Regis
      eLife (2017), 6 (), e26526/1-e26526/21CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)
      The protein elastin imparts extensibility, elastic recoil, and resilience to tissues including arterial walls, skin, lung alveoli, and the uterus. Elastin and elastin-like peptides are hydrophobic, disordered, and undergo liq.-liq. phase sepn. upon self-assembly. Despite extensive study, the structure of elastin remains controversial. We use mol. dynamics simulations on a massive scale to elucidate the structural ensemble of aggregated elastin-like peptides. Consistent with the entropic nature of elastic recoil, the aggregated state is stabilized by the hydrophobic effect. However, self-assembly does not entail formation of a hydrophobic core. The polypeptide backbone forms transient, sparse hydrogen-bonded turns and remains significantly hydrated even as self-assembly triples the extent of non-polar side chain contacts. Individual chains in the assembly approach a maximally-disordered, melt-like state which may be called the liq. state of proteins. These findings resolve long-standing controversies regarding elastin structure and function and afford insight into the phase sepn. of disordered proteins.
    52. 52
      Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF Chimera--a Visualization System for Exploratory Research and Analysis. J. Comput. Chem. 2004, 25 (13), 1605– 1612,  DOI: 10.1002/jcc.20084
      52
      UCSF Chimera-A visualization system for exploratory research and analysis
      Pettersen, Eric F.; Goddard, Thomas D.; Huang, Conrad C.; Couch, Gregory S.; Greenblatt, Daniel M.; Meng, Elaine C.; Ferrin, Thomas E.
      Journal of Computational Chemistry (2004), 25 (13), 1605-1612CODEN: JCCHDD; ISSN:0192-8651. (John Wiley & Sons, Inc.)
      The design, implementation, and capabilities of an extensible visualization system, UCSF Chimera, are discussed. Chimera is segmented into a core that provides basic services and visualization, and extensions that provide most higher level functionality. This architecture ensures that the extension mechanism satisfies the demands of outside developers who wish to incorporate new features. Two unusual extensions are presented: Multiscale, which adds the ability to visualize large-scale mol. assemblies such as viral coats, and Collab., which allows researchers to share a Chimera session interactively despite being at sep. locales. Other extensions include Multalign Viewer, for showing multiple sequence alignments and assocd. structures; ViewDock, for screening docked ligand orientations; Movie, for replaying mol. dynamics trajectories; and Vol. Viewer, for display and anal. of volumetric data. A discussion of the usage of Chimera in real-world situations is given, along with anticipated future directions. Chimera includes full user documentation, is free to academic and nonprofit users, and is available for Microsoft Windows, Linux, Apple Mac OS X, SGI IRIX, and HP Tru64 Unix from http://www.cgl.ucsf.edu/chimera/.
    53. 53
      Hofweber, M.; Hutten, S.; Bourgeois, B.; Spreitzer, E.; Niedner-Boblenz, A.; Schifferer, M.; Ruepp, M.-D.; Simons, M.; Niessing, D.; Madl, T.; Dormann, D. Phase Separation of FUS Is Suppressed by Its Nuclear Import Receptor and Arginine Methylation. Cell 2018, 173 (3), 706– 719,  DOI: 10.1016/j.cell.2018.03.004
      53
      Phase Separation of FUS Is Suppressed by Its Nuclear Import Receptor and Arginine Methylation
      Hofweber, Mario; Hutten, Saskia; Bourgeois, Benjamin; Spreitzer, Emil; Niedner-Boblenz, Annika; Schifferer, Martina; Ruepp, Marc-David; Simons, Mikael; Niessing, Dierk; Madl, Tobias; Dormann, Dorothee
      Cell (Cambridge, MA, United States) (2018), 173 (3), 706-719.e13CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      Cytoplasmic FUS aggregates are a pathol. hallmark in a subset of patients with frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS). A key step that is disrupted in these patients is nuclear import of FUS mediated by the import receptor Transportin/Karyopherin-β2. In ALS-FUS patients, this is caused by mutations in the nuclear localization signal (NLS) of FUS that weaken Transportin binding. In FTD-FUS patients, Transportin is aggregated, and post-translational arginine methylation, which regulates the FUS-Transportin interaction, is lost. Here, we show that Transportin and arginine methylation have a crucial function beyond nuclear import-namely to suppress RGG/RG-driven phase sepn. and stress granule assocn. of FUS. ALS-assocd. FUS-NLS mutations weaken the chaperone activity of Transportin and loss of FUS arginine methylation, as seen in FTD-FUS, promote phase sepn., and stress granule partitioning of FUS. Our findings reveal two regulatory mechanisms of liq.-phase homeostasis that are disrupted in FUS-assocd. neurodegeneration.
    54. 54
      Alexander, E. J.; Ghanbari Niaki, A.; Zhang, T.; Sarkar, J.; Liu, Y.; Nirujogi, R. S.; Pandey, A.; Myong, S.; Wang, J. Ubiquilin 2 Modulates ALS/FTD-Linked FUS–RNA Complex Dynamics and Stress Granule Formation. Proc. Natl. Acad. Sci. U. S. A. 2018, 115 (49), E11485– E11494,  DOI: 10.1073/pnas.1811997115
      54
      Ubiquilin 2 modulates ALS/FTD-linked FUS-RNA complex dynamics and stress granule formation
      Alexander, Elizabeth J.; Niaki, Amirhossein Ghanbari; Zhang, Tao; Sarkar, Jaya; Liu, Yang; Nirujogi, Raja Sekhar; Pandey, Akhilesh; Myong, Sua; Wang, Jiou
      Proceedings of the National Academy of Sciences of the United States of America (2018), 115 (49), E11485-E11494CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The ubiquitin-like protein ubiquilin 2 (UBQLN2) has been genetically and pathol. linked to the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but its normal cellular functions are not well understood. In a search for UBQLN2-interacting proteins, we found an enrichment of stress granule (SG) components, including ALS/FTD-linked heterogeneous ribonucleoprotein fused in sarcoma (FUS). Through the use of an optimized SG detection method, we obsd. UBQLN2 and its interactors at SGs. A low complexity, Sti1-like repeat region in UBQLN2 was sufficient for its localization to SGs. Functionally, UBQLN2 neg. regulated SG formation. UBQLN2 increased the dynamics of FUS-RNA interaction and promoted the fluidity of FUS-RNA complexes at a single-mol. level. This solubilizing effect corresponded to a dispersal of FUS liq. droplets in vitro and a suppression of FUS SG formation in cells. ALS-linked mutations in UBQLN2 reduced its assocn. with FUS and impaired its function in regulating FUS-RNA complex dynamics and SG formation. These results reveal a previously unrecognized role for UBQLN2 in regulating the early stages of liq.-liq. phase sepn. by directly modulating the fluidity of protein-RNA complexes and the dynamics of SG formation.
