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Convergent Alterations of a Protein Hub Produce Divergent Effects within a Binding Site
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  • Ali Imran
    Ali Imran
    Department of Physics, Syracuse University, 201 Physics Building, Syracuse, New York 13244-1130, United States
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  • Brandon S. Moyer
    Brandon S. Moyer
    Ichor Life Sciences, Inc., 2651 US Route 11, LaFayette, New York 13084, United States
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  • Dan Kalina
    Dan Kalina
    Ichor Life Sciences, Inc., 2651 US Route 11, LaFayette, New York 13084, United States
    Department of Chemistry, State University of New York College of Environmental Science and Forestry, 1 Forestry Dr., Syracuse, New York 13210, United States
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  • Thomas M. Duncan
    Thomas M. Duncan
    Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, 4249 Weiskotten Hall, 766 Irving Avenue, Syracuse, New York 13210, United States
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  • Kelsey J. Moody
    Kelsey J. Moody
    Department of Physics, Syracuse University, 201 Physics Building, Syracuse, New York 13244-1130, United States
    Ichor Life Sciences, Inc., 2651 US Route 11, LaFayette, New York 13084, United States
    Department of Chemistry, State University of New York College of Environmental Science and Forestry, 1 Forestry Dr., Syracuse, New York 13210, United States
    Lewis School of Health Sciences, Clarkson University, 8 Clarkson Avenue, Potsdam, New York 13699, United States
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  • Aaron J. Wolfe
    Aaron J. Wolfe
    Department of Physics, Syracuse University, 201 Physics Building, Syracuse, New York 13244-1130, United States
    Ichor Life Sciences, Inc., 2651 US Route 11, LaFayette, New York 13084, United States
    Department of Chemistry, State University of New York College of Environmental Science and Forestry, 1 Forestry Dr., Syracuse, New York 13210, United States
    Lewis School of Health Sciences, Clarkson University, 8 Clarkson Avenue, Potsdam, New York 13699, United States
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  • Michael S. Cosgrove*
    Michael S. Cosgrove
    Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, 4249 Weiskotten Hall, 766 Irving Avenue, Syracuse, New York 13210, United States
    *Email: [email protected]
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  • Liviu Movileanu*
    Liviu Movileanu
    Department of Physics, Syracuse University, 201 Physics Building, Syracuse, New York 13244-1130, United States
    Department of Biomedical and Chemical Engineering, Syracuse University, 329 Link Hall, Syracuse, New York 13244, United States
    The BioInspired Institute, Syracuse University, Syracuse, New York 13244, United States
    *Email: [email protected]
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    Orcidhttps://orcid.org/0000-0002-2525-3341
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ACS Chemical Biology

Cite this: ACS Chem. Biol. 2022, 17, 6, 1586–1597
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https://doi.org/10.1021/acschembio.2c00273
Published May 25, 2022

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Abstract

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Progress in tumor sequencing and cancer databases has created an enormous amount of information that scientists struggle to sift through. While several research groups have created computational methods to analyze these databases, much work still remains in distinguishing key implications of pathogenic mutations. Here, we describe an approach to identify and evaluate somatic cancer mutations of WD40 repeat protein 5 (WDR5), a chromatin-associated protein hub. This multitasking protein maintains the functional integrity of large multi-subunit enzymatic complexes of the six human SET1 methyltransferases. Remarkably, the somatic cancer mutations of WDR5 preferentially distribute within and around an essential cavity, which hosts the WDR5 interaction (Win) binding site. Hence, we assessed the real-time binding kinetics of the interactions of key clustered WDR5 mutants with the Win motif peptide ligands of the SET1 family members (SET1Win). Our measurements highlight that this subset of mutants exhibits divergent perturbations in the kinetics and strength of interactions not only relative to those of the native WDR5 but also among various SET1Win ligands. These outcomes could form a fundamental basis for future drug discovery and other developments in medical biotechnology.

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Introduction

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WD40 repeat proteins (WDRs) are among the most abundant protein–protein interaction domains in the human proteome. (1−3) WDRs are either implicated in numerous cell signaling pathways (4,5) or in scaffolding large multi-subunit enzymatic complexes. (6,7) Notably, WD40 repeat protein 5 (WDR5) is a highly conserved nuclear hub, which is primarily known for its regulatory role in histone 3 lysine 4 (H3K4) mono- and di-methylation. (8−13) In this process, WDR5 bridges the interaction between the catalytic domain of mixed lineage leukemia MLL/SET1 family proteins and other subunits of the large methyltransferase complex. The assembly and stability of this enzymatic complex is necessary for optimal methyltransferase activity. (14−16) In addition, WDR5 interacts with other protein partners, such as transcription factor MYC (17−20) and 3-phosphoinositide-dependent protein kinase 1 (PDPK1). (21) Two highly conserved motifs of these protein binders, the WDR5 interaction (Win) motif (22−24) and WDR5-binding motif (WBM), (18,25,26) are deemed responsible for the vast majority of their interactions with WDR5. Interactions corresponding to these motifs are mediated by the Win and WBM sites, respectively (Figure 1a).

Figure 1

Figure 1. Two binding sites of WDR5 and the structure of the WDR5-MLL3Win complex. (a) Representations of the Win and WBM binding sites of WDR5. Orientations of WDR5 in the two cartoons are 180° with respect to each other. (b) Top view of the WDR5-MLL3Win complex. (c) Side view of the WDR5-MLL3Win complex. (d) Side view of the interaction sites between MLL3Win (green) and WDR5 (gray). All presented residues are within 5 Å of the other binding partner. The residues corresponding to the WDR5 mutations explored in this study are marked in dark gray. MLL3Win residues are labeled as well. Potential hydrogen bonds between the two binding partners are shown as yellow dotted lines. (e) Key residues of the WDR5 binding cavity involved in hydrogen bonding with the evolutionarily conserved Arg residue (R4710) of MLL3Win at position P0 (Table S1). The hydrogen bonds are indicated by thick dashed lines marked in yellow. The cutoff distance for identifying these hydrogen bonds was 4.0 Å. WDR5 was represented using pdb entry 4ERY. (35)

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For oncoproteins, the driver cancer mutations preferentially populate either within an active site or on their binding surface. (27,28) Based upon this argument, we postulated that missense somatic cancer mutations of WDR5 form a dense cluster either within one or both binding sites. Databases, such as Catalogue of Somatic Mutations in Cancer (COSMIC), (29,30) have become instrumental resources for unraveling the influential roles of specific proteins in different cancers. (31−33) However, by determining the density and location of known mutations, their important subsets under disease-like conditions can potentially be identified. Using the clustering of mutations in protein structures (CLUMPS) method, (34) we were able to determine, in accord with our hypothesis, that the high-density distribution of WDR5 missense alterations occurs within and around the Win binding site.
The Win binding site is located within a central cavity and facilitates high-affinity interactions of WDR5 with each of the six human histone methyltransferases (HMTs; MLL1-4 and SETd1A-B), participating in the formation of corresponding six SET1 enzymatic complexes. (35,36) Rearrangements in the MLL1 gene lead to solid tumors and aggressive lymphocytic leukemias in humans. (37) Moreover, WDR5 is overexpressed under various oncogenic conditions and its upregulation catalyzes cancer development. (38−42) In recent years, the multitasking Win binding site has received a lot of interest (21,43−45) because it is a promising target for anti-cancer drug discovery. (46−53) Therefore, a quantitative understanding of WDR5’s interactions with other Win motif partners has wide-ranging fundamental significance. (50,54−57) For example, the kinetic fingerprints and affinities of the interactions of WDR5 with Win motif peptides of SET1 family members (SET1Win) have been previously reported. (35,36)
Stimulated by our finding using the CLUMPS method, (34) we explored the impact of somatic cancer mutations of WDR5 on its interactions with 14-residue SET1Win peptide ligands of the six SET1 proteins (Figure 1b,c; Table S1). The WDR5-SET1 interaction requires the precise insertion of a highly conserved Arg residue of SET1 proteins into the Win binding site (Figure 1d,e). (23) This key interaction is a prerequisite for the structural and functional integrity of the C-terminal catalytic domain of SET1 proteins. (15,22,23) SET1Win ligands recapitulate the native interactions of the six SET1 proteins with WDR5 through the Win binding site. (35,36) Therefore, we utilized the benefit of biolayer interferometry (BLI) (44,58−60) for high-throughput settings and immobilized these SET1Win ligands onto the sensor surface. In this way, we probed the real-time kinetics and dynamics of their interactions with a subset of these WDR5 mutants, whose missense alterations are located within and around the Win binding site. Remarkably, while these clustered mutations feature spatial proximity, they exhibit divergent effects on interactions with each of the six SET1Win ligands. Finally, the results of this scalable kinetic platform were confirmed by orthogonal determinations of affinity constants of these interactions using steady-state fluorescence polarization (FP) spectroscopy. (61,62)

Results and Discussion

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Use of CLUMPS for the Identification of Mutation Clustering in WDR5

We employed the CLUMPS method (34) to investigate the three-dimensional (3D) clustering of 68 WDR5 mutations identified in 68 tumors. The missense mutations were comprehensively compiled using the COSMIC database. (29,30,63) Information collected for each mutation included the residue number, the number of tumor samples, in which a certain mutation was noted, and the total number of mutations, N, in a tumor sample. N was used as a measure of the accumulation of genetic damage in a tumor sample, in which a certain mutation was sequenced. We hypothesize the lower the accumulated genetic damage in a tumor sample, the less noisy signature (e.g., populated by numerous insignificant mutations) would be in that sample. Hence, a mutation selected from a subset of mutations of that tumor sample exhibits an increased likelihood of being an important mutation. Four overlapping subsets of mutations were created from the total set of known mutations with the following conditions: N < 10 000, N < 5000, N < 1000, and N < 500 (Table 1). For each subset, we calculated a weighted average proximity (WAP) score and the corresponding P-value (Materials and Methods; Figure S1). We found that a subset of WDR5 mutations with a relatively low N (N < 500) is more likely to show mutation clustering because P-value was smaller than 0.03. Notably, the low-N subset also showed a substantial presence of mutations within and around the Win binding site (Tables S2–S3). Therefore, a subset of seven mutations was selected from all known WDR5 somatic cancer mutations within and around the Win binding site (Table S4). This approach allowed us to study the effects of these mutations on the kinetics and dynamics of WDR5-SET1Win interactions.
Table 1. Results of Mutation Clustering of WDR5 for Different Subsets of Na
NmWAP scoreP-value
<10 000512.2580.403
<5000330.9310.206
<1000110.0950.111
<50080.0720.025
a

WAP scores were calculated using four different subsets of mutations divided on the basis of the genetic damage, N, in their corresponding tumors. The P-values were calculated by comparison to configurations with random permutations of the distribution of mutations. 106 configurations were used for each subset. N is the total number of mutations in a given tumor sample. m is the total number of mutations that met the condition N < Nmax, where Nmax is the upper limit of the number of mutations in a given tumor sample. Nmax values are listed below for four data subsets on the first column. m was kept constant for all configurations of a subset.

Biolayer Interferometry Measurements

In this study, targeted mutations have locations either within the WDR5 cavity (F133L, S175L, S218F, and D92N) or on the external surface and near the cavity (D172A, Y260H, and P216L) (Figure 2a; Table S4). These mutants were chosen based on their proximal locations to residues deemed to play key roles in SET1Win interactions with the native WDR5 protein (Figure S2, Tables S5–S6). (22,23,35,36) BLI measurements were used to determine the association (kon) and dissociation (koff) rate constants of WDR5-SET1Win interactions. (58,59) 14-residue SET1Win peptide ligands, namely, MLL1Win, MLL2Win, MLL3Win, MLL4Win, SETd1AWin, and SETd1BWin, were biotinylated at the N terminus and amidated at the C-terminus (Table S1). A 9-residue Gly/Ser-rich peptide spacer was inserted between the biotinylated site and the SET1Win sequence to avoid any steric hindrance of WDR5-SET1Win interactions from the sensor surface. Biotinylated SET1Win peptides were then tethered to the surface of streptavidin-coated sensors. Binding interactions of WDR5 with SET1Win ligands attached to the sensor surface were monitored through changes in the optical interference pattern generated by reflected light waves at the sensor surface (Figure 2b). The association binding curves were acquired by placing the BLI sensors in distinct wells of varying WDR5 concentration. The dissociation binding curves were collected by placing the BLI sensors in wells containing WDR5-free buffer. It should be mentioned that all association and dissociation phases obeyed single-exponential fits, suggesting bimolecular association processes and unimolecular dissociation mechanisms of these binding phases, respectively.

Figure 2

Figure 2. Label-free optical BLI sensorgrams of the WDR5 mutant-MLL3Win interactions. (a) Locations of the surface and cavity WDR5 mutations are shown in blue using surface and cross-sectional views of WDR5, respectively. (b) BLI sensorgrams showing the association and dissociation phases. For each WDR5 mutation, sensors with immobilized MLL3Win ligand were immersed in buffers containing different WDR5 concentrations (listed on sensorgrams) to monitor association kinetics. Sensors were then transferred to buffer alone to monitor dissociation kinetics.

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Interestingly, we noted very weak binding interactions of all SET1Win peptides with D92N, a cavity WDR5 mutant (Figures 2a and S3). While these interactions are detectable, they cannot be accurately quantified using BLI likely due to either a very low kon or a very high koff, or both. A couple of possibilities could explain this interesting outcome. First, Asn-92 might interfere with the two hydrogen bonds between the Arg residue at the P0 position of the SET1Win ligand (Table S1) and S91, a neighboring residue of WDR5 (Table S5). Second, the positively charged guanidinium group of Arg at P0 of SET1Win might make an N–O salt bridge with the negatively charged carboxyl group of Asp-92 (Table S6). (64) In addition, Asp-92 forms a salt bridge with Lys-52 located between β strands. Therefore, the absence of Asp-92 might alter the local conformation of the binding pocket.

Surface Mutants

We then looked at surface mutants and the effects of these mutations on the WDR5-SET1Win interaction. The normalized values of kon (Figure S4), koff (Figure 3), and dissociation constant KD-BLI (Figure 4) for these WDR5 mutants are the values of these parameters of the SET1Win-WDR5 mutant pair interactions divided by those values corresponding to the SET1Win-native WDR5 pair interactions. In general, surface mutants D172A, P216L, and Y260H exhibited closely similar values of kon, koff, and KD-BLI to those obtained for the native WDR5 protein (Tables S7–S9). (44) Again, we were not able to obtain a quantifiable kon for the MLL1Win-WDR5 mutant pair interactions due to limited time resolution of BLI. Interestingly, kon followed the same trend with respect to SET1Win peptides, as established in our previous study, (44) with the lowest values for the neutrally charged MLL4Win, the highest values for the acidic SETd1AWin and SETd1BWin, and the intermediate values for the positively charged MLL2Win and MLL3Win. For example, for P216L-MLL4Win interactions, kon was (1.9 ± 0.3) × 104 M–1 s–1. Yet, for the interactions of P216 L with MLL2Win, MLL3Win, SETd1AWin, and SETd1BWinkon were (5.6 ± 0.8) × 104 M–1 s–1, (5.3 ± 0.7) × 104 M–1 s–1, (8.6 ± 0.8) × 104 M–1 s–1, and (8.0 ± 0.8) × 104 M–1 s–1, respectively. We conclude that kon (0) < kon (+1) < kon (−1) for surface mutants, where the number between parentheses is the overall charge of the SET1Win peptides (Table S1). In other words, kon (MLL1Win, MLL4Win) < kon (MLL2Win, MLL3Win) < kon (SETd1AWin, SETd1BWin) for surface mutants. This kon rule is likely determined by an asymmetric charge distribution in SET1Win with respect to the highly conserved 6-residue Win motif peptide segment (P–3 through P2). Specifically, this is because of a positive charge located on the C-terminal flanking side in P4 (MLL2Win and MLL3Win) and a negative charge located on the N-terminal flanking side in P–7 (SETd1AWin and SETd1BWin). Asp-172 is located within the A pocket of WDR5 (Figure S2). SET1Win ligands show no difference in their interactions with D172A as compared to the native WDR5 protein. We did not see any significant changes in the kon and koff for this pocket mutant. In addition, we noted a significantly weakened interaction of P216L with MLL4Win. Pro-216 is located within the B pocket.

Figure 3

Figure 3. Normalized dissociation rate constants of the WDR5 mutant-SET1Win interactions using BLI sensorgrams. The koff values for each SET1Win ligand’s interaction with mutants have been divided by the koff of that SET1Win ligand’s interaction with the native WDR5 protein. (a) MLL2Win, (b) MLL3Win, (c) MLL4Win, (d) SETd1AWin, and (e) SETd1BWin. ND stands for “Not Determined”. Using a BLI measurement, the interaction between F133L and MLL4Win was detectable, but not quantifiable.