    55. 55
      Ahlers, J.; Adams, E. M.; Bader, V.; Pezzotti, S.; Winklhofer, K. F.; Tatzelt, J.; Havenith, M. The Key Role of Solvent in Condensation: Mapping Water in Liquid-Liquid Phase-Separated FUS. Biophys. J. 2021, 120, 1266,  DOI: 10.1016/j.bpj.2021.01.019
      55
      The key role of solvent in condensation: mapping water in liquid-liquid phase-separated FUS
      Ahlers, Jonas; Adams, Ellen M.; Bader, Verian; Pezzotti, Simone; Winklhofer, Konstanze F.; Tatzelt, Joerg; Havenith, Martina
      Biophysical Journal (2021), 120 (7), 1266-1275CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
      Formation of biomol. condensates through liq.-liq. phase sepn. (LLPS) has emerged as a pervasive principle in cell biol., allowing compartmentalization and spatiotemporal regulation of dynamic cellular processes. Proteins that form condensates under physiol. conditions often contain intrinsically disordered regions with low-complexity domains. Among them, the RNA-binding proteins FUS and TDP-43 have been a focus of intense investigation because aberrant condensation and aggregation of these proteins is linked to neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia. LLPS occurs when protein-rich condensates form surrounded by a dil. aq. soln. LLPS is per se entropically unfavorable. Energetically favorable multivalent protein-protein interactions are one important aspect to offset entropic costs. Another proposed aspect is the release of entropically unfavorable preordered hydration water into the bulk. We used attenuated total reflection spectroscopy in the terahertz frequency range to characterize the changes in the hydrogen bonding network accompanying the FUS enrichment in liq.-liq. phase-sepd. droplets to provide exptl. evidence for the key role of the solvent as a thermodn. driving force. The FUS concn. inside LLPS droplets was detd. to be increased to 2.0 mM independent of the initial protein concn. (5 or 10μM solns.) by fluorescence measurements. With terahertz spectroscopy, we revealed a dewetting of hydrophobic side chains in phase-sepd. FUS. Thus, the release of entropically unfavorable water populations into the bulk goes hand in hand with enthalpically favorable protein-protein interaction. Both changes are energetically favorable, and our study shows that both contribute to the thermodn. driving force in phase sepn.
    56. 56
      Li, X.; Romero, P.; Rani, M.; Dunker, A. K.; Obradovic, Z. Predicting Protein Disorder for N-, C-, and Internal Regions. Genome Inform. Workshop Genome Inform. 1999, 10, 30– 40
      56
      Predicting Protein Disorder for N-, C-, and Internal Regions
      Li; Romero; Rani; Dunker; Obradovic
      Genome informatics. Workshop on Genome Informatics (1999), 10 (), 30-40 ISSN:.
      Logistic regression (LR), discriminant analysis (DA), and neural networks (NN) were used to predict ordered and disordered regions in proteins. Training data were from a set of non-redundant X-ray crystal structures, with the data being partitioned into N-terminal, C-terminal and internal (I) regions. The DA and LR methods gave almost identical 5-cross validation accuracies that averaged to the following values: 75.9 +/- 3.1% (N-regions), 70.7 +/- 1.5% (I-regions), and 74.6 +/- 4.4% (C-regions). NN predictions gave slightly higher scores: 78.8 +/- 1.2% (N-regions), 72.5 +/- 1.2% (I-regions), and 75.3 +/- 3.3% (C-regions). Predictions improved with length of the disordered regions. Averaged over the three methods, values ranged from 52% to 78% for length = 9-14 to >/= 21, respectively, for I-regions, from 72% to 81% for length = 5 to 12-15, respectively, for N-regions, and from 70% to 80% for length = 5 to 12-15, respectively, for C-regions. These data support the hypothesis that disorder is encoded by the amino acid sequence.
    57. 57
      Boeynaems, S.; Holehouse, A. S.; Weinhardt, V.; Kovacs, D.; Van Lindt, J.; Larabell, C.; Van Den Bosch, L.; Das, R.; Tompa, P. S.; Pappu, R. V.; Gitler, A. D. Spontaneous Driving Forces Give Rise to Protein–RNA Condensates with Coexisting Phases and Complex Material Properties. Proc. Natl. Acad. Sci. U. S. A. 2019, 116, 7889– 7898,  DOI: 10.1073/pnas.1821038116
      57
      Spontaneous driving forces give rise to protein-RNA condensates with coexisting phases and complex material properties
      Boeynaems, Steven; Holehouse, Alex S.; Weinhardt, Venera; Kovacs, Denes; Van Lindt, Joris; Larabell, Carolyn; Van Den Bosch, Ludo; Das, Rhiju; Tompa, Peter S.; Pappu, Rohit V.; Gitler, Aaron D.
      Proceedings of the National Academy of Sciences of the United States of America (2019), 116 (16), 7889-7898CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Phase sepn. of multivalent protein and RNA mols. underlies the biogenesis of biomol. condensates such as membraneless organelles. In vivo, these condensates encompass hundreds of distinct types of mols. that typically organize into multilayered structures supporting the differential partitioning of mols. into distinct regions with distinct material properties. The interplay between driven (active) vs. spontaneous (passive) processes that are required for enabling the formation of condensates with coexisting layers of distinct material properties remains unclear. Here, the authors deploy systematic expts. and simulations based on coarse-grained models to show that the collective interactions among the simplest, biol. relevant proteins and archetypal RNA mols. are sufficient for driving the spontaneous emergence of multilayered condensates with distinct material properties. These studies yield a set of rules regarding homotypic and heterotypic interactions that are likely to be relevant for understanding the interplay between active and passive processes that control the formation of functional biomol. condensates.
    58. 58
      Kaur, T.; Raju, M.; Alshareedah, I.; Davis, R. B.; Potoyan, D. A.; Banerjee, P. R. Sequence-Encoded and Composition-Dependent Protein-RNA Interactions Control Multiphasic Condensate Morphologies. Nat. Commun. 2021, 12 (1), 872,  DOI: 10.1038/s41467-021-21089-4
      58
      Sequence-encoded and composition-dependent protein-RNA interactions control multiphasic condensate morphologies
      Kaur, Taranpreet; Raju, Muralikrishna; Alshareedah, Ibraheem; Davis, Richoo B.; Potoyan, Davit A.; Banerjee, Priya R.
      Nature Communications (2021), 12 (1), 872CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Multivalent protein-protein and protein-RNA interactions are the drivers of biol. phase sepn. Biomol. condensates typically contain a dense network of multiple proteins and RNAs, and their competing mol. interactions play key roles in regulating the condensate compn. and structure. Employing a ternary system comprising of a prion-like polypeptide (PLP), arginine-rich polypeptide (RRP), and RNA, we show that competition between the PLP and RNA for a single shared partner, the RRP, leads to RNA-induced demixing of PLP-RRP condensates into stable coexisting phases-homotypic PLP condensates and heterotypic RRP-RNA condensates. The morphol. of these biphasic condensates (non-engulfing/ partial engulfing/ complete engulfing) is detd. by the RNA-to-RRP stoichiometry and the hierarchy of intermol. interactions, providing a glimpse of the broad range of multiphasic patterns that are accessible to these condensates. Our findings provide a minimal set of phys. rules that govern the compn. and spatial organization of multicomponent and multiphasic biomol. condensates.
    59. 59
      Dignon, G. L.; Zheng, W.; Kim, Y. C.; Mittal, J. Temperature-Controlled Liquid–Liquid Phase Separation of Disordered Proteins. ACS Cent. Sci. 2019, 5 (5), 821– 830,  DOI: 10.1021/acscentsci.9b00102
      59
      Temperature-Controlled Liquid-Liquid Phase Separation of Disordered Proteins
      Dignon, Gregory L.; Zheng, Wenwei; Kim, Young C.; Mittal, Jeetain
      ACS Central Science (2019), 5 (5), 821-830CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
      The liq.-liq. phase sepn. (LLPS) of intrinsically disordered proteins (IDPs) is a commonly obsd. phenomenon within the cell, and such condensates are also highly attractive for applications in biomaterials and drug delivery. A better understanding of the sequence-dependent thermoresponsive behavior is of immense interest as it will aid in the design of protein sequences with desirable properties and in the understanding of cellular response to heat stress. In this work, the authors use a transferable coarse-grained model to directly probe the sequence-dependent thermoresponsive phase behavior of IDPs. To achieve this goal, the authors develop a unique knowledge-based amino acid potential that accounts for the temp.-dependent effects on solvent-mediated interactions for different types of amino acids. Remarkably, the authors are able to distinguish between more than 35 IDPs with upper or lower crit. soln. temps. at exptl. conditions, thus providing direct evidence that incorporating the temp.-dependent solvent-mediated interactions to IDP assemblies can capture the difference in the shape of the resulting phase diagrams. Given the success of the model in predicting exptl. behavior, the authors use it as a high-throughput screening framework to scan through millions of disordered sequences to characterize the compn. dependence of protein phase sepn.
    60. 60
      Zwicker, D.; Seyboldt, R.; Weber, C. A.; Hyman, A. A.; Jülicher, F. Growth and Division of Active Droplets Provides a Model for Protocells. Nat. Phys. 2017, 13 (4), 408– 413,  DOI: 10.1038/nphys3984
      60
      Growth and division of active droplets provides a model for protocells
      Zwicker, David; Seyboldt, Rabea; Weber, Christoph A.; Hyman, Anthony A.; Juelicher, Frank
      Nature Physics (2017), 13 (4), 408-413CODEN: NPAHAX; ISSN:1745-2473. (Nature Publishing Group)
      It has been proposed that during the early steps in the origin of life, small droplets could have formed via the segregation of mols. from complex mixts. by phase sepn. These droplets could have provided chem. reaction centers. However, whether these droplets could divide and propagate is unclear. Here we examine the behavior of droplets in systems that are maintained away from thermodn. equil. by an external supply of energy. In these systems, droplets grow by the addn. of droplet material generated by chem. reactions. Surprisingly, we find that chem. driven droplet growth can lead to shape instabilities that trigger the division of droplets into two smaller daughters. Therefore, chem. active droplets can exhibit cycles of growth and division that resemble the proliferation of living cells. Dividing active droplets could serve as a model for prebiotic protocells, where chem. reactions in the droplet play the role of a prebiotic metab.
    61. 61
      Wang, J.; Choi, J.-M.; Holehouse, A. S.; Lee, H. O.; Zhang, X.; Jahnel, M.; Maharana, S.; Lemaitre, R.; Pozniakovsky, A.; Drechsel, D.; Poser, I.; Pappu, R. V.; Alberti, S.; Hyman, A. A. A Molecular Grammar Governing the Driving Forces for Phase Separation of Prion-like RNA Binding Proteins. Cell 2018, 174 (3), 688– 699,  DOI: 10.1016/j.cell.2018.06.006
      61
      A Molecular Grammar Governing the Driving Forces for Phase Separation of Prion-like RNA Binding Proteins
      Wang, Jie; Choi, Jeong-Mo; Holehouse, Alex S.; Lee, Hyun O.; Zhang, Xiaojie; Jahnel, Marcus; Maharana, Shovamayee; Lemaitre, Regis; Pozniakovsky, Andrei; Drechsel, David; Poser, Ina; Pappu, Rohit V.; Alberti, Simon; Hyman, Anthony A.
      Cell (Cambridge, MA, United States) (2018), 174 (3), 688-699.e16CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      Proteins such as FUS (Fused in Sarcoma) phase sep. to form liq.-like condensates that can harden into less dynamic structures. However, how these properties emerge from the collective interactions of many amino acids remains largely unknown. Here, we use extensive mutagenesis to identify a sequence-encoded mol. grammar underlying the driving forces of phase sepn. of proteins in the FUS family and test aspects of this grammar in cells. Phase sepn. is primarily governed by multivalent interactions among tyrosine residues from prion-like domains (PLDs) and arginine residues from RNA-binding domains (RBDs), which are modulated by neg. charged residues. Glycine residues enhance the fluidity, whereas glutamine and serine residues promote hardening. We develop a model to show that the measured satn. concns. of phase sepn. are inversely proportional to the product of the nos. of arginine and tyrosine residues. These results suggest it is possible to predict phase-sepn. properties based on amino acid sequences.
    62. 62
      Lu, Y.; Lim, L.; Song, J. RRM Domain of ALS/FTD-Causing FUS Characteristic of Irreversible Unfolding Spontaneously Self-Assembles into Amyloid Fibrils. Sci. Rep. 2017, 7 (1), 1– 14,  DOI: 10.1038/s41598-017-01281-7
      62
      Transgenic Nicotiana benthamiana plants expressing a hairpin RNAi construct of a nematode Rs-cps gene exhibit enhanced resistance to Radopholus similis
      Li, Yu; Wang, Ke; Lu, Qisen; Du, Juan; Wang, Zhenyue; Wang, Desen; Sun, Bingjian; Li, Honglian
      Scientific Reports (2017), 7 (1), 1-11CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)
      Burrowing nematodes (Radopholus similis) cause severe harm in many agronomic and horticultural crops and are very difficult to manage. Cathepsin S is one of the most important cysteine proteinases and plays key roles in nematodes and many other parasites. To evaluate the effect of in planta RNAi on the control of this nematode, a specific fragment from the protease gene, cathepsin S (Rs-cps), was cloned into the binary vector pFGC5941 in the forward and reverse orientations to construct recombinant plant RNAi vectors. Transgenic Nicotiana benthamiana plants expressing Rs-cps dsRNA were obtained and studied. The transcript abundance of Rs-cps dsRNA appeared to be diverse in the different transgenic lines. Moreover, the bioassay results revealed that Rs-cps transgenic N. benthamiana plants were resistant to R. similis and the transcription level of Rs-cps in R. similis was drastically decreased. In addn., the reprodn. and hatching rate of R. similis isolated from the Rs-cps transgenic plants were also significantly reduced. Our results suggest that Rs-cps is essential for the reprodn. and pathogenicity of R. similis. This is the first study to employ in planta RNAi approach to target the Rs-cps gene for the control of plant parasitic nematodes.
    63. 63
      Li, S.; Yoshizawa, T.; Yamazaki, R.; Fujiwara, A.; Kameda, T.; Kitahara, R. Pressure and Temperature Phase Diagram for Liquid–Liquid Phase Separation of the RNA-Binding Protein Fused in Sarcoma. J. Phys. Chem. B 2021, 125 (25), 6821– 6829,  DOI: 10.1021/acs.jpcb.1c01451
      63
      Pressure and Temperature Phase Diagram for Liquid-Liquid Phase Separation of the RNA-Binding Protein Fused in Sarcoma
      Li, Shujie; Yoshizawa, Takuya; Yamazaki, Ryota; Fujiwara, Ayano; Kameda, Tomoshi; Kitahara, Ryo
      Journal of Physical Chemistry B (2021), 125 (25), 6821-6829CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
      Liq.-liq. phase sepn. (LLPS) of proteins and nucleic acids to form membraneless cellular compartments is considered to be involved in various biol. functions. The RNA-binding protein fused in sarcoma (FUS) undergoes LLPS in vivo and in vitro. Here, the authors studied the effects of pressure and temp. on the LLPS of FUS by high-pressure microscopy and high-pressure UV/visible spectroscopy. The phase-sepd. condensate of FUS was obliterated with increasing pressure but was obsd. again at a higher pressure. The authors generated a pressure-temp. phase diagram that describes the phase sepn. of FUS and provides a general understanding of the thermodn. properties of self-assembly and phase sepn. of proteins. FUS has two types of condensed phases, obsd. at low pressure (LP-LLPS) and high pressure (HP-LLPS). The HP-LLPS state was more condensed and exhibited lower susceptibility to dissoln. by 1,6-hexanediol and karyopherin-β2 than the LP-LLPS state. Moreover, mol. dynamic simulations revealed that electrostatic interactions were destabilized, whereas cation-π, π-π, and hydrophobic interactions were stabilized in HP-LLPS. When cation-π, π-π, and hydrophobic interactions were transiently stabilized in the cellular environment, the phase transition to HP-LLPS occurred; this might be correlated to the aberrant enrichment of cytoplasmic ribonucleoprotein granules, leading to amyotrophic lateral sclerosis.