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Figure 4

Figure 4. Normalized KD of the WDR5 mutant-SET1Win interactions using BLI sensorgrams. The KD values for each SET1Win ligand’s interaction with WDR5 mutants have been divided by the KD of that SET1Win ligand’s interaction with the native WDR5 protein. (a) MLL2Win, (b) MLL3Win, (c) MLL4Win, (d) SETd1AWin, and (e) SETd1BWin. ND stands for “Not Determined”. Using a BLI measurement, the interaction between F133L and MLL4Win was detectable, but not quantifiable.

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Cavity Mutants

In addition to D92N, we examined three WDR5 mutations within the WDR5 cavity, such as F133L, S175L, and S218F (Figure 2b). It has been previously reported that F133A significantly deteriorates the strength of the interactions of the MLL1 subunit with the WDR5-RbBP5-Ash2L subcomplex in vitro. (23) Phe-133 is a critical neighboring WDR5 residue of the evolutionarily conserved Arg at P0 of SET1Win ligands, contributing to a potentially strong cation−π interaction. Very weak interactions of F133L with MLL1Win and MLL4Win were not quantifiable using BLI. Here, F133L showed a decreased normalized kon with MLL2Win, MLL3Win, SETd1AWin, and SETd1BWin (Figure S4). As expected, F133L exhibited a noteworthy change in the koff with respect to the native WDR5 protein (Figure 3), leading to a significant increase in the KD-BLI. For MLL2Win, MLL3Win, SETd1AWin, and SETd1BWin, these increased values spanned a range between 1 and 2 orders of magnitude (Figure 4; Table S9). This outcome indirectly confirms their close similarity in sequence and interaction mechanisms with WDR5. (35,36) Their distinctions in binding affinities with respect to the other SET1Win ligands can be attributed to the interaction of their flanking sides with the WDR5 surface.
However, the most interesting mutational effect is that of S175L, which has a more divergent impact on interactions of the SET1Win peptides with respect to the native WDR5 protein. For example, S175L selectively weakens the interactions with MLL3Win, MLL4Win, and SETd1AWin, while substantially strengthening the interactions with SETd1BWin (Figures 3 and 4; Table S9). Moreover, this change is primarily associated with a change in the koff. Ser-175 is part of a cluster of neighboring residues that co-participate in an array of hydrogen bonds, π–π, cation−π, and hydrophobic interactions with the conserved Arg in P0. These include Ser-91, Phe-133, Ser-175, Ser-218, Cys-261, Phe-263, and Ile-305. (35) For example, Arg at P0 makes a water-mediated hydrogen bond with the Ser-175 backbone carbonyl group. (36)
Known SETd1B-WDR5 crystal structures suggest that replacing Ser-175 with Leu creates steric clashes that affect the structure of the B pocket (Figure S5). (35,36) Specifically, it could displace Tyr-191 and make the pocket more hydrophobic, which would explain the increased affinity with SETd1B. It is worth mentioning that SETd1B is unique because it has a Phe residue at P4 (Table S1) that inserts into the hydrophobic B-pocket, while the other B-pocket binders have a more polar residue (Lys or Tyr) in that position. Interestingly, the increased affinity is made possible through a 4-fold decrease in the dissociation rate constant with no change in the association rate constant. Given the importance of slow dissociation rates for effective therapeutics, (50) we predict molecules designed to take advantage of this interaction will improve dwell times and make more effective inhibitors. (21)
In agreement with prior crystallographic studies, (35,36) S218F exhibited weakened interactions with MLL2Win, MLL3Win, and MLL4Win. However, its interactions with SETd1AWin and SETd1BWin were closely similar to those with the native WDR5 protein (Figures 3 and 4; Table S9). This finding is in accordance with a different mechanism of binding interactions of SETd1AWin and SETd1BWin with respect to the other SET1Win ligands, likely due to an intermediate orientation of the C-terminal ends of SETd1AWin and SETd1BWin on the surface of blades 4 and 5.

Validations of BLI Data and Qualitative Comparisons between Competing Techniques

To validate the outcomes of BLI measurements, we next used steady-state FP spectroscopy as an orthogonal technique (Figure S6) to determine binding affinities, KD-FP, of the interactions of SET1Win ligands with WDR5 mutants (Figure 5; Table S10). 14-residue SET1Win peptide ligands were fluorescently labeled with Sulforhodamine B at the N terminus and amidated at the C terminus. A 3 nm long Gly/Ser-rich peptide spacer was inserted between the fluorophore site and the SET1Win sequence. Then, steady-state FP anisotropy, r, values were collected at increasing WDR5 concentrations. Dose-response FP measurements enabled determinations of the KD-FP. Remarkably, the FP experiments validate all qualitative findings using BLI. These include the confirmation of very weak binding interactions of D92N with all SET1Win ligands (Figure S7). In addition, we always found that the absolute KD values (i.e., not normalized) obeyed the following inequality: KD-BLI > KD-FP. This outcome confirms our previous results, indicating that measured interactions are stronger in unrestricted conditions than those corresponding to restrained conditions (Table S11). (44) Because these WDR5 mutants have been examined using BLI and FP, we can also compare these approaches qualitatively. (65) For example, using BLI, we can determine the kinetic fingerprint of these interactions. Yet, this cannot be inferred using steady-state FP spectroscopy. BLI is an immobilization-based technique, whereas FP is a method that probes the binding affinity in solution under unrestricted conditions. This is likely the reason why the KD-BLI is always about 1 order of magnitude greater than the KD-FP (Tables S9 and S10). In addition, FP measurements enabled us to measure some weaker interactions, which had kinetics that were too fast for the BLI time resolution (e.g., for MLL1Win). Furthermore, these approaches probe distinctive physical processes. On one hand, BLI is a real-time technique that samples both the association and dissociation phases based on alterations in the interference pattern of white light reflected on the sensor surface. On the other hand, steady-state FP is a time-independent technique that monitors changes in the rotational diffusion of a fluorescently labeled molecule upon its binding to another molecule.

Figure 5

Figure 5. Normalized KD of the WDR5 mutant-SET1Win interactions using steady-state FP spectroscopy. The KD values for each SET1Win ligand’s interaction with WDR5 mutants have been divided by the KD of that SET1Win ligand’s interaction with the native WDR5 protein. (a) MLL1Win, (b) MLL2Win, (c) MLL3Win, (d) MLL4Win, (e) SETd1AWin, and (f) SETd1BWin. For vertical bars marked by “*”, the KD of those interactions could not be determined. Those values represent the lower-limit of the KD based on the highest WDR5 mutant concentrations used in this study.

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We then calculated the ratio KD-BLI/KD-FP (Figure S8). The variability of the KD-BLI/KD-FP ratio for different interacting pairs was likely caused by two determinants: (i) the difference in mobility of each SET1Win ligand with respect to WDR5 mutants, and (ii) the distinction in the physical processes probed by the two methods. To cancel the effect of these two determinants, we calculated another dimensionless parameter, the ratio of normalized KD-BLI/normalized KD-FP, which spanned a much narrower spectrum, between 0.36 and 2.15 (Figure 6; Table S12). This finding illuminates the qualitative agreement of data resulting from BLI and FP measurements, fortifying our conclusions on the effect of introducing these missense mutations on SET1Win-WDR5 interactions. Moreover, these BLI and FP data are in accordance with a recent single-molecule study using an engineered protein nanopore, (45) which indicated unaffected D172A-MLL4Win interactions and weak D92N-MLL4Win interactions with respect to those of the native WDR5 protein. Taken together, these findings demonstrate the critical role of a negative charge located within the acidic WDR5 cavity for the strength of WDR5-SET1Win interactions.

Figure 6

Figure 6. Quantitative comparison between affinity data resulting from BLI and FP measurements. (a) 3D graph of the ratio of the normalized KD-BLI to the normalized KD-FP. (b) Two-dimensional heat map of the ratio of the normalized KD-BLI to the normalized KD-FP. Normalized KD values are the KD measured for a specific WDR5 mutant-SET1Win interaction pair divided by the KD value corresponding to the native WDR5 protein.

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Implications of Win Binding Site Mutants

Alterations in MLL/SET1 family enzymes are associated with genome-wide aberrations in the patterns of H3K4 methylation, which are linked to abnormal transcriptional programs that promote malignancy. (66−69) WDR5 is a key component of MLL/SET1 family complexes and mutations in the Win binding site disrupt MLL complex assembly and enzymatic activity. (22,23,35,70) Knockdown of WDR5 also alters global H3K4 methylation patterns resulting in developmental defects in vertebrates. (71) Recent exome sequencing projects have uncovered numerous missense mutations in WDR5 and other subunits of MLL/SET1 family complexes, suggesting that loss-of-function may underly malignancy. Cell- and animal-based studies are needed to determine whether these mutations result in disease. Yet, prioritizing such costly experiments would benefit greatly from reliable preliminary information that is beyond the capabilities of most current computational methods. (72) Thus, experimental studies to delineate how cancer-associated mutations impact the biochemical function of WDR5 remain an attractive approach to prioritize mutations for further study.
While the impact of these WDR5 mutations on WDR5-SET1Win interactions is readily distinguishable, their effect on the overall assembly of the SET1 complexes and their functional features is a bit more nuanced. Given our understanding of SET1 family complex behavior, (16,48) we can say that these inspected WDR5 mutations have a divergent impact. The absence of WDR5 (16) and/or the inhibition of SET1Win-WDR5 interactions (48) downregulates the H3K4 di-methylation function of MLL1 and SETd1A, while this upregulates the H3K4 mono-methylation function of MLL3. (16,70) Therefore, mutations that significantly disrupt SET1Win-WDR5 interactions are likely to have similar effects. Consequently, F133L and D92N should disrupt the di-methylation by MLL1 and SETd1A. Furthermore, the mono-methylation of MLL3 would be upregulated in the case of F133L, S218F, S175L, and D92N. Our results show that even within the Win binding site, given their effect on SET1win-WDR5 interactions, cavity mutations are more likely to be driver mutations instead of passenger mutations. This holds especially true for D92N, F133L, and S175L. For example, Ali and co-workers (2014) found that F133L disrupts the mitotic progression in the cell cycle process. (73)
Information that concerns the KD values of mutations within the B pocket is critical for future drug development. Precision medicine depends on understanding the unique biophysical impacts of each missense mutation on the structure and function of putative oncogene proteins. Our data suggest that this knowledge would help researchers in deciding which inhibitors to use as potential therapeutic approaches. For example, this work indicates that individuals harboring a breast cancer S175L mutation in WDR5 are more likely to respond to inhibitors targeting the hydrophobic interactions in the B pocket than other inhibitors. Furthermore, the unique impact of S175L in SETd1B also implies that those cancers are due to perturbations in the SETd1B-catalyzed H3K4 methylation pathway. This type of information would greatly enhance our ability to prioritize cellular and animal-based follow-up studies that can address more specific hypotheses.

Concluding Remarks

In this study, we evaluated key somatic cancer mutations of WDR5. Specifically, we used the CLUMPS approach to identify that these mutations accumulate within the Win binding site and extracted a representative subset of WDR5 mutants for determining the real-time kinetics of their interactions using high-throughput biosensing techniques. Our work shows that the total number of mutations in a tumor sample can be used as a parameter to filter out mutations. Furthermore, we noted that the Win site shows a substantial presence of low-N mutations, while the WBM site shows none. This helped us to exclusively focus on Win binding site mutants for further biophysical measurements. Therefore, we explored the effect of mutations in this binding site by evaluating a detailed kinetic fingerprint of corresponding interactions with various SET1Win ligands. We provide experimental evidence for influential roles of the residues within the WDR5 cavity on the strength of these interactions. Steady-state FP spectroscopy measurements also confirmed outcomes resulting from BLI experiments. Finally, the interactions of WDR5 cavity mutants depended on the nature of the SET1Win ligands. These divergent effects have distinctive impacts on H3K4 methylation, and therefore for the downstream expression of genes. This is a finding that can impact future strategies for the design, development, and optimization of inhibitors that are aimed at targeting the multitasking high-affinity Win binding site under oncogenic conditions. In the future, it would be interesting to examine the effect of the Win binding site cancer mutations on the kinetics and strength of the interactions of WDR5 with other protein partners and Win motif ligands.

Materials and Methods

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Clustering of Mutations in Protein Structures

This approach was used as previously reported. (34) WDR5 mutations were obtained using the COSMIC database (29,30,63) and available X-ray crystallographic information (PDB code 4ERY). (35) A WAP score was generated for the distribution of mutations using the following equation
(1)
where i and j iterated over all residues of WDR5. Here, di,j is the Euclidean distance between residues i and j in Angstroms, and r denotes the distance threshold set to a constant value of 6 Å. (34)si represents the normalized number of samples, in which the residue i was mutated. This parameter is given by
(2)
where ni represents the number of samples, in which the residue i was mutated. The P-value was determined by calculating the WAP score for 106 random distributions of mutations, and then by comparing it with that value of the known distribution.

Protein Expression and Purification

All expression plasmids were synthesized, codon optimized, and sequence verified by GenScript. Human WDR5 (UniProtKB─P61964; WDR5_HUMAN) and its mutants were expressed and purified as described previously. (22,23,44)

Peptide Synthesis, Labeling, Purification, and Analysis

For BLI measurements, 14-residue SET1Win peptide ligands were biotinylated at their N terminus and amidated at their C terminus. They were synthesized and purified to ≥95% purity by GenScript. Purity confirmation, amino acid analysis, and solubility testing were conducted and provided by GenScript. For steady-state FP measurements, details on peptide synthesis, labeling, purification, and analysis were previously published. (44) In brief, peptides were synthesized using a Biotage Syro I peptide synthesizer (Biotage). Then, the peptides were purified using reversed-phase chromatography in two steps: (i) flash chromatography using a Biotage Isolera One (Biotage), and (ii) semi-preparative HPLC using a Waters 2695 separations module equipped with a Waters 2996 photodiode array detector. Fluorophore-containing peptide fractions were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy for the identity and purity tests.

Biolayer Interferometry

Octet RED384 (FortéBio) was employed for the BLI studies. (44,58−60) The assay buffer contained 150 mM NaCl, 20 mM Tris–HCl, 1 mM TCEP, 1 mg/mL bovine serum albumin, pH 7.5. Streptavidin-coated biosensors were incubated for 15 min with 5 nM biotin-tagged SET1Win peptide to specifically immobilize an optimal level of peptide. Sensors were then rinsed briefly in an assay buffer to remove unbound peptides. Next, sensors were exposed to threefold serial dilutions of WDR5 for the association process. The dissociation phase was initiated by transferring the BLI sensors into WDR5-free buffer. For all WDR5 concentrations, binding curves were recorded by subtracting the baseline and the drift in the sensorgrams acquired with unloaded sensors. These BLI measurements were conducted at 24 °C. For various WDR5 concentrations, [C], the association phases were fitted using the following equation (74)
(3)
where Y0 and Y∞ are the response signals at the initial time and infinity, respectively. t is the cumulative time of the association phase, whereas kobs denotes the apparent first-order reaction rate constant of the association phase. The dissociation phases were fitted using the following equation
(4)
where Y0 and Y∞ denote the responses at the initial time and infinity, respectively. koff is the dissociation rate constant. The association rate constant, kon, was determined using the slope of the linear curve (62,75)
(5)
Using several WDR5 concentrations, we also conducted global fittings, which provided the corresponding kon and koff values. The equilibrium dissociation constants, KD, were indirectly determined using the kon and koff values (KD = koff/kon). In each case, three independent BLI recordings were acquired for further determinations of the kinetics and dynamics of WDR5-SET1Win interactions.

Steady-State Fluorescence Polarization (Steady-State FP) Measurements

Steady-state FP recordings were performed using a SpectraMax i3x plate reader (Molecular Devices). (61,76) All steady-state FP measurements were conducted using a buffer that contained 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM TCEP, 0.005% Tween 20, and 96-well black untreated polystyrene microplates (Corning Inc). Other details of steady-state FP measurements were previously reported. (44) 100 μL of each 20 nM labeled SET1Win peptide was added to individual wells at a final concentration of 10 nM. The steady-state FP anisotropy was measured on the plates after a 1 h incubation at room temperature in the dark. WDR5-dependent dose-response data were averaged and then fitted using a four-parameter logistic function to acquire the binding affinity (KD) for each interaction pairs.

Molecular Graphics

In this study, molecular graphics was conducted using the PyMOL Molecular Graphics System (Version 2.4.0 Schrödinger, LLC).

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschembio.2c00273.