    64. 64
      Gnutt, D.; Brylski, O.; Edengeiser, E.; Havenith, M.; Ebbinghaus, S. Imperfect Crowding Adaptation of Mammalian Cells towards Osmotic Stress and Its Modulation by Osmolytes. Mol. BioSyst. 2017, 13 (11), 2218– 2221,  DOI: 10.1039/C7MB00432J
      64
      Imperfect crowding adaptation of mammalian cells towards osmotic stress and its modulation by osmolytes
      Gnutt, David; Brylski, Oliver; Edengeiser, Eugen; Havenith, Martina; Ebbinghaus, Simon
      Molecular BioSystems (2017), 13 (11), 2218-2221CODEN: MBOIBW; ISSN:1742-2051. (Royal Society of Chemistry)
      Changes of the extracellular milieu could affect cellular crowding. To prevent detrimental effects, cells use adaptation mechanisms to react to such conditions. Using fluorescent crowding sensors, we show that the initial response to osmotic stress is fast but imperfect, while the slow response renders cells more tolerant to stress, particularly in the presence of osmolytes.
    65. 65
      Gnutt, D.; Sistemich, L.; Ebbinghaus, S. Protein Folding Modulation in Cells Subject to Differentiation and Stress. Front. Mol. Biosci. 2019, 6, 38,  DOI: 10.3389/fmolb.2019.00038
      65
      Protein folding modulation in cells subject to differentiation and stress
      Gnutt, Dav; Sistemich, Linda; Ebbinghaus, Simon
      Frontiers in Molecular Biosciences (2019), 6 (), 38CODEN: FMBRBS; ISSN:2296-889X. (Frontiers Media S.A.)
      Cytomimetic media are used to mimic the physicochem. properties of the cellular milieu in an in vitro expt. Themotivation is that compared to entire cells, they can be used efficiently in combination with a broad range of exptl. techniques. However, the development and use of cytomimetic media is hampered by the lack of in-cell data that could be used as a hallmark to directly evaluate and improve the performance of cytomimetic media in different applications. Such data must include the study of specific biomol. reactions in different cell types, different compartments of a single cells and different cellular conditions. In previous studies, model systems such as cancer cell lines, bacteria or oocytes were used. Here we studied how the environment of cells that undergo neuronal differentiation or proteostasis stress modulates the protein folding equil. We found that NGF induced differentiation leads to a decrease of the melting temp. and a change of the folding mechanism. Proteomic changes that occur upon differentiation could explain this effect, however, we found that the crowding effect remained unchanged. Using MG132, a common proteasome inhibitor and inducer of the unfolded protein response, we show that changes to the quality control machinery modulate the folding equil., leading to protein destabilization at prolonged stress exposure. Our study explores the range of protein folding modulation within cells subject to differentiation or stress that must be encountered in the development of cytomimetic media.
    66. 66
      Senske, M.; Törk, L.; Born, B.; Havenith, M.; Herrmann, C.; Ebbinghaus, S. Protein Stabilization by Macromolecular Crowding through Enthalpy Rather than Entropy. J. Am. Chem. Soc. 2014, 136 (25), 9036– 9041,  DOI: 10.1021/ja503205y
      66
      Protein stabilization by macromolecular crowding through enthalpy rather than entropy
      Senske, Michael; Toerk, Lisa; Born, Benjamin; Havenith, Martina; Herrmann, Christian; Ebbinghaus, Simon
      Journal of the American Chemical Society (2014), 136 (25), 9036-9041CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The interior of the cell is a densely crowded environment in which protein stability is affected differently than in dil. soln. Macromol. crowding is commonly understood in terms of an entropic vol. exclusion effect based on hardcore repulsions among the macromols. Here, the authors studied the thermal unfolding of ubiquitin in the presence of different cosolutes (glucose, dextran, poly(ethylene glycol), KCl, and urea). The results showed that for a correct dissection of the cosolute-induced changes of the free energy into its enthalpic and entropic contributions, the temp. dependence of the heat capacity change needs to be explicitly taken into account. In contrast to the prediction by excluded vol. theory, the authors obsd. an enthalpic stabilization and an entropic destabilization for glucose, dextran, and poly(ethylene glycol). The enthalpic stabilization mechanism induced by the macromol. crowder, dextran, was similar to the enthalpic stabilization mechanism of its monomeric building block, glucose. In the case of poly(ethylene glycol), entropy was dominating over enthalpy leading to an overall destabilization. The authors propose a new model to classify cosolute effects in terms of their enthalpic contributions to protein stability.
    67. 67
      Smith, A. E.; Zhou, L. Z.; Gorensek, A. H.; Senske, M.; Pielak, G. J. In-Cell Thermodynamics and a New Role for Protein Surfaces. Proc. Natl. Acad. Sci. U. S. A. 2016, 113 (7), 1725– 1730,  DOI: 10.1073/pnas.1518620113
      67
      In-cell thermodynamics and a new role for protein surfaces
      Smith, Austin E.; Zhou, Larry Z.; Gorensek, Annelise H.; Senske, Michael; Pielak, Gary J.
      Proceedings of the National Academy of Sciences of the United States of America (2016), 113 (7), 1725-1730CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      There is abundant, physiol. relevant knowledge about protein cores; they are hydrophobic, exquisitely well-packed, and nearly all H-bonds are satisfied. An equivalent understanding of protein surfaces has remained elusive because proteins are almost exclusively studied in vitro in simple aq. solns. Here, the authors established the essential physiol. roles played by protein surfaces by measuring the equil. thermodn. and kinetics of protein folding in the complex environment of living Escherichia coli cells, and under physiol. relevant in vitro conditions. Fluorine NMR data on the 7-kDa globular N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) showed that charge-charge interactions were fundamental to protein stability and folding kinetics in cells. These results contradicted predictions from accepted theories of macromol. crowding and showed that cosolutes commonly used to mimic the cellular interior did not yield physiol. relevant information. As such, the authors provide the foundation for a complete picture of protein chem. in cells.
    68. 68
      Monteith, W. B.; Cohen, R. D.; Smith, A. E.; Guzman-Cisneros, E.; Pielak, G. J. Quinary Structure Modulates Protein Stability in Cells. Proc. Natl. Acad. Sci. U. S. A. 2015, 112 (6), 1739– 1742,  DOI: 10.1073/pnas.1417415112
      68
      Quinary structure modulates protein stability in cells
      Monteith, William B.; Cohen, Rachel D.; Smith, Austin E.; Guzman-Cisneros, Emilio; Pielak, Gary J.
      Proceedings of the National Academy of Sciences of the United States of America (2015), 112 (6), 1739-1742CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Protein quinary interactions organize the cellular interior and its metab. Although the interactions stabilizing secondary, tertiary, and quaternary protein structure are well defined, details about the protein-matrix contacts that comprise quinary structure remain elusive. This gap exists because proteins function in a crowded cellular environment, but are traditionally studied in simple buffered solns. Here, the authors used NMR-detected H/D exchange to quantify quinary interactions between the B1 domain of protein G and the cytosol of Escherichia coli. The authors demonstrated that a surface mutation in this protein was 10-fold more destabilizing in cells than in buffer, a surprising result that firmly establishes the significance of quinary interactions. Remarkably, the energy involved in these interactions could be as large as the energies that stabilize specific protein complexes. These results will drive the crit. task of implementing quinary structure into models for understanding the proteome.
    69. 69
      Cohen, R. D.; Pielak, G. J. Quinary Interactions with an Unfolded State Ensemble: Quinary Interactions & Unfolded State Ensemble. Protein Sci. 2017, 26 (9), 1698– 1703,  DOI: 10.1002/pro.3206
      69
      Quinary interactions with an unfolded state ensemble
      Cohen, Rachel D.; Pielak, Gary J.
      Protein Science (2017), 26 (9), 1698-1703CODEN: PRCIEI; ISSN:1469-896X. (Wiley-Blackwell)
      Anfinsen's thermodn. hypothesis states that the native three-dimensional fold of a protein represents the structure with the lowest Gibbs free energy. Changes in the free energy of denaturation can arise from changes to the folded state, the unfolded state, or both. It has been recently recognized that quinary interactions, transient contacts that take place only in cells, can modulate protein stability through interactions involving the folded state. Here we show that the cellular environment can also remodel the unfolded state ensemble.
    70. 70
      Samanta, N.; Mahanta, D. D.; Hazra, S.; Kumar, G. S.; Mitra, R. K. Short Chain Polyethylene Glycols Unusually Assist Thermal Unfolding of Human Serum Albumin. Biochimie 2014, 104, 81– 89,  DOI: 10.1016/j.biochi.2014.05.009
      70
      Short chain polyethylene glycols unusually assist thermal unfolding of human serum albumin
      Samanta, Nirnay; Mahanta, Debasish Das; Hazra, Soumitra; Kumar, Gopinatha Suresh; Mitra, Rajib Kumar
      Biochimie (2014), 104 (), 81-89CODEN: BICMBE; ISSN:0300-9084. (Elsevier Masson SAS)
      In the present study we have investigated the thermal stability of the globular transport protein human serum albumin (HSA), in the presence of two small chain polyethylene glycols (namely PEG 200 and PEG 400). Both near- and far-UV CD (CD) study reveal that addn. of PEG moderately increases the α-helical content of the protein without abruptly changing its tertiary structure. The hydration structure at the protein surface experiences a notable change at 30% PEG (vol./vol.) concn. as evidenced from compressibility and dynamic light scattering (DLS) measurements. Thermal denaturation of HSA in the presence of PEG has been studied by CD and fluorescence spectroscopy using the intrinsic fluorophore tryptophan and it has been found that addn. of PEG makes the protein more prone towards unfolding, which is in contrary to what has been obsd. in case of larger mol. wt. polymers. The energetics of the thermal unfolding process has been obtained using differential scanning calorimetry (DSC) measurements. Our study concludes that both the indirect excluded vol. principle as well as interaction of the polymer at the protein surface is responsible for the obsd. change of the unfolding process.
    71. 71
      Sörensen, T.; Leeb, S.; Danielsson, J.; Oliveberg, M. Polyanions Cause Protein Destabilization Similar to That in Live Cells. Biochemistry 2021, 60 (10), 735– 746,  DOI: 10.1021/acs.biochem.0c00889
      71
      Polyanions Cause Protein Destabilization Similar to That in Live Cells
      Sorensen Therese; Leeb Sarah; Danielsson Jens; Oliveberg Mikael
      Biochemistry (2021), 60 (10), 735-746 ISSN:.
      The structural stability of proteins is found to markedly change upon their transfer to the crowded interior of live cells. For some proteins, the stability increases, while for others, it decreases, depending on both the sequence composition and the type of host cell. The mechanism seems to be linked to the strength and conformational bias of the diffusive in-cell interactions, where protein charge is found to play a decisive role. Because most proteins, nucleotides, and membranes carry a net-negative charge, the intracellular environment behaves like a polyanionic (Z:1) system with electrostatic interactions different from those of standard 1:1 ion solutes. To determine how such polyanion conditions influence protein stability, we use negatively charged polyacetate ions to mimic the net-negatively charged cellular environment. The results show that, per Na(+) equivalent, polyacetate destabilizes the model protein SOD1(barrel) significantly more than monoacetate or NaCl. At an equivalent of 100 mM Na(+), the polyacetate destabilization of SOD1(barrel) is similar to that observed in live cells. By the combined use of equilibrium thermal denaturation, folding kinetics, and high-resolution nuclear magnetic resonance, this destabilization is primarily assigned to preferential interaction between polyacetate and the globally unfolded protein. This interaction is relatively weak and involves mainly the outermost N-terminal region of unfolded SOD1(barrel). Our findings point thus to a generic influence of polyanions on protein stability, which adds to the sequence-specific contributions and needs to be considered in the evaluation of in vivo data.
    72. 72
      Kaur, T.; Alshareedah, I.; Wang, W.; Ngo, J.; Moosa, M. M.; Banerjee, P. R. Molecular Crowding Tunes Material States of Ribonucleoprotein Condensates. Biomolecules 2019, 9 (2), 71,  DOI: 10.3390/biom9020071
      72
      Molecular crowding tunes material states of ribonucleoprotein condensates
      Kaur, Taranpreet; Alshareedah, Ibraheem; Wang, Wei; Ngo, Jason; Moosa, Mahdi Muhammad; Banerjee, Priya R.
      Biomolecules (2019), 9 (2), 71CODEN: BIOMHC; ISSN:2218-273X. (MDPI AG)
      Ribonucleoprotein (RNP) granules are membraneless liq. condensates that dynamically form, dissolve, and mature into a gel-like state in response to a changing cellular environment. RNP condensation is largely governed by promiscuous attractive inter-chain interactions mediated by low-complexity domains (LCDs). Using an archetypal disordered RNP, fused in sarcoma (FUS), here we study how mol. crowding impacts the RNP liq. condensation. We observe that the liq.-liq. coexistence boundary of FUS is lowered by polymer crowders, consistent with an excluded vol. model. With increasing bulk crowder concn., the RNP partition increases and the diffusion rate decreases in the condensed phase. Furthermore, we show that RNP condensates undergo substantial hardening wherein protein-dense droplets transition from viscous fluid to viscoelastic gel-like states in a crowder concn.-dependent manner. Utilizing two distinct LCDs that broadly represent commonly occurring sequence motifs driving RNP phase transitions, we reveal that the impact of crowding is largely independent of LCD charge and sequence patterns. These results are consistent with a thermodn. model of crowder-mediated depletion interaction, which suggests that inter-RNP attraction is enhanced by mol. crowding. The depletion force is likely to play a key role in tuning the phys. properties of RNP condensates within the crowded cellular space.
    73. 73
      Park, S.; Barnes, R.; Lin, Y.; Jeon, B.; Najafi, S.; Delaney, K. T.; Fredrickson, G. H.; Shea, J.-E.; Hwang, D. S.; Han, S. Dehydration Entropy Drives Liquid-Liquid Phase Separation by Molecular Crowding. Commun. Chem. 2020, 3 (1), 1– 12,  DOI: 10.1038/s42004-020-0328-8
      There is no corresponding record for this reference.
    74. 74
      Nguemaha, V.; Zhou, H.-X. Liquid-Liquid Phase Separation of Patchy Particles Illuminates Diverse Effects of Regulatory Components on Protein Droplet Formation. Sci. Rep. 2018, 8 (1), 6728,  DOI: 10.1038/s41598-018-25132-1
      74
      Liquid-Liquid Phase Separation of Patchy Particles Illuminates Diverse Effects of Regulatory Components on Protein Droplet Formation
      Nguemaha Valery; Zhou Huan-Xiang; Zhou Huan-Xiang
      Scientific reports (2018), 8 (1), 6728 ISSN:.