  • Sequence of SET1Win peptide ligands; location of somatic cancer mutations from tumor samples with N < 500; results of mutation clustering for different mutation subsets; list of tumor locations associated with missense WDR5 mutations within and around the Win binding site; list of noncovalent bonds at the WDR5-SET1Win interface; location of surface WDR5 mutations within the A and B pockets; BLI measurements of very weak interactions; kinetic rate constants of association and dissociation of WDR5 mutants with SET1Win ligands; equilibrium dissociation constants of WDR5 mutants with SET1Win ligands; structural information on the effect of the S175L mutation; steady-state FP spectroscopy curves for the interactions of WDR5 mutants with SET1win ligands; equilibrium dissociation constants of WDR5 mutants with SET1Win ligands using steady-state FP spectroscopy; quantitative comparisons of affinity data acquired with BLI and FP; and supporting references (PDF)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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  • Corresponding Authors
    • Michael S. Cosgrove - Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, 4249 Weiskotten Hall, 766 Irving Avenue, Syracuse, New York 13210, United States;  Email: [email protected]
    • Liviu Movileanu - Department of Physics, Syracuse University, 201 Physics Building, Syracuse, New York 13244-1130, United States;  Department of Biomedical and Chemical Engineering, Syracuse University, 329 Link Hall, Syracuse, New York 13244, United States;  The BioInspired Institute, Syracuse University, Syracuse, New York 13244, United States;  Orcidhttps://orcid.org/0000-0002-2525-3341;  Email: [email protected]
  • Authors
    • Ali Imran - Department of Physics, Syracuse University, 201 Physics Building, Syracuse, New York 13244-1130, United States
    • Brandon S. Moyer - Ichor Life Sciences, Inc., 2651 US Route 11, LaFayette, New York 13084, United States
    • Dan Kalina - Ichor Life Sciences, Inc., 2651 US Route 11, LaFayette, New York 13084, United States;  Department of Chemistry, State University of New York College of Environmental Science and Forestry, 1 Forestry Dr., Syracuse, New York 13210, United States
    • Thomas M. Duncan - Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, 4249 Weiskotten Hall, 766 Irving Avenue, Syracuse, New York 13210, United States
    • Kelsey J. Moody - Department of Physics, Syracuse University, 201 Physics Building, Syracuse, New York 13244-1130, United States;  Ichor Life Sciences, Inc., 2651 US Route 11, LaFayette, New York 13084, United States;  Department of Chemistry, State University of New York College of Environmental Science and Forestry, 1 Forestry Dr., Syracuse, New York 13210, United States;  Lewis School of Health Sciences, Clarkson University, 8 Clarkson Avenue, Potsdam, New York 13699, United States
    • Aaron J. Wolfe - Department of Physics, Syracuse University, 201 Physics Building, Syracuse, New York 13244-1130, United States;  Ichor Life Sciences, Inc., 2651 US Route 11, LaFayette, New York 13084, United States;  Department of Chemistry, State University of New York College of Environmental Science and Forestry, 1 Forestry Dr., Syracuse, New York 13210, United States;  Lewis School of Health Sciences, Clarkson University, 8 Clarkson Avenue, Potsdam, New York 13699, United States
  • Author Contributions

    A.I., D.K., M.S.C., and L.M. designed research. A.I., B.M., and D.K. performed research. A.I. analyzed data and worked on data interpretations. T.M.D., K.J.M., A.J.W., M.S.C., and L.M. provided reagents, supervision, and funding. A.I., M.S.C., and L.M. wrote the paper.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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We are grateful to our colleagues in the Movileanu and Cosgrove laboratories and at Ichor Life Sciences Laboratories for their comments on the paper and stimulating discussions as well as for their assistance during the early stage of this project. This work was supported by the National Cancer Institute of the U.S. National Institutes of Health grant R01 CA140522 (to M.S.C.) and by the National Institute of General Medical Sciences of the U.S. National Institutes of Health grant R01 GM129429 (to L.M.).