      Recently many cellular functions have been associated with membraneless organelles, or protein droplets, formed by liquid-liquid phase separation (LLPS). Proteins in these droplets often contain RNA-binding domains, but the effects of RNA on LLPS have been controversial. To gain better understanding on the roles of RNA and other macromolecular regulators, here we used Gibbs-ensemble simulations to determine phase diagrams of two-component patchy particles, as models for mixtures of proteins with regulatory components. Protein-like particles have four patches, with attraction strength εPP; regulatory particles experience mutual steric repulsion but have two attractive patches toward proteins, with the strength εPR tunable. At low εPR, the regulator, due to steric repulsion, preferentially partitions in the dispersed phase, thereby displacing the protein into the droplet phase and promoting LLPS. At moderate εPR, the regulator starts to partition and displace the protein in the droplet phase, but only to weaken bonding networks and thereby suppress LLPS. At εPR > εPP, the enhanced bonding ability of the regulator initially promotes LLPS, but at higher amounts, the resulting displacement of the protein suppresses LLPS. These results illustrate how RNA can have disparate effects on LLPS, thus able to perform diverse functions in different organelles.
    75. 75
      Protter, D. S. W.; Rao, B. S.; Van Treeck, B.; Lin, Y.; Mizoue, L.; Rosen, M. K.; Parker, R. Intrinsically Disordered Regions Can Contribute Promiscuous Interactions to RNP Granule Assembly. Cell Rep. 2018, 22 (6), 1401– 1412,  DOI: 10.1016/j.celrep.2018.01.036
      75
      Intrinsically Disordered Regions Can Contribute Promiscuous Interactions to RNP Granule Assembly
      Protter, David S. W.; Rao, Bhalchandra S.; Van Treeck, Briana; Lin, Yuan; Mizoue, Laura; Rosen, Michael K.; Parker, Roy
      Cell Reports (2018), 22 (6), 1401-1412CODEN: CREED8; ISSN:2211-1247. (Cell Press)
      Eukaryotic cells contain large RNA-protein assemblies referred to as RNP granules, whose assembly is promoted by both traditional protein interactions and intrinsically disordered protein domains. Using RNP granules as an example, we provide evidence for an assembly mechanism of large cellular structures wherein specific protein-protein or protein-RNA interactions act together with promiscuous interactions of intrinsically disordered regions (IDRs). This synergistic assembly mechanism illuminates RNP granule assembly and explains why many components of RNP granules, and other large dynamic assemblies, contain IDRs linked to specific protein-protein or protein-RNA interaction modules. We suggest assemblies based on combinations of specific interactions and promiscuous IDRs are common features of eukaryotic cells.
    76. 76
      Danielsson, J.; Mu, X.; Lang, L.; Wang, H.; Binolfi, A.; Theillet, F.-X.; Bekei, B.; Logan, D. T.; Selenko, P.; Wennerström, H.; Oliveberg, M. Thermodynamics of Protein Destabilization in Live Cells. Proc. Natl. Acad. Sci. U. S. A. 2015, 112 (40), 12402– 12407,  DOI: 10.1073/pnas.1511308112
      76
      Thermodynamics of protein destabilization in live cells
      Danielsson, Jens; Mu, Xin; Lang, Lisa; Wang, Huabing; Binolfi, Andres; Theillet, Francois-Xavier; Bekei, Beata; Logan, Derek T.; Selenko, Philipp; Wennerstroem, Haakan; Oliveberg, Mikael
      Proceedings of the National Academy of Sciences of the United States of America (2015), 112 (40), 12402-12407CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at at. resoln. the thermal unfolding of a β-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37° but with a conspicuous twist: while the melting temp. goes down the cold unfolding moves into the physiol. regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, detd. by the individual protein's interplay with the functionally optimized "interaction landscape" of the cellular interior.
    77. 77
      Ghosh, A.; Mazarakos, K.; Zhou, H.-X. Three Archetypical Classes of Macromolecular Regulators of Protein Liquid–Liquid Phase Separation. Proc. Natl. Acad. Sci. U. S. A. 2019, 116 (39), 19474– 19483,  DOI: 10.1073/pnas.1907849116
      77
      Three archetypical classes of macromolecular regulators of protein liquid-liquid phase separation
      Ghosh, Archishman; Mazarakos, Konstantinos; Zhou, Huan-Xiang
      Proceedings of the National Academy of Sciences of the United States of America (2019), 116 (39), 19474-19483CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Membraneless organelles, corresponding to the droplet phase upon liq.-liq. phase sepn. (LLPS) of protein or protein-RNA mixts., mediate myriad cellular functions. Cells use a variety of biochem. signals such as expression level and posttranslational modification to regulate droplet formation and dissoln., but the phys. basis of the regulatory mechanisms remains ill-defined and quant. assessment of the effects is largely lacking. Our computational study predicted that the strength of attraction by droplet-forming proteins dictates whether and how macromol. regulators promote or suppress LLPS. We exptl. tested this prediction, using the pentamers of SH3 domains and proline-rich motifs (SH35 and PRM5) as droplet-forming proteins. Detn. of the changes in phase boundary and the partition coeffs. in the droplet phase over a wide range of regulator concns. yielded both a quant. measure and a mechanistic understanding of the regulatory effects. Three archetypical classes of regulatory effects were obsd. Ficoll 70 at high concns. indirectly promoted SH35-PRM5 LLPS, by taking up vol. in the bulk phase and thereby displacing SH35 and PRM5 into the droplet phase. Lysozyme had a moderate partition coeff. and suppressed LLPS by substituting weaker attraction with SH35 for the stronger SH35-PRM5 attraction in the droplet phase. By forming even stronger attraction with PRM5, heparin at low concns. partitioned heavily into the droplet phase and promoted LLPS. These characteristics were recapitulated by computational results of patchy particle models, validating the identification of the 3 classes of macromol. regulators as vol.-exclusion promotors, weak-attraction suppressors, and strong-attraction promotors.
    78. 78
      André, A. A. M.; Spruijt, E. Liquid–Liquid Phase Separation in Crowded Environments. Int. J. Mol. Sci. 2020, 21 (16), 5908,  DOI: 10.3390/ijms21165908
      78
      Liquid-liquid phase separation in crowded environments
      Andre, Alain A. M.; Spruijt, Evan
      International Journal of Molecular Sciences (2020), 21 (16), 5908CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
      A review. Biomol. condensates play a key role in organizing cellular fluids such as the cytoplasm and nucleoplasm. Most of these non-membranous organelles show liq.-like properties both in cells and when studied in vitro through liq.-liq. phase sepn. (LLPS) of purified proteins. In general, LLPS of proteins is known to be sensitive to variations in pH, temp. and ionic strength, but the role of crowding remains underappreciated. Several decades of research have shown that macromol. crowding can have profound effects on protein interactions, folding and aggregation, and it must, by extension, also impact LLPS. However, the precise role of crowding in LLPS is far from trivial, as most condensate components have a disordered nature and exhibit multiple weak attractive interactions. Here, we discuss which factors det. the scope of LLPS in crowded environments, and we review the evidence for the impact of macromol. crowding on phase boundaries, partitioning behavior and condensate properties. Based on a comparison of both in vivo and in vitro LLPS studies, we propose that phase sepn. in cells does not solely rely on attractive interactions, but shows important similarities to segregative phase sepn.
    79. 79
      Banani, S. F.; Rice, A. M.; Peeples, W. B.; Lin, Y.; Jain, S.; Parker, R.; Rosen, M. K. Compositional Control of Phase-Separated Cellular Bodies. Cell 2016, 166 (3), 651– 663,  DOI: 10.1016/j.cell.2016.06.010
      79
      Compositional control of phase-separated cellular bodies
      Banani, Salman F.; Rice, Allyson M.; Peeples, William B.; Lin, Yuan; Jain, Saumya; Parker, Roy; Rosen, Michael K.
      Cell (Cambridge, MA, United States) (2016), 166 (3), 651-663CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      Cellular bodies such as P bodies and PML nuclear bodies (PML NBs) appear to be phase-sepd. liqs. organized by multivalent interactions among proteins and RNA mols. Although many components of various cellular bodies are known, general principles that define body compn. are lacking. Here, the authors modeled cellular bodies using several engineered multivalent proteins and RNA. In vitro and in cells, these scaffold mols. formed phase-sepd. liqs. that concd. low valency client proteins. Clients partitioned differently depending on the ratio of scaffolds, with a sharp switch across the phase diagram diagonal. The compn. could switch rapidly through changes in scaffold concn. or valency. Natural PML NBs and P bodies showed analogous partitioning behavior, suggesting how their compns. could be controlled by levels of PML SUMOylation or cellular mRNA concn., resp. The data suggested a conceptual framework for considering the compn. and control thereof of cellular bodies assembled through heterotypic multivalent interactions.
    80. 80
      Sterpone, F.; Melchionna, S.; Tuffery, P.; Pasquali, S.; Mousseau, N.; Cragnolini, T.; Chebaro, Y.; St-Pierre, J.-F.; Kalimeri, M.; Barducci, A.; Laurin, Y.; Tek, A.; Baaden, M.; Nguyen, P. H.; Derreumaux, P. The OPEP Protein Model: From Single Molecules, Amyloid Formation, Crowding and Hydrodynamics to DNA/RNA Systems. Chem. Soc. Rev. 2014, 43 (13), 4871– 4893,  DOI: 10.1039/C4CS00048J
      80
      The OPEP protein model: from single molecules, amyloid formation, crowding and hydrodynamics to DNA/RNA systems
      Sterpone, Fabio; Melchionna, Simone; Tuffery, Pierre; Pasquali, Samuela; Mousseau, Normand; Cragnolini, Tristan; Chebaro, Yassmine; St-Pierre, Jean-Francois; Kalimeri, Maria; Barducci, Alessandro; Laurin, Yoann; Tek, Alex; Baaden, Marc; Nguyen, Phuong Hoang; Derreumaux, Philippe
      Chemical Society Reviews (2014), 43 (13), 4871-4893CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      A review. The OPEP coarse-grained protein model has been applied to a wide range of applications since its first release 15 years ago. The model, which combines energetic and structural accuracy and chem. specificity, allows the study of single protein properties, DNA-RNA complexes, amyloid fibril formation and protein suspensions in a crowded environment. Here we first review the current state of the model and the most exciting applications using advanced conformational sampling methods. We then present the current limitations and a perspective on the ongoing developments.
    81. 81
      Stirnemann, F.; Derreumaux, P.; Melchionna, S. Protein Simulations in Fluids: Coupling the OPEP Coarse-Grained Force Field with Hydrodynamics. J. Chem. Theory Comput. 2015, 11 (4), 1843– 1853,  DOI: 10.1021/ct501015h
      There is no corresponding record for this reference.
    82. 82
      Claes, F.; Rudyak, S.; Laird, A. S.; Louros, N.; Beerten, J.; Debulpaep, M.; Michiels, E.; van der Kant, R.; Van Durme, J.; De Baets, G.; Houben, B.; Ramakers, M.; Yuan, K.; Gwee, S. S. L.; Hernandez, S.; Broersen, K.; Oliveberg, M.; Moahamed, B.; Kirstein, J.; Robberecht, W.; Rousseau, F.; Schymkowitz, J. Exposure of a Cryptic Hsp70 Binding Site Determines the Cytotoxicity of the ALS-Associated SOD1-Mutant A4V. Protein Eng., Des. Sel. 2019, 32 (10), 443– 457,  DOI: 10.1093/protein/gzaa008
      82
      Exposure of a cryptic Hsp70 binding site determines the cytotoxicity of the ALS-associated SOD1-mutant A4V
      Claes, Filip; Rudyak, Stanislav; Laird, Angela S.; Louros, Nikolaos; Beerten, Jacinte; Debulpaep, Maja; Michiels, Emiel; van der Kant, Rob; Van Durme, Joost; De Baets, Greet; Houben, Bert; Ramakers, Meine; Yuan, Kristy; Gwee, Serene S. L.; Hernandez, Sara; Broersen, Kerensa; Oliveberg, Mikael; Moahamed, Barbara; Kirstein, Janine; Robberecht, Wim; Rousseau, Frederic; Schymkowitz, Joost
      Protein Engineering, Design & Selection (2019), 32 (10), 443-457CODEN: PEDSBR; ISSN:1741-0134. (Oxford University Press)
      The accumulation of toxic protein aggregates is thought to play a key role in a range of degenerative pathologies, but it remains unclear why aggregation of polypeptides into non-native assemblies is toxic and why cellular clearance pathways offer ineffective protection. We here study the A4V mutant of SOD1, which forms toxic aggregates in motor neurons of patients with familial amyotrophic lateral sclerosis (ALS). A comparison of the location of aggregation prone regions (APRs) and Hsp70 binding sites in the denatured state of SOD1 reveals that ALS-assocd. mutations promote exposure of the APRs more than the strongest Hsc/Hsp70 binding site that we could detect. Mutations designed to increase the exposure of this Hsp70 interaction site in the denatured state promote aggregation but also display an increased interaction with Hsp70 chaperones. Depending on the cell type, in vitro this resulted in cellular inclusion body formation or increased clearance, accompanied with a suppression of cytotoxicity. The latter was also obsd. in a zebrafish model in vivo. Our results suggest that the uncontrolled accumulation of toxic SOD1A4V aggregates results from insufficient detection by the cellular surveillance network.
    83. 83
      Imamoglu, R.; Balchin, D.; Hayer-Hartl, M.; Hartl, F. U. Bacterial Hsp70 Resolves Misfolded States and Accelerates Productive Folding of a Multi-Domain Protein. Nat. Commun. 2020, 11 (1), 1– 13,  DOI: 10.1038/s41467-019-14245-4
      There is no corresponding record for this reference.
    84. 84
      Gal, J.; Ström, A.-L.; Kilty, R.; Zhang, F.; Zhu, H. P62 Accumulates and Enhances Aggregate Formation in Model Systems of Familial Amyotrophic Lateral Sclerosis*. J. Biol. Chem. 2007, 282 (15), 11068– 11077,  DOI: 10.1074/jbc.M608787200
      84
      p62 Accumulates and Enhances Aggregate Formation in Model Systems of Familial Amyotrophic Lateral Sclerosis
      Gal, Jozsef; Stroem, Anna-Lena; Kilty, Renee; Zhang, Fujian; Zhu, Haining
      Journal of Biological Chemistry (2007), 282 (15), 11068-11077CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by motor neuron death. A hallmark of the disease is the appearance of protein aggregates in the affected motor neurons. We have found that p62, a protein implicated in protein aggregate formation, accumulated progressively in the G93A mouse spinal cord. The accumulation of p62 was in parallel to the increase of polyubiquitinated proteins and mutant SOD1 aggregates. Immunostaining studies showed that p62, ubiquitin, and mutant SOD1 co-localized in the protein aggregates in affected cells in G93A mouse spinal cord. The p62 protein selectively interacted with familial ALS mutants, but not WT SOD1. When p62 was co-expressed with SOD1 in NSC34 cells, it greatly enhanced the formation of aggregates of the ALS-linked SOD1 mutants, but not wild-type SOD1. Cell viability was measured in the presence and absence of overexpressed p62, and the results suggest that the large aggregates facilitated by p62 were not directly toxic to cells under the conditions in this study. Deletion of the ubiquitin-assocn. (UBA) domain of p62 significantly decreased the p62-facilitated aggregate formation, but did not completely inhibit it. Further protein interaction expts. also showed that the truncated p62 with the UBA domain deletion remained capable of interacting with mutant SOD1. The findings of this study show that p62 plays a crit. role in forming protein aggregates in familial ALS, likely by linking misfolded mutant SOD1 mols. and other cellular proteins together.