References

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    Patel, Anamika; Dharmarajan, Venkatasubramanian; Vought, Valarie E.; Cosgrove, Michael S.
    Journal of Biological Chemistry (2009), 284 (36), 24242-24256CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Transcription in eukaryotic genomes depends on enzymes that regulate the degree of histone H3 lysine 4 (H3K4) methylation. The mixed lineage leukemia protein-1 (MLL1) is a member of the SET1 family of H3K4 methyltransferases and is frequently rearranged in acute leukemias. Despite sequence comparisons that predict that SET1 family enzymes should only monomethylate their substrates, mono-, di-, and trimethylation of H3K4 has been attributed to SET1 family complexes in vivo and in vitro. To better understand this paradox, we have biochem. reconstituted and characterized a five-component 200-kDa MLL1 core complex contg. human MLL1, WDR5, RbBP5, Ash2L, and DPY-30. We demonstrate that the isolated MLL1 SET domain is a slow monomethyltransferase and that tyrosine 3942 of MLL1 prevents di- and trimethylation of H3K4. In contrast, a complex contg. the MLL1 SET domain, WDR5, RbBP5, Ash2L, and DPY-30, displays a marked ∼600-fold increase in enzymic activity but only to the di-Me form of H3K4. Single turnover kinetic expts. reveal that the reaction leading to H3K4 dimethylation involves the transient accumulation of a monomethylated species, suggesting that the MLL1 core complex uses a non-processive mechanism to catalyze multiple lysine methylation. We have also discovered that the non-SET domain components of the MLL1 core complex possess a previously unrecognized methyltransferase activity that catalyzes H3K4 dimethylation within the MLL1 core complex. Our results suggest that the mechanism of multiple lysine methylation by the MLL1 core complex involves the sequential addn. of two Me groups at two distinct active sites within the complex.
  16. 16
    Shinsky, S. A.; Monteith, K. E.; Viggiano, S.; Cosgrove, M. S. Biochemical reconstitution and phylogenetic comparison of human SET1 family core complexes involved in histone methylation. J. Biol. Chem. 2015, 290, 6361– 6375,  DOI: 10.1074/jbc.m114.627646
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    Biochemical Reconstitution and Phylogenetic Comparison of Human SET1 Family Core Complexes Involved in Histone Methylation
    Shinsky, Stephen A.; Monteith, Kelsey E.; Viggiano, Susan; Cosgrove, Michael S.
    Journal of Biological Chemistry (2015), 290 (10), 6361-6375CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Mixed lineage leukemia protein-1 (MLL1) is a member of the SET1 family of histone H3 lysine 4 (H3K4) methyltransferases that are required for metazoan development. MLL1 is the best characterized human SET1 family member, which includes MLL1-4 and SETd1A/B. MLL1 assembles with WDR5, RBBP5, ASH2L, DPY-30 (WRAD) to form the MLL1 core complex, which is required for H3K4 dimethylation and transcriptional activation. Because all SET1 family proteins interact with WRAD in vivo, it is hypothesized they are regulated by similar mechanisms. However, recent evidence suggests differences among family members that may reflect unique regulatory inputs in the cell. Missing is an understanding of the intrinsic enzymic activities of different SET1 family complexes under std. conditions. In this investigation, we reconstituted each human SET1 family core complex and compared subunit assembly and enzymic activities. We found that in the absence of WRAD, all but one SET domain catalyzes at least weak H3K4 monomethylation. In the presence of WRAD, all SET1 family members showed stimulated monomethyltransferase activity but differed in their di- and trimethylation activities. We found that these differences are correlated with evolutionary lineage, suggesting these enzyme complexes have evolved to accomplish unique tasks within metazoan genomes. To understand the structural basis for these differences, we employed a "phylogenetic scanning mutagenesis" assay and identified a cluster of amino acid substitutions that confer a WRAD-dependent gain-of-function dimethylation activity on complexes assembled with the MLL3 or Drosophila trithorax proteins. These results form the basis for understanding how WRAD differentially regulates SET1 family complexes in vivo.
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    Thomas, L. R.; Wang, Q.; Grieb, B. C.; Phan, J.; Foshage, A. M.; Sun, Q.; Olejniczak, E. T.; Clark, T.; Dey, S.; Lorey, S.; Alicie, B.; Howard, G. C.; Cawthon, B.; Ess, K. C.; Eischen, C. M.; Zhao, Z.; Fesik, S. W.; Tansey, W. P. Interaction with WDR5 promotes target gene recognition and tumorigenesis by MYC. Mol. Cell. Biochem. 2015, 58, 440– 452,  DOI: 10.1016/j.molcel.2015.02.028
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    Thomas, L. R.; Adams, C. M.; Wang, J.; Weissmiller, A. M.; Creighton, J.; Lorey, S. L.; Liu, Q.; Fesik, S. W.; Eischen, C. M.; Tansey, W. P. Interaction of the oncoprotein transcription factor MYC with its chromatin cofactor WDR5 is essential for tumor maintenance. Proc. Natl. Acad. Sci. U.S.A. 2019, 116, 25260– 25268,  DOI: 10.1073/pnas.1910391116
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    Interaction of the oncoprotein transcription factor MYC with its chromatin cofactor WDR5 is essential for tumor maintenance
    Thomas, Lance R.; Adams, Clare M.; Wang, Jing; Weissmiller, April M.; Creighton, Joy; Lorey, Shelly L.; Liu, Qi; Fesik, Stephen W.; Eischen, Christine M.; Tansey, William P.
    Proceedings of the National Academy of Sciences of the United States of America (2019), 116 (50), 25260-25268CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The oncoprotein transcription factor MYC is overexpressed in the majority of cancers. Key to its oncogenic activity is the ability of MYC to regulate gene expression patterns that drive and maintain the malignant state. MYC is also considered a validated anticancer target, but efforts to pharmacol. inhibit MYC have failed. The dependence of MYC on cofactors creates opportunities for therapeutic intervention, but for any cofactor this requires structural understanding of how the cofactor interacts with MYC, knowledge of the role it plays in MYC function, and demonstration that disrupting the cofactor interaction will cause existing cancers to regress. One cofactor for which structural information is available is WDR5, which interacts with MYC to facilitate its recruitment to chromatin. To explore whether disruption of the MY-WDR5 interaction could potentially become a viable anticancer strategy, we developed a Burkitt's lymphoma system that allows replacement of wild-type MYC for mutants that are defective for WDR5 binding or all known nuclear MYC functions. Using this system, we show that WDR5 recruits MYC to chromatin to control the expression of genes linked to biomass accumulation. We further show that disrupting the MYC-WDR5 interaction within the context of an existing cancer promotes rapid and comprehensive tumor regression in vivo. These observations connect WDR5 to a core tumorigenic function of MYC and establish that, if a therapeutic window can be established, MYC-WDR5 inhibitors could be developed as anticancer agents.
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    Thomas, L. R.; Adams, C. M.; Fesik, S. W.; Eischen, C. M.; Tansey, W. P. Targeting MYC through WDR5. Mol. Cell. Oncol. 2020, 7, 1709388,  DOI: 10.1080/23723556.2019.1709388
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    Targeting MYC through WDR5
    Thomas Lance R; Tansey William P; Adams Clare M; Eischen Christine M; Fesik Stephen W; Tansey William P
    Molecular & cellular oncology (2020), 7 (2), 1709388 ISSN:2372-3556.
    The oncoprotein transcription factor MYC is overexpressed in most cancers and is responsible for hundreds of thousands of cancer deaths worldwide every year. MYC is also a highly validated - but currently undruggable - anti-cancer target. We recently showed that breaking the interaction of MYC with its chromatin co-factor WD repeat-containing protein 5 (WDR5) promotes tumor regression in mouse xenografts, laying the foundation for a new strategy to inhibit MYC in the clinic.
  20. 20
    Thomas, L. R.; Foshage, A. M.; Weissmiller, A. M.; Tansey, W. P. The MYC-WDR5 Nexus and Cancer. Cancer Res. 2015, 75, 4012– 4015,  DOI: 10.1158/0008-5472.can-15-1216
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    The MYC-WDR5 Nexus and Cancer
    Thomas, Lance R.; Foshage, Audra M.; Weissmiller, April M.; Tansey, William P.
    Cancer Research (2015), 75 (19), 4012-4015CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)
    The MYC oncogenes encode a family of transcription factors that feature prominently in cancer. MYC proteins are overexpressed or deregulated in a majority of malignancies and drive tumorigenesis by inducing widespread transcriptional reprogramming that promotes cell proliferation, metab., and genomic instability. The ability of MYC to regulate transcription depends on its dimerization with MAX, which creates a DNA-binding domain that recognizes specific sequences in the regulatory elements of MYC target genes. Recently, we discovered that recognition of target genes by MYC also depends on its interaction with WDR5, a WD40-repeat protein that exists as part of several chromatin-regulatory complexes. Here, we discuss how interaction of MYC with WDR5 could create an avidity-based chromatin recognition mechanism that allows MYC to select its target genes in response to both genetic and epigenetic determinants. We rationalize how the MYC-WDR5 interaction provides plasticity in target gene selection by MYC and speculate on the biochem. and genomic contexts in which this interaction occurs. Finally, we discuss how properties of the MYC-WDR5 interface make it an attractive point for discovery of small-mol. inhibitors of MYC function in cancer cells.
  21. 21
    Guarnaccia, A. D.; Rose, K. L.; Wang, J.; Zhao, B.; Popay, T. M.; Wang, C. E.; Guerrazzi, K.; Hill, S.; Woodley, C. M.; Hansen, T. J.; Lorey, S. L.; Shaw, J. G.; Payne, W. G.; Weissmiller, A. M.; Olejniczak, E. T.; Fesik, S. W.; Liu, Q.; Tansey, W. P. Impact of WIN site inhibitor on the WDR5 interactome. Cell Rep. 2021, 34, 108636,  DOI: 10.1016/j.celrep.2020.108636
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    Impact of WIN site inhibitor on the WDR5 interactome
    Guarnaccia, Alissa D.; Rose, Kristie L.; Wang, Jing; Zhao, Bin; Popay, Tessa M.; Wang, Christina E.; Guerrazzi, Kiana; Hill, Salisha; Woodley, Chase M.; Hansen, Tyler J.; Lorey, Shelly L.; Shaw, J. Grace; Payne, William G.; Weissmiller, April M.; Olejniczak, Edward T.; Fesik, Stephen W.; Liu, Qi; Tansey, William P.
    Cell Reports (2021), 34 (3), 108636CODEN: CREED8; ISSN:2211-1247. (Cell Press)
    The chromatin-assocd. protein WDR5 is a promising pharmacol. target in cancer, with most drug discovery efforts directed against an arginine-binding cavity in WDR5 called the WIN site. Despite a clear expectation that WIN site inhibitors will alter the repertoire of WDR5 interaction partners, their impact on the WDR5 interactome remains unknown. Here, we use quant. proteomics to delineate how the WDR5 interactome is changed by WIN site inhibition. We show that the WIN site inhibitor alters the interaction of WDR5 with dozens of proteins, including those linked to phosphatidylinositol 3-kinase (PI3K) signaling. As proof of concept, we demonstrate that the master kinase PDPK1 is a bona fide high-affinity WIN site binding protein that engages WDR5 to modulate transcription of genes expressed in the G2 phase of the cell cycle. This dataset expands our understanding of WDR5 and serves as a resource for deciphering the action of WIN site inhibitors.
  22. 22
    Patel, A.; Dharmarajan, V.; Cosgrove, M. S. Structure of WDR5 bound to mixed lineage leukemia protein-1 peptide. J. Biol. Chem. 2008, 283, 32158– 32161,  DOI: 10.1074/jbc.c800164200
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    Structure of WDR5 Bound to Mixed Lineage Leukemia Protein-1 Peptide
    Patel, Anamika; Dharmarajan, Venkatasubramanian; Cosgrove, Michael S.
    Journal of Biological Chemistry (2008), 283 (47), 32158-32161CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    The mixed lineage leukemia protein-1 (MLL1) catalyzes histone H3 lysine 4 methylation and is regulated by interaction with WDR5 (WD-repeat protein-5), RbBP5 (retinoblastoma-binding protein-5), and the Ash2L (absent, small, homeotic disks-2-like) oncoprotein. In the accompanying investigation, we describe the identification of a conserved arginine contg. motif, called the "Win" or WDR5 interaction motif, that is essential for the assembly and H3K4 dimethylation activity of the MLL1 core complex. Here we present a 1.7-Å crystal structure of WDR5 bound to a peptide derived from the MLL1 Win motif. Our results show that Arg-3765 of MLL1 is bound in the same arginine binding pocket on WDR5 that was previously suggested to bind histone H3. Thermodn. binding expts. show that the MLL1 Win peptide is preferentially recognized by WDR5. These results are consistent with a model in which WDR5 recognizes Arg-3765 of MLL1, which is essential for the assembly and enzymic activity of the MLL1 core complex.
  23. 23
    Patel, A.; Vought, V. E.; Dharmarajan, V.; Cosgrove, M. S. A conserved arginine-containing motif crucial for the assembly and enzymatic activity of the mixed lineage leukemia protein-1 core complex. J. Biol. Chem. 2008, 283, 32162– 32175,  DOI: 10.1074/jbc.m806317200
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    23
    A Conserved Arginine-containing Motif Crucial for the Assembly and Enzymatic Activity of the Mixed Lineage Leukemia Protein-1 Core Complex
    Patel, Anamika; Vought, Valarie E.; Dharmarajan, Venkatasubramanian; Cosgrove, Michael S.
    Journal of Biological Chemistry (2008), 283 (47), 32162-32175CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    The mixed lineage leukemia protein-1 (MLL1) belongs to the SET1 family of histone H3 lysine 4 methyltransferases. Recent studies indicate that the catalytic subunits of SET1 family members are regulated by interaction with a conserved core group of proteins that include the WD repeat protein-5 (WDR5), retinoblastoma-binding protein-5 (RbBP5), and the absent small homeotic-2-like protein (Ash2L). It has been suggested that WDR5 functions to bridge the interactions between the catalytic and regulatory subunits of SET1 family complexes. However, the mol. details of these interactions are unknown. To gain insight into the interactions among these proteins, we have detd. the biophys. basis for the interaction between the human WDR5 and MLL1. Our studies reveal that WDR5 preferentially recognizes a previously unidentified and conserved arginine-contg. motif, called the "Win" or WDR5 interaction motif, which is located in the N-SET region of MLL1 and other SET1 family members. Surprisingly, our structural and functional studies show that WDR5 recognizes arginine 3765 of the MLL1 Win motif using the same arginine-binding pocket on WDR5 that was previously shown to bind histone H3. We demonstrate that WDR5's recognition of arginine 3765 of MLL1 is essential for the assembly and enzymic activity of the MLL1 core complex in vitro.
  24. 24
    Song, J.-J.; Kingston, R. E. WDR5 interacts with mixed lineage leukemia (MLL) protein via the histone H3-binding pocket. J. Biol. Chem. 2008, 283, 35258– 35264,  DOI: 10.1074/jbc.m806900200
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    WDR5 Interacts with Mixed Lineage Leukemia (MLL) Protein via the Histone H3-binding Pocket
    Song, Ji-Joon; Kingston, Robert E.
    Journal of Biological Chemistry (2008), 283 (50), 35258-35264CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    WDR5 is a component of the mixed lineage leukemia (MLL) complex, which methylates lysine 4 of histone H3, and was identified as a methylated Lys-4 histone H3-binding protein. Here, we present a crystal structure of WDR5 bound to an MLL peptide. Surprisingly, we find that WDR5 utilizes the same pocket shown to bind histone H3 for this MLL interaction. Furthermore, the WDR5-MLL interaction is disrupted preferentially by mono- and di-methylated Lys-4 histone H3 over unmodified and tri-methylated Lys-4 histone H3. These data implicate a delicate interplay between the effector, WDR5, the catalytic subunit, MLL, and the substrate, histone H3, of the MLL complex. We suggest that the activity of the MLL complex might be regulated through this interplay.
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    Odho, Z.; Southall, S. M.; Wilson, J. R. Characterization of a novel WDR5-binding site that recruits RbBP5 through a conserved motif to enhance methylation of histone H3 lysine 4 by mixed lineage leukemia protein-1. J. Biol. Chem. 2010, 285, 32967– 32976,  DOI: 10.1074/jbc.m110.159921
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    Characterization of a novel WDR5-binding site that recruits RbBP5 through a conserved motif to enhance methylation of histone H3 lysine 4 by mixed lineage leukemia protein-1
    Odho, Zain; Southall, Stacey M.; Wilson, Jon R.
    Journal of Biological Chemistry (2010), 285 (43), 32967-32976CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Histone modification is well established as a fundamental mechanism driving the regulation of transcription, replication, and DNA repair through the control of chromatin structure. Likewise, it is apparent that incorrect targeting of histone modifications contributes to misregulated gene expression and hence to developmental disorders and diseases of genomic instability such as cancer. The KMT2 family of SET domain methyltransferases, typified by mixed lineage leukemia protein-1 (MLL1), is responsible for histone H3 lysine 4 methylation, a marker of active genes. To ensure that this modification is correctly targeted, a multiprotein complex assocs. with the methyltransferase and directs activity. We have identified a novel interaction site on the core complex protein WD repeat protein-5 (WDR5), and we mapped the complementary site on its partner retinoblastoma-binding protein-5 (RbBP5). We have characterized this interaction by x-ray crystallog. and show how it is fundamental to the assembly of the complex and to the regulation of methyltransferase activity. We show which region of RbBP5 contributes directly to mixed lineage leukemia activation, and we combine our structural and biochem. data to produce a model to show how WDR5 and RbBP5 act cooperatively to stimulate activity.
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    Chacón Simon, S.; Wang, F.; Thomas, L. R.; Phan, J.; Zhao, B.; Olejniczak, E. T.; Macdonald, J. D.; Shaw, J. G.; Schlund, C.; Payne, W.; Creighton, J.; Stauffer, S. R.; Waterson, A. G.; Tansey, W. P.; Fesik, S. W. Discovery of WD Repeat-Containing Protein 5 (WDR5)-MYC Inhibitors Using Fragment-Based Methods and Structure-Based Design. J. Med. Chem. 2020, 63, 4315– 4333,  DOI: 10.1021/acs.jmedchem.0c00224
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    Discovery of WD Repeat-Containing Protein 5 (WDR5)-MYC Inhibitors Using Fragment-Based Methods and Structure-Based Design
    Chacon Simon, Selena; Wang, Feng; Thomas, Lance R.; Phan, Jason; Zhao, Bin; Olejniczak, Edward T.; MacDonald, Jonathan D.; Shaw, J. Grace; Schlund, Caden; Payne, William; Creighton, Joy; Stauffer, Shaun R.; Waterson, Alex G.; Tansey, William P.; Fesik, Stephen W.
    Journal of Medicinal Chemistry (2020), 63 (8), 4315-4333CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    The frequent deregulation of MYC and its elevated expression via multiple mechanisms drives cells to a tumorigenic state. Indeed, MYC is overexpressed in up to ~ 50% of human cancers and is considered a highly validated anticancer target. Recently, we discovered that WD repeat-contg. protein 5 (WDR5) binds to MYC and is a crit. cofactor required for the recruitment of MYC to its target genes and reported the first small mol. inhibitors of the WDR5-MYC interaction using structure-based design. These compds. display high binding affinity, but have poor physicochem. properties and are hence not suitable for in vivo studies. Herein, we conducted an NMR-based fragment screening to identify addnl. chem. matter and, using a structure-based approach, we merged a fragment hit with the previously reported sulfonamide series. Compds. in this series can disrupt the WDR5-MYC interaction in cells, and as a consequence, we obsd. a redn. of MYC localization to chromatin.
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    Gao, J.; Chang, M. T.; Johnsen, H. C.; Gao, S. P.; Sylvester, B. E.; Sumer, S. O.; Zhang, H.; Solit, D. B.; Taylor, B. S.; Schultz, N.; Sander, C. 3D clusters of somatic mutations in cancer reveal numerous rare mutations as functional targets. Genome Med. 2017, 9, 4,  DOI: 10.1186/s13073-016-0393-x
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    3D clusters of somatic mutations in cancer reveal numerous rare mutations as functional targets
    Gao, Jianjiong; Chang, Matthew T.; Johnsen, Hannah C.; Gao, Sizhi Paul; Sylvester, Brooke E.; Sumer, Selcuk Onur; Zhang, Hongxin; Solit, David B.; Taylor, Barry S.; Schultz, Nikolaus; Sander, Chris
    Genome Medicine (2017), 9 (), 4/1-4/13CODEN: GMEECG; ISSN:1756-994X. (BioMed Central Ltd.)
    Many mutations in cancer are of unknown functional significance. Std. methods use statistically significant recurrence of mutations in tumor samples as an indicator of functional impact. We extend such analyses into the long tail of rare mutations by considering recurrence of mutations in clusters of spatially close residues in protein structures. Analyzing 10,000 tumor exomes, we identify more than 3000 rarely mutated residues in proteins as potentially functional and exptl. validate several in RAC1 and MAP2K1. These potential driver mutations (web resources: 3dhotspots.org and cBioPortal.org) can extend the scope of genomically informed clin. trials and of personalized choice of therapy.
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    Acuner, S. E.; Sumbul, F.; Torun, H.; Haliloglu, T. Oncogenic mutations on Rac1 affect global intrinsic dynamics underlying GTP and PAK1 binding. Biophys. J. 2021, 120, 866– 876,  DOI: 10.1016/j.bpj.2021.01.016
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    Oncogenic mutations on Rac1 affect global intrinsic dynamics underlying GTP and PAK1 binding
    Acuner, Saliha Ece; Sumbul, Fidan; Torun, Hamdi; Haliloglu, Turkan
    Biophysical Journal (2021), 120 (5), 866-876CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
    Rac1 is a small member of the Rho GTPase family. One of the most important downstream effectors of Rac1 is a serine/threonine kinase, p21-activated kinase 1 (PAK1). Mutational activation of PAK1 by Rac1 has oncogenic signaling effects. Here, although we focus on Rac1-PAK1 interaction by at.-force-microscopy-based single-mol. force spectroscopy expts., we explore the effect of active mutations on the intrinsic dynamics and binding interactions of Rac1 by Gaussian network model anal. and mol. dynamics simulations. We observe that Rac1 oncogenic mutations are at the hinges of three global modes of motion, suggesting the mech. changes as potential markers of oncogenicity. Indeed, the dissocn. of wild-type Rac1-PAK1 complex shows two distinct unbinding dynamic states that are reduced to one with constitutively active Q61L and oncogenic Y72C mutant Rac1, as revealed by single-mol. force spectroscopy expts. Q61L and Y72C mutations change the mechanics of the Rac1-PAK1 complex by increasing the elasticity of the protein and slowing down the transition to the unbound state. On the other hand, Rac1's intrinsic dynamics reveal more flexible GTP and PAK1-binding residues on switches I and II with Q61L, Y72C, oncogenic P29S and Q61R, and neg. T17N mutations. The cooperativity in the fluctuations of GTP-binding sites around the p-loop and switch I decreases in all mutants, mostly in Q61L, whereas some PAK1-binding residues display enhanced coupling with GTP-binding sites in Q61L and Y72C and within each other in P29S. The predicted binding free energies of the modeled Rac1-PAK1 complexes show that the change in the dynamic behavior likely means a more favorable PAK1 interaction. Overall, these findings suggest that the active mutations affect intrinsic functional dynamic events and alter the mechanics underlying the binding of Rac1 to GTP and upstream and downstream partners including PAK1.
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    Forbes, S. A.; Beare, D.; Bindal, N.; Bamford, S.; Ward, S.; Cole, C. G.; Jia, M.; Kok, C.; Boutselakis, H.; De, T.; Sondka, Z.; Ponting, L.; Stefancsik, R.; Harsha, B.; Tate, J.; Dawson, E.; Thompson, S.; Jubb, H.; Campbell, P. J. COSMIC: High-Resolution Cancer Genetics Using the Catalogue of Somatic Mutations in Cancer. Curr. Protoc. Hum. Genet. 2016, 91, 10– 37,  DOI: 10.1002/cphg.21
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    COSMIC: High-Resolution Cancer Genetics Using the Catalogue of Somatic Mutations in Cancer
    Forbes S A; Beare D; Bindal N; Bamford S; Ward S; Cole C G; Jia M; Kok C; Boutselakis H; De T; Sondka Z; Ponting L; Stefancsik R; Harsha B; Tate J; Dawson E; Thompson S; Jubb H; Campbell P J
    Current protocols in human genetics (2016), 91 (), 10.11.1-10.11.37 ISSN:.
    COSMIC (http://cancer.sanger.ac.uk) is an expert-curated database of somatic mutations in human cancer. Broad and comprehensive in scope, recent releases in 2016 describe over 4 million coding mutations across all human cancer disease types. Mutations are annotated across the entire genome, but expert curation is focused on over 400 key cancer genes. Now encompassing the majority of molecular mutation mechanisms in oncogenetics, COSMIC additionally describes 10 million non-coding mutations, 1 million copy-number aberrations, 9 million gene-expression variants, and almost 8 million differentially methylated CpGs. This information combines a consistent interpretation of the data from the major cancer genome consortia and cancer genome literature with exhaustive hand curation of over 22,000 gene-specific literature publications. This unit describes the graphical Web site in detail; alternative protocols overview other ways the entire database can be accessed, analyzed, and downloaded. © 2016 by John Wiley & Sons, Inc.
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    Tate, J. G.; Bamford, S.; Jubb, H. C.; Sondka, Z.; Beare, D. M.; Bindal, N.; Boutselakis, H.; Cole, C. G.; Creatore, C.; Dawson, E.; Fish, P.; Harsha, B.; Hathaway, C.; Jupe, S. C.; Kok, C. Y.; Noble, K.; Ponting, L.; Ramshaw, C. C.; Rye, C. E.; Speedy, H. E.; Stefancsik, R.; Thompson, S. L.; Wang, S.; Ward, S.; Campbell, P. J.; Forbes, S. A. COSMIC: the Catalogue Of Somatic Mutations In Cancer. Nucleic Acids Res. 2019, 47, D941– d947,  DOI: 10.1093/nar/gky1015
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    COSMIC: the Catalogue Of Somatic Mutations In Cancer
    Tate, John G.; Bamford, Sally; Jubb, Harry C.; Sondka, Zbyslaw; Beare, David M.; Bindal, Nidhi; Boutselakis, Harry; Cole, Charlotte G.; Creatore, Celestino; Dawson, Elisabeth; Fish, Peter; Harsha, Bhavana; Hathaway, Charlie; Jupe, Steve C.; Kok, Chai Yin; Noble, Kate; Ponting, Laura; Ramshaw, Christopher C.; Rye, Claire E.; Speedy, Helen E.; Stefancsik, Ray; Thompson, Sam L.; Wang, Shicai; Ward, Sari; Campbell, Peter J.; Forbes, Simon A.
    Nucleic Acids Research (2019), 47 (D1), D941-D947CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
    COSMIC, the Catalog Of Somatic Mutations In Cancer (https://cancer.sanger.ac.uk) is the most detailed and comprehensive resource for exploring the effect of somatic mutations in human cancer. The latest release, COSMIC v86 (August 2018), includes almost 6 million coding mutations across 1.4 million tumor samples, curated from over 26,000 publications. In addn. to coding mutations, COSMIC covers all the genetic mechanisms by which somatic mutations promote cancer, including non-coding mutations, gene fusions, copy-no. variants and drug resistance mutations. COSMIC is primarily handcurated, ensuring quality, accuracy and descriptive data capture. Building on our manual curation processes, we are introducing new initiatives that allow us to prioritize key genes and diseases, and to react more quickly and comprehensively to new findings in the literature. Alongside improvements to the public website and data-download systems, new functionality in COSMIC-3D allows exploration of mutations within three-dimensional protein structures, their protein structural and functional impacts, and implications for druggability. In parallel with COSMIC's deep and broad variant coverage, the Cancer Gene Census (CGC) describes a curated catalog of genes driving every form of human cancer. Currently describing 719 genes, the CGC has recently introduced functional descriptions of how each gene drives disease, summarized into the 10 cancer Hallmarks.
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    Torkamani, A.; Schork, N. J. Prediction of cancer driver mutations in protein kinases. Cancer Res. 2008, 68, 1675– 1682,  DOI: 10.1158/0008-5472.can-07-5283
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    Prediction of Cancer Driver Mutations in Protein Kinases
    Torkamani, Ali; Schork, Nicholas J.
    Cancer Research (2008), 68 (6), 1675-1682CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)
    A large no. of somatic mutations accumulate during the process of tumorigenesis. A subset of these mutations contribute to tumor progression (known as "driver" mutations) whereas the majority of these mutations are effectively neutral (known as "passenger" mutations). The ability to differentiate between drivers and passengers will be crit. to the success of upcoming large-scale cancer DNA resequencing projects. Here we show a method capable of discriminating between drivers and passengers in the most frequently cancer-assocd. protein family, protein kinases. We apply this method to multiple cancer data sets, validating its accuracy by showing that it is capable of identifying known drivers, has excellent agreement with previous statistical ests. of the frequency of drivers, and provides strong evidence that predicted drivers are under pos. selection by various sequence and structural analyses. Furthermore, we identify particular positions in protein kinases that seem to play a role in oncogenesis. Finally, we provide a ranked list of candidate driver mutations.
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    Ryslik, G. A.; Cheng, Y.; Modis, Y.; Zhao, H. Leveraging protein quaternary structure to identify oncogenic driver mutations. BMC Bioinf. 2016, 17, 137,  DOI: 10.1186/s12859-016-0963-3
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    Leveraging protein quaternary structure to identify oncogenic driver mutations
    Ryslik, Gregory A.; Cheng, Yuwei; Modis, Yorgo; Zhao, Hongyu
    BMC Bioinformatics (2016), 17 (), 137/1-137/13CODEN: BBMIC4; ISSN:1471-2105. (BioMed Central Ltd.)
    Background: Identifying key "driver" mutations which are responsible for tumorigenesis is crit. in the development of new oncol. drugs. Due to multiple pharmacol. successes in treating cancers that are caused by such driver mutations, a large body of methods have been developed to differentiate these mutations from the benign "passenger" mutations which occur in the tumor but do not further progress the disease. Under the hypothesis that driver mutations tend to cluster in key regions of the protein, the development of algorithms that identify these clusters has become a crit. area of research. Results: We have developed a novel methodol., QuartPAC (Quaternary Protein Amino acid Clustering), that identifies non-random mutational clustering while utilizing the protein quaternary structure in 3D space. By integrating the spatial information in the Protein Data Bank (PDB) and the mutational data in the Catalog of Somatic Mutations in Cancer (COSMIC), QuartPAC is able to identify clusters which are otherwise missed in a variety of proteins. The R package is available on Bioconductor at: http://bioconductor.jp/packages/3.1/bioc/html/QuartPAC.html. Conclusion:QuartPAC provides a unique tool to identify mutational clustering while accounting for the complete folded protein quaternary structure.
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    Kumar, S.; Clarke, D.; Gerstein, M. B. Leveraging protein dynamics to identify cancer mutational hotspots using 3D structures. Proc. Natl. Acad. Sci. U.S.A. 2019, 116, 18962– 18970,  DOI: 10.1073/pnas.1901156116
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    Leveraging protein dynamics to identify cancer mutational hotspots using 3D structures
    Kumar, Sushant; Clarke, Declan; Gerstein, Mark B.
    Proceedings of the National Academy of Sciences of the United States of America (2019), 116 (38), 18962-18970CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Large-scale exome sequencing of tumors has enabled the identification of cancer drivers using recurrence-based approaches. Some of these methods also employ 3D protein structures to identify mutational hotspots in cancer-assocd. genes. In detg. such mutational clusters in structures, existing approaches overlook protein dynamics, despite its essential role in protein function. We present a framework to identify cancer driver genes using a dynamics-based search of mutational hotspot communities. Mutations are mapped to protein structures, which are partitioned into distinct residue communities. These communities are identified in a framework where residue-residue contact edges are weighted by correlated motions (as inferred by dynamics-based models). We then search for signals of pos. selection among these residue communities to identify putative driver genes, while applying our method to the TCGA (The Cancer Genome Atlas) PanCancer Atlas missense mutation catalog. Overall, we predict 1 or more mutational hotspots within the resolved structures of proteins encoded by 434 genes. These genes were enriched among biol. processes assocd. with tumor progression. Addnl., a comparison between our approach and existing cancer hotspot detection methods using structural data suggests that including protein dynamics significantly increases the sensitivity of driver detection.
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    Kamburov, A.; Lawrence, M. S.; Polak, P.; Leshchiner, I.; Lage, K.; Golub, T. R.; Lander, E. S.; Getz, G. Comprehensive assessment of cancer missense mutation clustering in protein structures. Proc. Natl. Acad. Sci. U.S.A. 2015, 112, E5486– E5495,  DOI: 10.1073/pnas.1516373112
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    Comprehensive assessment of cancer missense mutation clustering in protein structures
    Kamburov, Atanas; Lawrence, Michael S.; Polak, Paz; Leshchiner, Ignaty; Lage, Kasper; Golub, Todd R.; Lander, Eric S.; Getz, Gad
    Proceedings of the National Academy of Sciences of the United States of America (2015), 112 (40), E5486-E5495CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Large-scale tumor sequencing projects enabled the identification of many new cancer gene candidates through computational approaches. Here, we describe a general method to detect cancer genes based on significant 3D clustering of mutations relative to the structure of the encoded protein products. The approach can also be used to search for proteins with an enrichment of mutations at binding interfaces with a protein, nucleic acid, or small mol. partner. We applied this approach to systematically analyze the PanCancer compendium of somatic mutations from 4,742 tumors relative to all known 3D structures of human proteins in the Protein Data Bank. We detected significant 3D clustering of missense mutations in several previously known oncoproteins including HRAS, EGFR, and PIK3CA. Although clustering of missense mutations is often regarded as a hallmark of oncoproteins, we obsd. that a no. of tumor suppressors, including FBXW7, VHL, and STK11, also showed such clustering. Beside these known cases, we also identified significant 3D clustering of missense mutations in NUF2, which encodes a component of the kinetochore, that could affect chromosome segregation and lead to aneuploidy. Anal. of interaction interfaces revealed enrichment of mutations in the interfaces between FBXW7-CCNE1, HRAS-RASA1, CUL4B-CAND1, OGT-HCFC1, PPP2R1A-PPP2R5C/PPP2R2A, DICER1-Mg2+, MAX-DNA, SRSF2-RNA, and others. Together, our results indicate that systematic consideration of 3D structure can assist in the identification of cancer genes and in the understanding of the functional role of their mutations.
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    Dharmarajan, V.; Lee, J.-H.; Patel, A.; Skalnik, D. G.; Cosgrove, M. S. Structural basis for WDR5 interaction (Win) motif recognition in human SET1 family histone methyltransferases. J. Biol. Chem. 2012, 287, 27275– 27289,  DOI: 10.1074/jbc.m112.364125
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    Structural Basis for WDR5 Interaction (Win) Motif Recognition in Human SET1 Family Histone Methyltransferases
    Dharmarajan, Venkatasubramanian; Lee, Jeong-Heon; Patel, Anamika; Skalnik, David G.; Cosgrove, Michael S.
    Journal of Biological Chemistry (2012), 287 (33), 27275-27289CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Translocations and amplifications of the mixed lineage leukemia-1 (MLL1) gene are assocd. with aggressive myeloid and lymphocytic leukemias in humans. MLL1 is a member of the SET1 family of histone H3 lysine 4 (H3K4) methyltransferases, which are required for transcription of genes involved in hematopoiesis and development. MLL1 assocs. with a subcomplex contg. WDR5, RbBP5, Ash2L, and DPY-30 (WRAD), which together form the MLL1 core complex that is required for sequential mono- and dimethylation of H3K4. We previously demonstrated that WDR5 binds the conserved WDR5 interaction (Win) motif of MLL1 in vitro, an interaction that is required for the H3K4 dimethylation activity of the MLL1 core complex. In this investigation, we demonstrate that arginine 3765 of the MLL1 Win motif is required to co-immunoppt. WRAD from mammalian cells, suggesting that the WDR5-Win motif interaction is important for the assembly of the MLL1 core complex in vivo. We also demonstrate that peptides that mimic SET1 family Win motif sequences inhibit H3K4 dimethylation by the MLL1 core complex with varying degrees of efficiency. To understand the structural basis for these differences, we detd. structures of WDR5 bound to six different naturally occurring Win motif sequences at resolns. ranging from 1.9 to 1.2 Å. Our results reveal that binding energy differences result from interactions between non-conserved residues C-terminal to the Win motif and to a lesser extent from subtle variation of residues within the Win motif. These results highlight a new class of methylation inhibitors that may be useful for the treatment of MLL1-related malignancies.
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    Zhang, P.; Lee, H.; Brunzelle, J. S.; Couture, J.-F. The plasticity of WDR5 peptide-binding cleft enables the binding of the SET1 family of histone methyltransferases. Nucleic Acids Res. 2012, 40, 4237– 4246,  DOI: 10.1093/nar/gkr1235
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    The plasticity of WDR5 peptide-binding cleft enables the binding of the SET1 family of histone methyltransferases
    Zhang, Pamela; Lee, Hwabin; Brunzelle, Joseph S.; Couture, Jean-Francois
    Nucleic Acids Research (2012), 40 (9), 4237-4246CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    In mammals, the SET1 family of lysine methyltransferases (KMTs), which includes MLL1-5, SET1A and SET1B, catalyzes the methylation of lysine-4 (Lys-4) on histone H3. Recent reports have demonstrated that a three-subunit complex composed of WD-repeat protein-5 (WDR5), retinoblastoma-binding protein-5 (RbBP5) and absent, small, homeotic disks-2-like (ASH2L) stimulates the methyltransferase activity of MLL1. On the basis of studies showing that this stimulation is in part controlled by an interaction between WDR5 and a small region located in close proximity of the MLL1 catalytic domain [referred to as the WDR5-interacting motif (Win)], it has been suggested that WDR5 might play an analogous role in scaffolding the other SET1 complexes. We herein provide biochem. and structural evidence showing that WDR5 binds the Win motifs of MLL2-4, SET1A and SET1B. Comparative anal. of WDR5-Win complexes reveals that binding of the Win motifs is achieved by the plasticity of WDR5 peptidyl-arginine-binding cleft allowing the C-terminal ends of the Win motifs to be maintained in structurally divergent conformations. Consistently, enzymic assays reveal that WDR5 plays an important role in the optimal stimulation of MLL2-4, SET1A and SET1B methyltransferase activity by the RbBP5-ASH2L heterodimer. Overall, our findings illustrate the function of WDR5 in scaffolding the SET1 family of KMTs and further emphasize on the important role of WDR5 in regulating global histone H3 Lys-4 methylation.