    85. 85
      Ciechanover, A.; Kwon, Y. T. Degradation of Misfolded Proteins in Neurodegenerative Diseases: Therapeutic Targets and Strategies. Exp. Mol. Med. 2015, 47 (3), e147– e147,  DOI: 10.1038/emm.2014.117
      85
      Degradation of misfolded proteins in neurodegenerative diseases: therapeutic targets and strategies
      Ciechanover, Aaron; Kwon, Yong Tae
      Experimental & Molecular Medicine (2015), 47 (3), e147CODEN: EMMEF3; ISSN:2092-6413. (NPG Nature Asia-Pacific)
      A review. Mammalian cells remove misfolded proteins using various proteolytic systems, including the ubiquitin (Ub)-proteasome system (UPS), chaperone mediated autophagy (CMA) and macroautophagy. The majority of misfolded proteins are degraded by the UPS, in which Ub-conjugated substrates are deubiquitinated, unfolded and cleaved into small peptides when passing through the narrow chamber of the proteasome. The substrates that expose a specific degrdn. signal, the KFERQ sequence motif, can be delivered to and degraded in lysosomes via the CMA. Aggregation-prone substrates resistant to both the UPS and the CMA can be degraded by macroautophagy, in which cargoes are segregated into autophagosomes before degrdn. by lysosomal hydrolases. Although most misfolded and aggregated proteins in the human proteome can be degraded by cellular protein quality control, some native and mutant proteins prone to aggregation into β-sheet-enriched oligomers are resistant to all known proteolytic pathways and can thus grow into inclusion bodies or extracellular plaques. The accumulation of protease-resistant misfolded and aggregated proteins is a common mechanism underlying protein misfolding disorders, including neurodegenerative diseases such as Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), prion diseases and Amyotrophic Lateral Sclerosis (ALS). In this review, we provide an overview of the proteolytic pathways in neurons, with an emphasis on the UPS, CMA and macroautophagy, and discuss the role of protein quality control in the degrdn. of pathogenic proteins in neurodegenerative diseases. Addnl., we examine existing putative therapeutic strategies to efficiently remove cytotoxic proteins from degenerating neurons.
    86. 86
      Vecchi, G.; Sormanni, P.; Mannini, B.; Vandelli, A.; Tartaglia, G. G.; Dobson, C. M.; Hartl, F. U.; Vendruscolo, M. Proteome-Wide Observation of the Phenomenon of Life on the Edge of Solubility. Proc. Natl. Acad. Sci. U. S. A. 2020, 117 (2), 1015– 1020,  DOI: 10.1073/pnas.1910444117
      86
      Proteome-wide observation of the phenomenon of life on the edge of solubility
      Vecchi, Giulia; Sormanni, Pietro; Mannini, Benedetta; Vandelli, Andrea; Tartaglia, Gian Gaetano; Dobson, Christopher M.; Hartl, F. Ulrich; Vendruscolo, Michele
      Proceedings of the National Academy of Sciences of the United States of America (2020), 117 (2), 1015-1020CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      To function effectively proteins must avoid aberrant aggregation, and hence they are expected to be expressed at concns. safely below their soly. limits. By analyzing proteome-wide mass spectrometry data of Caenorhabditis elegans, however, we show that the levels of about three-quarters of the nearly 4,000 proteins analyzed in adult animals are close to their intrinsic soly. limits, indeed exceeding them by about 10% on av. We next asked how aging and functional self-assembly influence these soly. limits. We found that despite the fact that the total quantity of proteins within the cellular environment remains approx. const. during aging, protein aggregation sharply increases between days 6 and 12 of adulthood, after the worms have reproduced, as individual proteins lose their stoichiometric balances and the cellular machinery that maintains soly. undergoes functional decline. These findings reveal that these proteins are highly prone to undergoing concn.-dependent phase sepn., which on aging is rationalized in a decrease of their effective solubilities, in particular for proteins assocd. with translation, growth, reprodn., and the chaperone system.
    87. 87
      Dobson, C. M. Principles of Protein Folding, Misfolding and Aggregation. Semin. Cell Dev. Biol. 2004, 15 (1), 3– 16,  DOI: 10.1016/j.semcdb.2003.12.008
      87
      Principles of protein folding, misfolding and aggregation
      Dobson, Christopher M.
      Seminars in Cell & Developmental Biology (2004), 15 (1), 3-16CODEN: SCDBFX; ISSN:1084-9521. (Elsevier Science B.V.)
      A review. Here, the author summarizes the current understanding of the underlying and universal mechanism by which newly synthesized proteins achieve their biol. functional states. Protein mols., however, all have a finite tendency either to misfold, or to fail to maintain their correctly folded states, under some circumstances. The author describes some of the consequences of such behavior, particularly in the context of the aggregation events that are frequently assocd. with aberrant folding. The authors focuses in particular on the emerging links between protein aggregation and the increasingly prevalent forms of debilitating disease with which it is now known to be assocd.
    88. 88
      Zbinden, A.; Pérez-Berlanga, M.; De Rossi, P.; Polymenidou, M. Phase Separation and Neurodegenerative Diseases: A Disturbance in the Force. Dev. Cell 2020, 55 (1), 45– 68,  DOI: 10.1016/j.devcel.2020.09.014
      88
      Phase Separation and Neurodegenerative Diseases: A Disturbance in the Force
      Zbinden, Aurelie; Perez-Berlanga, Manuela; De Rossi, Pierre; Polymenidou, Magdalini
      Developmental Cell (2020), 55 (1), 45-68CODEN: DCEEBE; ISSN:1534-5807. (Cell Press)
      Protein aggregation is the main hallmark of neurodegenerative diseases. Many proteins found in pathol. inclusions are known to undergo liq.-liq. phase sepn., a reversible process of mol. self-assembly. Emerging evidence supports the hypothesis that aberrant phase sepn. behavior may serve as a trigger of protein aggregation in neurodegeneration, and efforts to understand and control the underlying mechanisms are underway. Here, we review similarities and differences among four main proteins, α-synuclein, FUS, tau, and TDP-43, which are found aggregated in different diseases and were independently shown to phase sep. We discuss future directions in the field that will help shed light on the mol. mechanisms of aggregation and neurodegeneration.

  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.1c09589.

    • Description of materials and methods, visual and plot of agregates of Wt and A4V mutants in HeLaFUS-mCh cells; laser heating and FRET data; kinetic traces and amplitudes as a function of temperature; PCs of SOD1bar-Wt and the various mutants inside SGs as a function of protein expression levels; correlation plot for SOD1bar PCs; mobility of SOD1bar inside SGs and cytosol after 120 minutes of HS; SDS PAGE gel for purified FUS-MBP and SOD1bar constructs; DIC images of FUS droplets in the presence and absence of SOD1bar; line-ROI intensity plots; images of SOD1bar G41D-enriched into or depleted from FUS-Wt droplets; change of the cross section of FUS droplets after HS at 43 °C with time; difference in ΔGf°′ at 37 °C; PCs as a function of ΔGf°′; LBMD simulation snapshots; interaction energies and contacts; fractions of protein surface areas occupied by the different residue types; mean number of contacts between different categories of residues; Tables S1 and S2 (including all the data points shown in the graphics) (PDF)


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