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    Muntean, A. G.; Hess, J. L. The pathogenesis of mixed-lineage leukemia. Annu. Rev. Pathol.: Mech. Dis. 2012, 7, 283– 301,  DOI: 10.1146/annurev-pathol-011811-132434
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    The pathogenesis of mixed-lineage leukemia
    Muntean, Andrew G.; Hess, Jay L.
    Annual Review of Pathology: Mechanisms of Disease (2012), 7 (), 283-301CODEN: ARPMCU; ISSN:1553-4006. (Annual Reviews Inc.)
    A review. Aggressive leukemias arise in both children and adults as a result of rearrangements to the mixed-lineage leukemia gene (MLL) located on chromosome 11q23. MLL encodes a large histone methyltransferase that directly binds DNA and pos. regulates gene transcription, including homeobox (HOX) genes. MLL is involved in chromosomal translocations, partial tandem duplications, and amplifications, all of which result in hematopoietic malignancies due to sustained HOX expression and stalled differentiation. MLL lesions are assocd. with both acute myeloid leukemia and acute lymphoid leukemia and are usually assocd. with a relatively poor prognosis despite improved treatment options such as allogeneic hematopoietic stem cell transplantation, which underscores the need for new treatment regimens. Recent advances have begun to reveal the mol. mechanisms that drive MLL-assocd. leukemias, which, in turn, have provided opportunities for therapeutic development. Here, we discuss the etiol. of MLL leukemias and potential directions for future therapy.
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    Chen, X.; Xie, W.; Gu, P.; Cai, Q.; Wang, B.; Xie, Y.; Dong, W.; He, W.; Zhong, G.; Lin, T.; Huang, J. Upregulated WDR5 promotes proliferation, self-renewal and chemoresistance in bladder cancer via mediating H3K4 trimethylation. Sci. Rep. 2015, 5, 8293,  DOI: 10.1038/srep08293
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    Upregulated WDR5 promotes proliferation, self-renewal and chemoresistance in bladder cancer via mediating H3K4 trimethylation
    Chen, Xu; Xie, Weibin; Gu, Peng; Cai, Qingqing; Wang, Bo; Xie, Yun; Dong, Wen; He, Wang; Zhong, Guangzheng; Lin, Tianxin; Huang, Jian
    Scientific Reports (2015), 5 (), 8293CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
    WD repeat domain 5 (WDR5) plays an important role in various biol. functions through the epigenetic regulation of gene transcription; however, its role in bladder cancer remains largely unknown. Our study investigated the role of WDR5 in bladder cancer and demonstrated that WDR5 was upregulated in bladder cancer tissues, and elevated WDR5 protein levels pos. correlated with advanced tumor stage and poor survival. Through gain or loss of function, we demonstrated that WDR5 promoted proliferation, self-renewal and chemoresistance to cisplatin in bladder cancer cells in vitro, and tumor growth in vivo. Mechanistically, WDR5 regulated various functions in bladder cancer by mediating the transcription of cyclin B1, cyclin E1, cyclin E2, UHMK1, MCL1, BIRC3 and Nanog by histone H3 lysine 4 trimethylation. Therefore, we have discovered that WDR5 plays an important role in bladder cancer suggesting that WDR5 is a potential biomarker and a promising target in the treatment of bladder cancer.
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    Chen, X.; Gu, P.; Li, K.; Xie, W.; Chen, C.; Lin, T.; Huang, J. Gene expression profiling of WDR5 regulated genes in bladder cancer. Genomics Data 2015, 5, 27– 29,  DOI: 10.1016/j.gdata.2015.05.003
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    Gene expression profiling of WDR5 regulated genes in bladder cancer
    Chen Xu; Gu Peng; Lin Tianxin; Li Kuiqing; Xie Weibin; Chen Changhao; Huang Jian
    Genomics data (2015), 5 (), 27-9 ISSN:2213-5960.
    WD repeat domain 5 (WDR5) plays an important role in various biological functions through the epigenetic regulation of gene transcription (Wysocka et al., 2005 [1]; Sandstrom et al., 2014[2]; Ang et al., 2011[3]). Recently, our study found that WDR5 was upregulated in bladder cancer tissues, promoted bladder cancer cell proliferation, self-renewal and chemoresistance to cisplatin in bladder cancer cells in vitro, and tumor growth in vivo (Chen et al., 2015). To gain a molecular understanding of the role of WDR5 in promoting bladder cancer, we performed a genome-wide analysis on WDR5 knockdown by microarray gene expression profiling. Here we provide detailed experimental methods and analysis for the microarray data, which have been deposited into Gene Expression Omnibus (GEO): GSE59132.
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    Ge, Z.; Song, E. J.; Kawasawa, Y. I.; Li, J.; Dovat, S.; Song, C. WDR5 high expression and its effect on tumorigenesis in leukemia. Oncotarget 2016, 7, 37740– 37754,  DOI: 10.18632/oncotarget.9312
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    WDR5 high expression and its effect on tumorigenesis in leukemia
    Ge Zheng; Ge Zheng; Song Evelyn J; Dovat Sinisa; Song Chunhua; Ge Zheng; Li Jianyong; Kawasawa Yuka Imamura
    Oncotarget (2016), 7 (25), 37740-37754 ISSN:.
    WD repeat domain 5 (WDR5) plays an important role in various biological functions through the epigenetic regulation of gene transcription. However, the oncogenic effect of WDR5 in leukemia remains largely unknown. Here, we found WDR5 expression is increased in leukemia patients. High expression of WDR5 is associated with high risk leukemia; Patients with WDR5 and MLL1 high expression have poor complete remission rate. We further identified the global genomic binding of WDR5 in leukemic cells and found the genomic co-localization of WDR5 binding with H3K4me3 enrichment. Moreover, WDR5 knockdown by shRNA suppresses cell proliferation, induces apoptosis, inhibits the expression of WDR5 targets, and blocks the H3K4me3 enrichment on the promoter of its targets. We also observed the positive correlation of WDR5 expression with these targets in the cohort study of leukemia patients. Our data reveal that WDR5 may have oncogenic effect and WDR5-mediated H3K4 methylation plays an important role in leukemogenesis.
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    Sun, W.; Guo, F.; Liu, M. Up-regulated WDR5 promotes gastric cancer formation by induced cyclin D1 expression. J. Cell. Biochem. 2018, 119, 3304– 3316,  DOI: 10.1002/jcb.26491
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    Up-regulated WDR5 promotes gastric cancer formation by induced cyclin D1 expression
    Sun, Wei; Guo, Fuchao; Liu, Mingkai
    Journal of Cellular Biochemistry (2018), 119 (4), 3304-3316CODEN: JCEBD5; ISSN:0730-2312. (Wiley-Blackwell)
    Gastric cancer (GC) is the fourth common cancer and second leading cause of cancer-related mortality in the world. WD repeat domain 5 (WDR5) has been identified that its functions as an important role in various biol. functions through the epigenetic regulation of gene transcription. However, the oncogenic effect of WDR5 in gastric cancer remains largely unknown. In this study, we investigated the role of WDR5 in gastric cancer genesis. We found that WDR5 expression is increased in gastric cancer patients. Through survival anal., we found that high expression of WDR5 is assocd. with high risk gastric cancer; patients who with WDR5 high expression have poor survival rate compared with those who with WDR5 low expression. To make further investigation, we identified that WDR5 is targeted for cell cycle arrest by the Cyclin D1 in a process that is regulated by H3K4me3. Moreover, over-expression of WDR5 promotes cell proliferation, induces S/G2/M arrest in cell cycle, and promotes the expression of WDR5 targets, as well as that of H3K4me3 on the promoter of its targets. Inversely, WDR5 knockdown by shRNA inhibits cell proliferation, reverses S/G2/M arrest in cell cycle, and suppresses the expression of WDR5 targets, as well as that of H3K4me3. We also obsd. the pos. correlation of WDR5 expression with its target in the cohort study of gastric patients. Taken together, our data reveal that WDR5 may have oncogenic effect and WDR5-mediated H3K4 methylation plays an important role in gastric cancer.
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    Wang, F.; Zhang, J.; Ke, X.; Peng, W.; Zhao, G.; Peng, S.; Xu, J.; Xu, B.; Cui, H. WDR5-Myc axis promotes the progression of glioblastoma and neuroblastoma by transcriptional activating CARM1. Biochem. Biophys. Res. Commun. 2020, 523, 699– 706,  DOI: 10.1016/j.bbrc.2019.12.101
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    WDR5-Myc axis promotes the progression of glioblastoma and neuroblastoma by transcriptional activating CARM1
    Wang, Feng; Zhang, Jiayi; Ke, Xiaoxue; Peng, Wen; Zhao, Gaichao; Peng, Shihan; Xu, Jie; Xu, Bo; Cui, Hongjuan
    Biochemical and Biophysical Research Communications (2020), 523 (3), 699-706CODEN: BBRCA9; ISSN:0006-291X. (Elsevier B.V.)
    The WD repeat domain 5 (WDR5), also known as SWD3 and BIG-3, is often overexpressed in cancers, however its mol. function in cancer remains to be elucidated. In this study, we found that WDR5 promoted the proliferation and self-renewal of glioblastoma and neuroblastoma cells. The data from databases and Western blot assay showed that CARM1 is a downstream gene of WDR5-Myc axis. In addn., we obsd. that WDR5 promoted the binding of Myc to CARM1 promoter by interacting with Myc and inducing histone 3 lysine 4 trimethylation (H3K4me3). Dual luciferase reporter system indicated that Myc binds to the upstream region (-520 to -515) before transcription start site (TSS) of CARM1 promoter. These findings suggest a novel regulatory model for the proliferation and tumorigenesis of glioblastoma and neuroblastoma by WDR5-Myc axis.
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    Bryan, A. F.; Wang, J.; Howard, G. C.; Guarnaccia, A. D.; Woodley, C. M.; Aho, E. R.; Rellinger, E. J.; Matlock, B. K.; Flaherty, D. K.; Lorey, S. L.; Chung, D. H.; Fesik, S. W.; Liu, Q.; Weissmiller, A. M.; Tansey, W. P. WDR5 is a conserved regulator of protein synthesis gene expression. Nucleic Acids Res. 2020, 48, 2924– 2941,  DOI: 10.1093/nar/gkaa051
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    WDR5 is a conserved regulator of protein synthesis gene expression
    Bryan, Audra F.; Wang, Jing; Howard, Gregory C.; Guarnaccia, Alissa D.; Woodley, Chase M.; Aho, Erin R.; Rellinger, Eric J.; Matlock, Brittany K.; Flaherty, David K.; Lorey, Shelly L.; Chung, Dai H.; Fesik, Stephen W.; Liu, Qi; Weissmiller, April M.; Tansey, William P.
    Nucleic Acids Research (2020), 48 (6), 2924-2941CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
    WDR5 is a highly-conserved nuclear protein that performs multiple scaffolding functions in the context of chromatin. WDR5 is also a promising target for pharmacol. inhibition in cancer, with small mol. inhibitors of an arginine-binding pocket of WDR5 (the 'WIN' site) showing efficacy against a range of cancer cell lines in vitro. Efforts to understand WDR5, or establish the mechanism of action of WIN site inhibitors, however, are stymied by its many functions in the nucleus, and a lack of knowledge of the conserved gene networks--if any--that are under its control. Here, we have performed comparative genomic analyses to identify the conserved sites of WDR5 binding to chromatin, and the conserved genes regulated by WDR5, across a diverse panel of cancer cell lines. We show that a specific cohort of protein synthesis genes (PSGs) are invariantly bound by WDR5, demonstrate that the WIN site anchors WDR5 to chromatin at these sites, and establish that PSGs are bona fide, acute, and persistent targets of WIN site blockade. Together, these data reveal that WDR5 plays a predominant transcriptional role in biomass accumulation and provide further evidence that WIN site inhibitors act to repress gene networks linked to protein synthesis homeostasis. In Saccharomyces cerevisiae, ribosomal protein gene (RPG) promoters display binding sites for either Rap1 or Abf1 transcription factors. Unlike Rap1-assocd. promoters, the small cohort of Abf1-dependent RPGs (Abf1-RPGs) has not been extensively investigated. We show that RPL3, RPL4B, RPP1A, RPS22B and RPS28A/B share a common promoter architecture, with an Abf1 site upstream of a conserved element matching the sequence recognized by Fhl1, a transcription factor which together with Ifh1 orchestrates Rap1-assocd. RPG regulation. Abf1 and Fhl1 promoter assocn. was confirmed by ChIP and/or gel retardation assays. Mutational anal. revealed a more severe requirement of Abf1 than Fhl1 binding sites for RPG transcription. In the case of RPS22B an unusual Tbf1 binding site promoted both RPS22B and intron-hosted SNR44 expression. Abf1-RPG down-regulation upon TOR pathway inhibition was much attenuated at defective mutant promoters unable to bind Abf1. TORC1 inactivation caused the expected redn. of Ifh1 occupancy at RPS22B and RPL3 promoters, but unexpectedly it entailed largely increased Abf1 assocn. with Abf1-RPG promoters. We present evidence that Abf1 recruitment upon nutritional stress, also obsd. for representative ribosome biogenesis genes, favors RPG transcriptional rescue upon nutrient replenishment, thus pointing to nutrient-regulated Abf1 dynamics at promoters as a novel mechanism in ribosome biogenesis control.
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    Imran, A.; Moyer, B. S.; Canning, A. J.; Kalina, D.; Duncan, T. M.; Moody, K. J.; Wolfe, A. J.; Cosgrove, M. S.; Movileanu, L. Kinetics of the multitasking high-affinity Win binding site of WDR5 in restricted and unrestricted conditions. Biochem. J. 2021, 478, 2145– 2161,  DOI: 10.1042/bcj20210253
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    Kinetics of the multitasking high-affinity Win binding site of WDR5 in restricted and unrestricted conditions
    Imran, Ali; Moyer, Brandon S.; Canning, Ashley J.; Kalina, Dan; Duncan, Thomas M.; Moody, Kelsey J.; Wolfe, Aaron J.; Cosgrove, Michael S.; Movileanu, Liviu
    Biochemical Journal (2021), 478 (11), 2145-2161CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
    Recent advances in quant. proteomics show that WD40 proteins play a pivotal role in numerous cellular networks. Yet, they have been fairly unexplored and their phys. assocns. with other proteins are ambiguous. A quant. understanding of these interactions has wide-ranging significance. WD40 repeat protein 5 (WDR5) interacts with all members of human SET1/MLL methyltransferases, which regulate methylation of the histone 3 lysine 4 (H3K4). Here, using real-time binding measurements in a high-throughput setting, we identified the kinetic fingerprint of transient assocns. between WDR5 and 14-residue WDR5 interaction (Win) motif peptides of each SET1 protein (SET1Win). Our results reveal that the high-affinity WDR5-SET1Win interactions feature slow assocn. kinetics. This finding is likely due to the requirement of SET1Win to insert into the narrow WDR5 cavity, also named the Win binding site. Furthermore, our explorations indicate fairly slow dissocn. kinetics. This conclusion is in accordance with the primary role of WDR5 in maintaining the functional integrity of a large multisubunit complex, which regulates the histone methylation. Because the Win binding site is considered a key therapeutic target, the immediate outcomes of this study could form the basis for accelerated developments in medical biotechnol.
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    Mayse, L. A.; Imran, A.; Larimi, M. G.; Cosgrove, M. S.; Wolfe, A. J.; Movileanu, L. Disentangling the recognition complexity of a protein hub using a nanopore. Nat. Commun. 2022, 13, 978,  DOI: 10.1038/s41467-022-28465-8
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    Disentangling the recognition complexity of a protein hub using a nanopore
    Mayse, Lauren Ashley; Imran, Ali; Larimi, Motahareh Ghahari; Cosgrove, Michael S.; Wolfe, Aaron James; Movileanu, Liviu
    Nature Communications (2022), 13 (1), 978CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)
    WD40 repeat proteins are frequently involved in processing cell signaling and scaffolding large multi-subunit machineries. Despite their significance in physiol. and disease-like conditions, their reversible interactions with other proteins remain modestly examd. Here, we show the development and validation of a protein nanopore for the detection and quantification of WD40 repeat protein 5 (WDR5), a chromatin-assocd. hub involved in epigenetic regulation of histone methylation. Our nanopore sensor is equipped with a 14-residue Win motif of mixed lineage leukemia 4 methyltransferase (MLL4Win), a WDR5 ligand. Our approach reveals a broad dynamic range of MLL4Win-WDR5 interactions and three distant subpopulations of binding events, representing three modes of protein recognition. The three binding events are confirmed as specific interactions using a weakly binding WDR5 deriv. and various environmental contexts. These outcomes demonstrate the substantial sensitivity of our nanopore sensor, which can be utilized in protein analytics.
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    Karatas, H.; Townsend, E. C.; Cao, F.; Chen, Y.; Bernard, D.; Liu, L.; Lei, M.; Dou, Y.; Wang, S. High-affinity, small-molecule peptidomimetic inhibitors of MLL1/WDR5 protein-protein interaction. J. Am. Chem. Soc. 2013, 135, 669– 682,  DOI: 10.1021/ja306028q
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    High-Affinity, Small-Molecule Peptidomimetic Inhibitors of MLL1/WDR5 Protein-Protein Interaction
    Karatas, Hacer; Townsend, Elizabeth C.; Cao, Fang; Chen, Yong; Bernard, Denzil; Liu, Liu; Lei, Ming; Dou, Yali; Wang, Shaomeng
    Journal of the American Chemical Society (2013), 135 (2), 669-682CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Mixed lineage leukemia 1 (MLL1) is a histone H3 lysine 4 (H3K4) methyltransferase, and targeting the MLL1 enzymic activity has been proposed as a novel therapeutic strategy for the treatment of acute leukemia harboring MLL1 fusion proteins. The MLL1/WDR5 protein-protein interaction is essential for MLL1 enzymic activity. In the present study, we designed a large no. of peptidomimetics to target the MLL1/WDR5 interaction based upon -CO-ARA-NH-, the min. binding motif derived from MLL1. Our study led to the design of high-affinity peptidomimetics, which bind to WDR5 with Ki < 1 nM and function as potent antagonists of MLL1 activity in a fully reconstituted in vitro H3K4 methyltransferase assay. Detn. of co-crystal structures of two potent peptidomimetics in complex with WDR5 establishes their structural basis for high-affinity binding to WDR5. Evaluation of one such peptidomimetic, MM-102, in bone marrow cells transduced with MLL1-AF9 fusion construct shows that the compd. effectively decreases the expression of HoxA9 and Meis-1, two crit. MLL1 target genes in MLL1 fusion protein mediated leukemogenesis. MM-102 also specifically inhibits cell growth and induces apoptosis in leukemia cells harboring MLL1 fusion proteins. Our study provides the first proof-of-concept for the design of small-mol. inhibitors of the WDR5/MLL1 protein-protein interaction as a novel therapeutic approach for acute leukemia harboring MLL1 fusion proteins.
  47. 47
    Cao, F.; Townsend, E. C.; Karatas, H.; Xu, J.; Li, L.; Lee, S.; Liu, L.; Chen, Y.; Ouillette, P.; Zhu, J.; Hess, J. L.; Atadja, P.; Lei, M.; Qin, Z. S.; Malek, S.; Wang, S.; Dou, Y. Targeting MLL1 H3K4 methyltransferase activity in mixed-lineage leukemia. Mol. Cell 2014, 53, 247– 261,  DOI: 10.1016/j.molcel.2013.12.001
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    Targeting MLL1 H3K4 Methyltransferase Activity in Mixed-Lineage Leukemia
    Cao, Fang; Townsend, Elizabeth C.; Karatas, Hacer; Xu, Jing; Li, Li; Lee, Shirley; Liu, Liu; Chen, Yong; Ouillette, Peter; Zhu, Jidong; Hess, Jay L.; Atadja, Peter; Lei, Ming; Qin, Zhaohui S.; Malek, Sami; Wang, Shaomeng; Dou, Yali
    Molecular Cell (2014), 53 (2), 247-261CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)
    Here we report a comprehensive characterization of our recently developed inhibitor MM-401 that targets the MLL1 H3K4 methyltransferase activity. MM-401 is able to specifically inhibit MLL1 activity by blocking MLL1-WDR5 interaction and thus the complex assembly. This targeting strategy does not affect other mixed-lineage leukemia (MLL) family histone methyltransferases (HMTs), revealing a unique regulatory feature for the MLL1 complex. Using MM-401 and its enantiomer control MM-NC-401, we show that inhibiting MLL1 methyltransferase activity specifically blocks proliferation of MLL cells by inducing cell-cycle arrest, apoptosis, and myeloid differentiation without general toxicity to normal bone marrow cells or non-MLL cells. More importantly, transcriptome analyses show that MM-401 induces changes in gene expression similar to those of MLL1 deletion, supporting a predominant role of MLL1 activity in regulating MLL1-dependent leukemia transcription program. We envision broad applications for MM-401 in basic and translational research.
  48. 48
    Alicea-Velázquez, N. L.; Shinsky, S. A.; Loh, D. M.; Lee, J. H.; Skalnik, D. G.; Cosgrove, M. S. Targeted Disruption of the Interaction between WD-40 Repeat Protein 5 (WDR5) and Mixed Lineage Leukemia (MLL)/SET1 Family Proteins Specifically Inhibits MLL1 and SETd1A Methyltransferase Complexes. J. Biol. Chem. 2016, 291, 22357– 22372,  DOI: 10.1074/jbc.M116.752626
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    48
    Targeted Disruption of the Interaction between WD-40 Repeat Protein 5 (WDR5) and Mixed Lineage Leukemia (MLL)/SET1 Family Proteins Specifically Inhibits MLL1 and SETd1A Methyltransferase Complexes
    Alicea-Velazquez, Nilda L.; Shinsky, Stephen A.; Loh, Daniel M.; Lee, Jeong-Heon; Skalnik, David G.; Cosgrove, Michael S.
    Journal of Biological Chemistry (2016), 291 (43), 22357-22372CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    MLL1 belongs to the SET1 family of histone H3 lysine 4 (H3K4) methyltransferases, composed of MLL1-4 and SETd1A/B. MLL1 translocations are present in acute leukemias, and mutations in several family members are assocd. with cancer and developmental disorders. MLL1 assocs. with a subcomplex contg. WDR5, RbBP5, ASH2L, and DPY-30 (WRAD), forming the MLL1 core complex required for H3K4 mono- and dimethylation and transcriptional activation. Core complex assembly requires interaction of WDR5 with the MLL1 Win (WDR5 interaction) motif, which is conserved across the SET1 family. Agents that mimic the SET1 family Win motif inhibit the MLL1 core complex and have become an attractive approach for targeting MLL1 in cancers. Like MLL1, other SET1 family members interact with WRAD, but the roles of the Win motif in complex assembly and enzymic activity remain unexplored. Here, we show that the Win motif is necessary for interaction of WDR5 with all members of the human SET1 family. Mutation of the Win motif-WDR5 interface severely disrupts assembly and activity of MLL1 and SETd1A complexes but only modestly disrupts MLL2/4 and SETd1B complexes without significantly altering enzymic activity in vitro. Notably, in the absence of WDR5, MLL3 interacts with RAD and shows enhanced activity. To further probe the role of the Win motif-WDR5 interaction, we designed a peptidomimetic that binds WDR5 (Kd ∼3 nm) and selectively inhibits activity of MLL1 and SETd1A core complexes within the SET1 family. Our results reveal that SET1 family complexes with the weakest Win motif-WDR5 interaction are more susceptible to Win motif-based inhibitors.
  49. 49
    Karatas, H.; Li, Y.; Liu, L.; Ji, J.; Lee, S.; Chen, Y.; Yang, J.; Huang, L.; Bernard, D.; Xu, J.; Townsend, E. C.; Cao, F.; Ran, X.; Li, X.; Wen, B.; Sun, D.; Stuckey, J. A.; Lei, M.; Dou, Y.; Wang, S. Discovery of a Highly Potent, Cell-Permeable Macrocyclic Peptidomimetic (MM-589) Targeting the WD Repeat Domain 5 Protein (WDR5)-Mixed Lineage Leukemia (MLL) Protein-Protein Interaction. J. Med. Chem. 2017, 60, 4818– 4839,  DOI: 10.1021/acs.jmedchem.6b01796
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    Discovery of a Highly Potent, Cell-Permeable Macrocyclic Peptidomimetic (MM-589) Targeting the WD Repeat Domain 5 Protein (WDR5)-Mixed Lineage Leukemia (MLL) Protein-Protein Interaction
    Karatas, Hacer; Li, Yangbing; Liu, Liu; Ji, Jiao; Lee, Shirley; Chen, Yong; Yang, Jiuling; Huang, Liyue; Bernard, Denzil; Xu, Jing; Townsend, Elizabeth C.; Cao, Fang; Ran, Xu; Li, Xiaoqin; Wen, Bo; Sun, Duxin; Stuckey, Jeanne A.; Lei, Ming; Dou, Yali; Wang, Shaomeng
    Journal of Medicinal Chemistry (2017), 60 (12), 4818-4839CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    We report herein the design, synthesis and evaluation of macrocyclic peptidomimetics that bind to WDR5 and block the WDR5-MLL protein-protein interaction. Compd. (I) (R1 = Me; n = 1) (MM-589) binds to WDR5 with an IC50 value of 0.90 nM (Ki value <1 nM), and inhibits the MLL H3K4 methyltransferase (HMT) activity with an IC50 value of 12.7 nM. Compd. I (R1 = Me; n = 1) potently and selectively inhibits cell growth in human leukemia cell lines harboring MLL translocations, and is >40 times better than the previously reported compd. MM-401. Co-crystal structures of I (R1 = H, n = 5) and (R1 = Me; n = 1) complexed with WDR5 provide structural basis for their high affinity binding to WDR5. Addnl., we have developed and optimized a new, AlphaLISA-based MLL HMT functional assay to facilitate the functional evaluation of these designed compds. Compd. I (R1 = Me; n = 1) represents the most potent inhibitor of the WDR5-MLL interaction reported to date and further optimization of I (R1 = Me; n = 1) may yield a new therapy for acute leukemia.
  50. 50
    Schapira, M.; Tyers, M.; Torrent, M.; Arrowsmith, C. H. WD40 repeat domain proteins: a novel target class?. Nat. Rev. Drug Discovery 2017, 16, 773– 786,  DOI: 10.1038/nrd.2017.179
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    WD40 repeat domain proteins: a novel target class?
    Schapira, Matthieu; Tyers, Mike; Torrent, Maricel; Arrowsmith, Cheryl H.
    Nature Reviews Drug Discovery (2017), 16 (11), 773-786CODEN: NRDDAG; ISSN:1474-1776. (Nature Research)
    Antagonism of protein-protein interactions (PPIs) with small mols. is becoming more feasible as a therapeutic approach. Successful PPI inhibitors tend to target proteins contg. deep peptide-binding grooves or pockets rather than the more common large, flat protein interaction surfaces. Here, we review one of the most abundant PPI domains in the human proteome, the WD40 repeat (WDR) domain, which has a central peptide-binding pocket and is a member of the β-propeller domain-contg. protein family. Recently, two WDR domain-contg. proteins, WDR5 and EED, as well as other β-propeller domains have been successfully targeted by potent, specific, cell-active, drug-like chem. probes. Could WDR domains be a novel target class for drug discovery. Although the research is at an early stage and therefore not clin. validated, cautious optimism is justified, as WDR domain-contg. proteins are involved in multiple disease-assocd. pathways. The druggability and structural diversity of WDR domain binding pockets suggest that understanding how to target this prevalent domain class will open up areas of disease biol. that have so far resisted drug discovery efforts.
  51. 51
    Aho, E. R.; Weissmiller, A. M.; Fesik, S. W.; Tansey, W. P. Targeting WDR5: A WINning Anti-Cancer Strategy?. Epigenet. Insights 2019, 12, 2516865719865282,  DOI: 10.1177/2516865719865282
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    Aho, E. R.; Wang, J.; Gogliotti, R. D.; Howard, G. C.; Phan, J.; Acharya, P.; Macdonald, J. D.; Cheng, K.; Lorey, S. L.; Lu, B.; Wenzel, S.; Foshage, A. M.; Alvarado, J.; Wang, F.; Shaw, J. G.; Zhao, B.; Weissmiller, A. M.; Thomas, L. R.; Vakoc, C. R.; Hall, M. D.; Hiebert, S. W.; Liu, Q.; Stauffer, S. R.; Fesik, S. W.; Tansey, W. P. Displacement of WDR5 from Chromatin by a WIN Site Inhibitor with Picomolar Affinity. Cell Rep. 2019, 26, 2916– 2928.e13,  DOI: 10.1016/j.celrep.2019.02.047
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    Displacement of WDR5 from Chromatin by a WIN Site Inhibitor with Picomolar Affinity
    Aho, Erin R.; Wang, Jing; Gogliotti, Rocco D.; Howard, Gregory C.; Phan, Jason; Acharya, Pankaj; Macdonald, Jonathan D.; Cheng, Ken; Lorey, Shelly L.; Lu, Bin; Wenzel, Sabine; Foshage, Audra M.; Alvarado, Joseph; Wang, Feng; Shaw, J. Grace; Zhao, Bin; Weissmiller, April M.; Thomas, Lance R.; Vakoc, Christopher R.; Hall, Matthew D.; Hiebert, Scott W.; Liu, Qi; Stauffer, Shaun R.; Fesik, Stephen W.; Tansey, William P.
    Cell Reports (2019), 26 (11), 2916-2928.e13CODEN: CREED8; ISSN:2211-1247. (Cell Press)
    The chromatin-assocd. protein WDR5 is a promising target for pharmacol. inhibition in cancer. Drug discovery efforts center on the blockade of the "WIN site" of WDR5, a well-defined pocket that is amenable to small mol. inhibition. Various cancer contexts have been proposed to be targets for WIN site inhibitors, but a lack of understanding of WDR5 target genes and of the primary effects of WIN site inhibitors hampers their utility. Here, by the discovery of potent WIN site inhibitors, we demonstrate that the WIN site links WDR5 to chromatin at a small cohort of loci, including a specific subset of ribosome protein genes. WIN site inhibitors rapidly displace WDR5 from chromatin and decrease the expression of assocd. genes, causing translational inhibition, nucleolar stress, and p53 induction. Our studies define a mode by which WDR5 engages chromatin and forecast that WIN site blockade could have utility against multiple cancer types.
  53. 53
    Dennis, M. L.; Morrow, B. J.; Dolezal, O.; Cuzzupe, A. N.; Stupple, A. E.; Newman, J.; Bentley, J.; Hattarki, M.; Nuttall, S. D.; Foitzik, R. C.; Street, I. P.; Stupple, P. A.; Monahan, B. J.; Peat, T. S. Fragment screening for a protein-protein interaction inhibitor to WDR5. Struct. Dyn. 2019, 6, 064701,  DOI: 10.1063/1.5122849
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    Fragment screening for a protein-protein interaction inhibitor to WDR5
    Dennis, Matthew L.; Morrow, Benjamin J.; Dolezal, Olan; Cuzzupe, Anthony N.; Stupple, Alexandra E.; Newman, Janet; Bentley, John; Hattarki, Meghan; Nuttall, Stewart D.; Foitzik, Richard C.; Street, Ian P.; Stupple, Paul A.; Monahan, Brendon J.; Peat, Thomas. S.
    Structural Dynamics (2019), 6 (6), 064701/1-064701/20CODEN: SDTYAE; ISSN:2329-7778. (American Institute of Physics)
    In this study, a fragment screen with Surface Plasmon Resonance (SPR) was used to identify a highly ligand-efficient imidazole-contg. compd. that is bound in the WIN site. The subsequent medicinal chem. campaign-guided by a suite of high-resoln. cocrystal structures with WDR5-progressed the initial hit to a low micromolar binder. One outcome from this study is a moiety that substitutes well for the side chain of arginine; a tripeptide contg. one such substitution was resolved in a high resoln. structure (1.5 Å) with a binding mode analogous to the native tripeptide. SPR furthermore indicates a similar residence time (kd = ∼0.06 s-1) for these two analogs. This novel scaffold therefore represents a possible means to overcome the potential permeability issues of WDR5 ligands that possess highly basic groups like guanidine. The series reported here furthers the understanding of the WDR5 WIN site and functions as a starting point for the development of more potent WDR5 inhibitors that may serve as cancer therapeutics. (c) 2019 American Institute of Physics.
  54. 54
    Bolshan, Y.; Getlik, M.; Kuznetsova, E.; Wasney, G. A.; Hajian, T.; Poda, G.; Nguyen, K. T.; Wu, H.; Dombrovski, L.; Dong, A.; Senisterra, G.; Schapira, M.; Arrowsmith, C. H.; Brown, P. J.; Al-Awar, R.; Vedadi, M.; Smil, D. Synthesis, Optimization, and Evaluation of Novel Small Molecules as Antagonists of WDR5-MLL Interaction. ACS Med. Chem. Lett. 2013, 4, 353– 357,  DOI: 10.1021/ml300467n
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    Synthesis, Optimization, and Evaluation of Novel Small Molecules as Antagonists of WDR5-MLL Interaction
    Bolshan, Yuri; Getlik, Matthaus; Kuznetsova, Ekaterina; Wasney, Gregory A.; Hajian, Taraneh; Poda, Gennadiy; Nguyen, Kong T.; Wu, Hong; Dombrovski, Ludmila; Dong, Aiping; Senisterra, Guillermo; Schapira, Matthieu; Arrowsmith, Cheryl H.; Brown, Peter J.; Al-awar, Rima; Vedadi, Masoud; Smil, David
    ACS Medicinal Chemistry Letters (2013), 4 (3), 353-357CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)
    The WD40-repeat protein WDR5 plays a crit. role in maintaining the integrity of MLL complexes and fully activating their methyltransferase function. MLL complexes, the trithorax-like family of SET1 methyltransferases, catalyze trimethylation of lysine 4 on histone 3, and they have been widely implicated in various cancers. Antagonism of WDR5 and MLL subunit interaction by small mols. has recently been presented as a practical way to inhibit activity of the MLL1 complex, and N-(2-(4-methylpiperazin-1-yl)-5-substituted-phenyl) benzamides were reported as potent and selective antagonists of such an interaction. Here, we describe the protein crystal structure guided optimization of prototypic compd. I (Kdis = 7 μM), leading to identification of more potent antagonist II (Kdis = 0.3 μM).
  55. 55
    Getlik, M.; Smil, D.; Zepeda-Velázquez, C.; Bolshan, Y.; Poda, G.; Wu, H.; Dong, A.; Kuznetsova, E.; Marcellus, R.; Senisterra, G.; Dombrovski, L.; Hajian, T.; Kiyota, T.; Schapira, M.; Arrowsmith, C. H.; Brown, P. J.; Vedadi, M.; Al-Awar, R. Structure-Based Optimization of a Small Molecule Antagonist of the Interaction Between WD Repeat-Containing Protein 5 (WDR5) and Mixed-Lineage Leukemia 1 (MLL1). J. Med. Chem. 2016, 59, 2478– 2496,  DOI: 10.1021/acs.jmedchem.5b01630
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    Structure-Based Optimization of a Small Molecule Antagonist of the Interaction Between WD Repeat-Containing Protein 5 (WDR5) and Mixed-Lineage Leukemia 1 (MLL1)
    Getlik, Matthaus; Smil, David; Zepeda-Velazquez, Carlos; Bolshan, Yuri; Poda, Gennady; Wu, Hong; Dong, Aiping; Kuznetsova, Ekaterina; Marcellus, Richard; Senisterra, Guillermo; Dombrovski, Ludmila; Hajian, Taraneh; Kiyota, Taira; Schapira, Matthieu; Arrowsmith, Cheryl H.; Brown, Peter J.; Vedadi, Masoud; Al-awar, Rima
    Journal of Medicinal Chemistry (2016), 59 (6), 2478-2496CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    WD repeat-contg. protein 5 (WDR5) is an important component of the multiprotein complex essential for activating mixed-lineage leukemia 1 (MLL1). Rearrangement of the MLL1 gene is assocd. with onset and progression of acute myeloid and lymphoblastic leukemias, and targeting the WDR5-MLL1 interaction may result in new cancer therapeutics. Our previous work showed that binding of small mol. ligands to WDR5 can modulate its interaction with MLL1, suppressing MLL1 methyltransferase activity. Initial structure-activity relationship studies identified N-(2-(4-methylpiperazin-1-yl)-5-substituted-phenyl) benzamides as potent and selective antagonists of this protein-protein interaction. Guided by crystal structure data and supported by in silico library design, we optimized the scaffold by varying the C-1 benzamide and C-5 substituents. This allowed us to develop the first highly potent (Kdisp < 100 nM) small mol. antagonists of the WDR5-MLL1 interaction and demonstrate that N-(4-(4-methylpiperazin-1-yl)-3'-(morpholinomethyl)-[1,1'-biphenyl]-3-yl)-6-oxo-4-(trifluoromethyl)-1,6-dihydropyridine-3-carboxamide 16d (OICR-9429) is a potent and selective chem. probe suitable to help dissect the biol. role of WDR5.
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    Schapira, M.; Arrowsmith, C. H. Methyltransferase inhibitors for modulation of the epigenome and beyond. Curr. Opin. Chem. Biol. 2016, 33, 81– 87,  DOI: 10.1016/j.cbpa.2016.05.030
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    Methyltransferase inhibitors for modulation of the epigenome and beyond
    Schapira, Matthieu; Arrowsmith, Cheryl H.
    Current Opinion in Chemical Biology (2016), 33 (), 81-87CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)
    Over the past two years tremendous progress has been made in the discovery of new inhibitors of protein lysine and arginine methyltransferases, establishing this class of epigenetic enzymes, along with DNA methyltransferases, as druggable protein families. New inhibitors of protein methyltransferases have been described with a variety of mechanisms of action including cofactor competitive, substrate competitive, allosteric inhibitors and disruptors of protein-protein interactions. Inhibitors have been used extensively in oncol. studies, and inhibitors of EZH2, and DOT1L are currently in clin. trials. Finally, advances in understanding the clin. mechanism of action of 5-azacytidine and related DNA hypomethylation agents were reported, revealing a likely role for a cell autonomous innate immune response.
  57. 57
    Gupta, A.; Xu, J.; Lee, S.; Tsai, S. T.; Zhou, B.; Kurosawa, K.; Werner, M. S.; Koide, A.; Ruthenburg, A. J.; Dou, Y.; Koide, S. Facile target validation in an animal model with intracellularly expressed monobodies. Nat. Chem. Biol. 2018, 14, 895– 900,  DOI: 10.1038/s41589-018-0099-z
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    Facile target validation in an animal model with intracellularly expressed monobodies
    Gupta, Ankit; Xu, Jing; Lee, Shirley; Tsai, Steven T.; Zhou, Bo; Kurosawa, Kohei; Werner, Michael S.; Koide, Akiko; Ruthenburg, Alexander J.; Dou, Yali; Koide, Shohei
    Nature Chemical Biology (2018), 14 (9), 895-900CODEN: NCBABT; ISSN:1552-4450. (Nature Research)
    Rapidly detg. the biol. effect of perturbing a site within a potential drug target could guide drug discovery efforts, but it remains challenging. Here, the authors describe a facile target validation approach that exploits monobodies, small synthetic binding proteins that can be fully functionally expressed in cells. The authors developed a potent and selective monobody to WDR5, a core component of the mixed lineage leukemia (MLL) methyltransferase complex. The monobody bound to the MLL interaction site of WDR5, the same binding site for small-mol. inhibitors whose efficacy has been demonstrated in cells but not in animals. As a genetically encoded reagent, the monobody inhibited proliferation of an MLL-AF9 cell line in vitro, suppressed its leukemogenesis and conferred a survival benefit in an in vivo mouse leukemia model. The capacity of this approach to readily bridge biochem., structural, cellular characterization and tests in animal models may accelerate discovery and validation of druggable sites.
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    Concepcion, J.; Witte, K.; Wartchow, C.; Choo, S.; Yao, D.; Persson, H.; Wei, J.; Li, P.; Heidecker, B.; Ma, W.; Varma, R.; Zhao, L.-S.; Perillat, D.; Carricato, G.; Recknor, M.; Du, K.; Ho, H.; Ellis, T.; Gamez, J.; Howes, M.; Phi-Wilson, J.; Lockard, S.; Zuk, R.; Tan, H. Label-free detection of biomolecular interactions using BioLayer interferometry for kinetic characterization. Comb. Chem. High Throughput Screen. 2009, 12, 791– 800,  DOI: 10.2174/138620709789104915
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    Label-free detection of biomolecular interactions using biolayer interferometry for kinetic characterization
    Concepcion, Joy; Witte, Krista; Wartchow, Charles; Choo, Sae; Yao, Danfeng; Persson, Henrik; Wei, Jing; Li, Pu; Heidecker, Bettina; Ma, Weilei; Varma, Ram; Zhao, Lian-She; Perillat, Donald; Carricato, Greg; Recknor, Michael; Du, Kevin; Ho, Huddee; Ellis, Tim; Gamez, Juan; Howes, Michael; Phi-Wilson, Janette; Lockard, Scott; Zuk, Robert; Tan, Hong
    Combinatorial Chemistry & High Throughput Screening (2009), 12 (8), 791-800CODEN: CCHSFU; ISSN:1386-2073. (Bentham Science Publishers Ltd.)
    A review. The anal. of biomol. interactions is key in the drug development process. Label-free biosensor methods provide information on binding, kinetics, concn., and the affinity of an interaction. These techniques provide real-time monitoring of interactions between an immobilized ligand (such as a receptor) to an analyte in soln. without the use of labels. Advances in biosensor design and detection using BioLayer Interferometry (BLI) provide a simple platform that enables label-free monitoring of biomol. interactions without the use of flow cells. We review the applications of BLI in a wide variety of research and development environments for quantifying antibodies and proteins and measuring kinetics parameters.
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    Weeramange, C. J.; Fairlamb, M. S.; Singh, D.; Fenton, A. W.; Swint-Kruse, L. The strengths and limitations of using biolayer interferometry to monitor equilibrium titrations of biomolecules. Protein Sci 2020, 29, 1018– 1034,  DOI: 10.1002/pro.3827
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    The strengths and limitations of using biolayer interferometry to monitor equilibrium titrations of biomolecules
    Weeramange, Chamitha J.; Fairlamb, Max S.; Singh, Dipika; Fenton, Aron W.; Swint-Kruse, Liskin
    Protein Science (2020), 29 (4), 1018-1034CODEN: PRCIEI; ISSN:1469-896X. (Wiley-Blackwell)
    Every method used to quantify biomol. interactions has its own strengths and limitations. To quantify protein-DNA binding affinities, nitrocellulose filter binding assays with 32P-labeled DNA quantify Kd values from 10-12 to 10-8 M but have several tech. limitations. Here, we considered the suitability of biolayer interferometry (BLI), which monitors assocn. and dissocn. of a sol. macromol. to an immobilized species; the ratio koff/kon dets. Kd. However, for lactose repressor protein (LacI) and an engineered repressor protein ("LLhF") binding immobilized DNA, complicated kinetic curves precluded this anal. Thus, we detd. whether the amplitude of the BLI signal at equil. related linearly to the fraction of protein bound to DNA. A key question was the effective concn. of immobilized DNA. Equil. titrn. expts. with DNA concns. below Kd (equil. binding regime) must be analyzed differently than those with DNA near or above Kd (stoichiometric binding regime). For ForteBio streptavidin tips, the most frequent effective DNA concn. was ∼2 x 10-9 M. Although variation occurred among different lots of sensor tips, binding events with Kd ≥ 10-8 M should reliably be in the equil. binding regime. We also obsd. effects from multi-valent interactions: Tetrameric LacI bound two immobilized DNAs whereas dimeric LLhF did not. We next used BLI to quantify the amt. of inducer sugars required to allosterically diminish protein-DNA binding and to assess the affinity of fructose-1-kinase for the DNA-LLhF complex. Overall, when exptl. design corresponded with appropriate data interpretation, BLI was convenient and reliable for monitoring equil. titrns. and thereby quantifying a variety of binding interactions.
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    Imran, A.; Moyer, B. S.; Wolfe, A. J.; Cosgrove, M. S.; Makarov, D. E.; Movileanu, L. Interplay of Affinity and Surface Tethering in Protein Recognition. J. Phys. Chem. Lett. 2022, 13, 4021– 4028,  DOI: 10.1021/acs.jpclett.2c00621
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    Interplay of Affinity and Surface Tethering in Protein Recognition
    Imran, Ali; Moyer, Brandon S.; Wolfe, Aaron J.; Cosgrove, Michael S.; Makarov, Dmitrii E.; Movileanu, Liviu
    Journal of Physical Chemistry Letters (2022), 13 (18), 4021-4028CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
    Surface-tethered ligand-receptor complexes are key components in biol. signaling and adhesion. They also find increasing utility in single-mol. assays and biotechnol. applications. Here, we study the real-time binding kinetics between various surface-immobilized peptide ligands and their unrestrained receptors. A long peptide tether increases the assocn. of ligand-receptor complexes, exptl. proving the fly casting mechanism where the disorder accelerates protein recognition. On the other hand, a short peptide tether enhances the complex dissocn. Notably, the rate consts. measured for the same receptor, but under different spatial constraints, are strongly correlated to one another. Furthermore, this correlation can be used to predict how surface tethering on a ligand-receptor complex alters its binding kinetics. Our results have immediate implications in the broad areas of biomol. recognition, intrinsically disordered proteins, and biosensor technol.
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    Wolfe, A. J.; Si, W.; Zhang, Z.; Blanden, A. R.; Hsueh, Y.-C.; Gugel, J. F.; Pham, B.; Chen, M.; Loh, S. N.; Rozovsky, S.; Aksimentiev, A.; Movileanu, L. Quantification of membrane protein-detergent complex interactions. J. Phys. Chem. B 2017, 121, 10228– 10241,  DOI: 10.1021/acs.jpcb.7b08045
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    Quantification of Membrane Protein-Detergent Complex Interactions
    Wolfe, Aaron J.; Si, Wei; Zhang, Zhengqi; Blanden, Adam R.; Hsueh, Yi-Ching; Gugel, Jack F.; Pham, Bach; Chen, Min; Loh, Stewart N.; Rozovsky, Sharon; Aksimentiev, Aleksei; Movileanu, Liviu
    Journal of Physical Chemistry B (2017), 121 (44), 10228-10241CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
    Although fundamentally significant in structural, chem., and membrane biol., the interfacial protein-detergent complex (PDC) interactions have been modestly examd. because of the complicated behavior of both detergents and membrane proteins in aq. phase. Membrane proteins are prone to unproductive aggregation resulting from poor detergent solvation, but the participating forces in this phenomenon remain ambiguous. Here, using rational membrane protein design, targeted chem. modification, and steady-state fluorescence polarization spectroscopy, the detergent desolvation of membrane proteins can be quant. evaluated. Depleting the detergent in the sample well produced a two-state transition of membrane proteins between a fully detergent-solvated state and a detergent-desolvated state, the nature of which depended on the interfacial PDC interactions. Using a panel of six membrane proteins of varying hydrophobic topog., structural fingerprint, and charge distribution on the solvent-accessible surface, the authors provide direct exptl. evidence for the contributions of the electrostatic and hydrophobic interactions to the protein solvation properties. Moreover, all-atom mol. dynamics simulations report the major contribution of the hydrophobic forces exerted at the PDC interface. This semiquant. approach might be extended in the future to include studies of the interfacial PDC interactions of other challenging membrane protein systems of unknown structure. This would have practical importance in protein extn., solubilization, stabilization, and crystn.
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    Wolfe, A. J.; Gugel, J. F.; Chen, M.; Movileanu, L. Kinetics of Membrane Protein-Detergent Interactions Depend on Protein Electrostatics. J. Phys. Chem. B 2018, 122, 9471– 9481,  DOI: 10.1021/acs.jpcb.8b07889
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    Kinetics of Membrane Protein-Detergent Interactions Depend on Protein Electrostatics
    Wolfe, Aaron J.; Gugel, Jack F.; Chen, Min; Movileanu, Liviu
    Journal of Physical Chemistry B (2018), 122 (41), 9471-9481CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
    Interactions of a membrane protein with a detergent micelle represent a fundamental process with practical implications in structural and chem. biol. Quant. assessment of the kinetics of protein-detergent complex (PDC) interactions has always been challenged by complicated behavior of both membrane proteins and solubilizing detergents in aq. phase. Here, we show the kinetic reads of the desorption of maltoside-contg. detergents from β-barrel membrane proteins. Using steady-state fluorescence polarization (FP) anisotropy measurements, we recorded real-time, specific signatures of the PDC interactions. The results of these measurements were used to infer the model-dependent rate consts. of assocn. and dissocn. of the proteomicelles. Remarkably, the kinetics of the PDC interactions depend on the overall protein charge despite the nonionic nature of the detergent monomers. In the future, this approach might be employed for high-throughput screening of kinetic fingerprints of different membrane proteins stabilized in micelles that contain mixts. of various detergents.
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    Forbes, S. A.; Beare, D.; Boutselakis, H.; Bamford, S.; Bindal, N.; Tate, J.; Cole, C. G.; Ward, S.; Dawson, E.; Ponting, L.; Stefancsik, R.; Harsha, B.; Kok, C. Y.; Jia, M.; Jubb, H.; Sondka, Z.; Thompson, S.; De, T.; Campbell, P. J. COSMIC: somatic cancer genetics at high-resolution. Nucleic Acids Res. 2017, 45, D777– d783,  DOI: 10.1093/nar/gkw1121
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    COSMIC: somatic cancer genetics at high-resolution
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    COSMIC, the Catalog of Somatic Mutations in Cancer (http://cancer.sanger.ac.uk) is a high-resoln. resource for exploring targets and trends in the genetics of human cancer. Currently the broadest database of mutations in cancer, the information in COSMIC is curated by expert scientists, primarily by scrutinizing large nos. of scientific publications. Over 4 million coding mutations are described in v78 (Sept. 2016), combining genome-wide sequencing results from 28 366 tumors with complete manual curation of 23 489 individual publications focused on 186 key genes and 286 key fusion pairs across all cancers. Mol. profiling of large tumor nos. has also allowed the annotation of more than 13 million non-coding mutations, 18 029 gene fusions, 187 429 genome rearrangements, 1 271 436 abnormal copy no. segments, 9 175 462 abnormal expression variants and 7 879 142 differentially methylated CpG dinucleotides. COSMIC now details the genetics of drug resistance, novel somatic gene mutations which allow a tumor to evade therapeutic cancer drugs. Focusing initially on highly characterized drugs and genes, COSMIC v78 contains wide resistance mutation profiles across 20 drugs, detailing the recurrence of 301 unique resistance alleles across 1934 drug-resistant tumors. All information from the COSMIC database is available freely on the COSMIC website.
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    The electrostatic free energy contribution of an ion pair in a protein depends on two factors, geometrical orientation of the side-chain charged groups with respect to each other and the structural context of the ion pair in the protein. Conformers in NMR ensembles enable studies of the relationship between geometry and electrostatic strengths of ion pairs, because the protein structural contexts are highly similar across different conformers. We have studied this relationship using a dataset of 22 unique ion pairs in 14 NMR conformer ensembles for 11 nonhomologous proteins. In different NMR conformers, the ion pairs are classified as salt bridges, nitrogen-oxygen (N-O) bridges and longer-range ion pairs on the basis of geometrical criteria. In salt bridges, centroids of the side-chain charged groups and at least a pair of side-chain nitrogen and oxygen atoms of the ion-pairing residues are within a 4 Å distance. In N-O bridges, at least a pair of the side-chain nitrogen and oxygen atoms of the ion-pairing residues are within 4 Å distance, but the distance between the side-chain charged group centroids is greater than 4 Å. In the longer-range ion pairs, the side-chain charged group centroids as well as the side-chain nitrogen and oxygen atoms are more than 4 Å apart. Continuum electrostatic calcns. indicate that most of the ion pairs have stabilizing electrostatic contributions when their side-chain charged group centroids are within 5 Å distance. Hence, most (∼92%) of the salt bridges and a majority (68%) of the N-O bridges are stabilizing. Most (∼89%) of the destabilizing ion pairs are the longer-range ion pairs. In the NMR conformer ensembles, the electrostatic interaction between side-chain charged groups of the ion-pairing residues is the strongest for salt bridges, considerably weaker for N-O bridges, and the weakest for longer-range ion pairs. These results suggest empirical rules for stabilizing electrostatic interactions in proteins.
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    Several biophys. approaches are available to study protein-protein interactions. Most approaches are conducted in bulk soln. and are therefore limited to an av. measurement of the ensemble of mol. interactions. Here, single-particle EM can enrich the understanding of protein-protein interactions at the single-mol. level and potentially capture states that are unobservable with ensemble methods because they are below the limit of detection or not conducted on an appropriate time scale. Using the HIV-1 envelope glycoprotein (Env) and its interaction with receptor CD4-binding site neutralizing antibodies as a model system, the authors both corroborate ensemble kinetics-derived parameters and demonstrate how time-course EM can further dissect stoichiometric states of complexes that are not readily observable with other methods. Visualization of the kinetics and stoichiometry of Env-antibody complexes demonstrated the applicability of this approach to qual. and semi-quant. differentiate two highly similar neutralizing antibodies. Furthermore, implementation of machine-learning techniques for sorting class avs. of these complexes into discrete subclasses of particles helped reduce human bias. The authors' data provide proof of concept that single-particle EM can be used to generate a "visual" kinetic profile that should be amenable to studying many other protein-protein interactions, is relatively simple and complementary to well-established biophys. approaches. Moreover, the authors' method provides crit. insights into broadly neutralizing antibody recognition of Env, which may inform vaccine immunogen design and immunotherapeutic development.
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    Wu, H.-T.; Liu, Y.-E.; Hsu, K.-W.; Wang, Y.-F.; Chan, Y.-C.; Chen, Y.; Chen, D.-R. MLL3 Induced by Luteolin Causes Apoptosis in Tamoxifen-Resistant Breast Cancer Cells through H3K4 Monomethylation and Suppression of the PI3K/AKT/mTOR Pathway. Am. J. Chin. Med. 2020, 48, 1221– 1241,  DOI: 10.1142/s0192415x20500603
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    American Journal of Chinese Medicine (2020), 48 (5), 1221-1241CODEN: AJCMBA; ISSN:0192-415X. (World Scientific Publishing Co. Pte. Ltd.)
    Tamoxifen is one of the most common hormone therapy drug for estrogen receptor pos. breast cancer. Tumor cells with drug resistance often cause recurrence and metastasis in cancer patients. Luteolin is a natural compd. found from various types of vegetables and exhibit anticancer activity in different cancers. This study demonstrated that luteolin inhibits the proliferation and induces apoptosis of tamoxifen-resistant ER-pos. breast cancer cells. Luteolin also causes cell cycle arrest at the G2/M phase and decreases mitochondrial membrane potential. Besides, luteolin reduces the levels of activated PI3K/AKT/mTOR signaling pathway. The combination treatment of luteolin and PI3K, AKT, or mTOR inhibitors synergistically increases apoptosis in tamoxifen-resistant ER-pos. breast cancer cells. Ras gene family (K-Ras, H-Ras, and N-Ras), an activator of PI3K, was transcriptionally repressed by luteolin via induction of tumor suppressor mixed-lineage leukemia 3 expression. MLL3 increases the level of monomethylation of Histone 3 Lysine 4 on the enhancer and promoter region of Ras genes, thus causes repression of Ras expressions. Our finding implies that luteolin was a promising natural agent against tamoxifen resistance of breast cancer.
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    Shinsky, S. A.; Cosgrove, M. S. Unique Role of the WD-40 Repeat Protein 5 (WDR5) Subunit within the Mixed Lineage Leukemia 3 (MLL3) Histone Methyltransferase Complex. J. Biol. Chem. 2015, 290, 25819– 25833,  DOI: 10.1074/jbc.m115.684142
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    Unique role of the WD-40 repeat protein 5 (WDR5) subunit within the mixed lineage leukemia 3 (MLL3) histone methyltransferase complex
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    The MLL3 (mixed lineage leukemia 3) protein is a member of the human SET1 family of histone H3 lysine 4 methyltransferases and contains the conserved WDR5 interaction (Win) motif and the catalytic suppressor of variegation, enhancer of zeste, trithorax (SET) domain. The human SET1 family includes MLL1-4 and SETd1A/B, which all interact with a conserved subcomplex contg. WDR5, RbBP5, Ash2L, and DPY-30 (WRAD) to form the minimal core complex required for full methyltransferase activity. However, recent evidence suggests that the WDR5 subunit may not be utilized in an identical manner within all SET1 family core complexes. Although the roles of WDR5 within the MLL1 core complex have been extensively studied, not much is known about the roles of WDR5 in other SET1 family core complexes. In this investigation, we set out to characterize the roles of the WDR5 subunit in the MLL3 core complex. We found that unlike MLL1, the MLL3 SET domain assembles with the RbBP5/Ash2L heterodimer independently of the Win motif-WDR5 interaction. Furthermore, we obsd. that WDR5 inhibits the monomethylation activity of the MLL3 core complex, which is dependent on the Win motif. We also found evidence suggesting that the WRAD subcomplex catalyzes weak H3K4 monomethylation within the context of the MLL3 core complex. Furthermore, soln. structures of the MLL3 core complex assembled with and without WDR5 by small angle x-ray scattering show similar overall topologies. Together, this work demonstrates a unique role for WDR5 in modulating the enzymic activity of the MLL3 core complex.
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    Histone H3 lysine 4 (K4) methylation has been linked to the transcriptional activation in a variety of eukaryotic species. Here authors show that a common component of MLL1, MLL2, and hSet1 H3 K4 methyltransferase complexes, the WD40-repeat protein WDR5, directly assocs. with histone H3 di- and trimethylated at K4 and with H3-K4-dimethylated nucleosomes. WDR5 is required for binding of the methyltransferase complex to the K4-dimethylated H3 tail as well as for global H3 K4 trimethylation and HOX gene activation in human cells. WDR5 is essential for vertebrate development, in that WDR5-depleted X. laevis tadpoles exhibit a variety of developmental defects and abnormal spatial Hox gene expression. These results are the first demonstration that a WD40-repeat protein acts as a module for recognition of a specific histone modification and suggest a mechanism for reading and writing an epigenetic mark for gene activation.
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    MLL, the trithorax ortholog, is a well-characterized histone 3 lysine 4 methyltransferase that is crucial for proper regulation of the Hox genes during embryonic development. Chromosomal translocations, disrupting the Mll gene, lead to aggressive leukemia with poor prognosis. However, the functions of MLL in cellular processes like cell-cycle regulation are not well studied. Here we show that the MLL has a regulatory role during multiple phases of the cell cycle. RNAi-mediated knockdown reveals that MLL regulates S-phase progression and, proper segregation and cytokinesis during M phase. Using deletions and mutations, we narrow the cell-cycle regulatory role to the C subunit of MLL. Our anal. reveals that the transactivation domain and not the SET domain is important for the S-phase function of MLL. Surprisingly, disruption of MLL-WRAD interaction is sufficient to disrupt proper mitotic progression. These mitotic functions of WRAD are independent of SET domain of MLL and, therefore, define a new role of WRAD in subset of MLL functions. Finally, we address the overlapping and unique roles of the different SET family members in the cell cycle.
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    How to measure and evaluate binding affinities
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    Quant. measurements of biomol. assocns. are central to biol. understanding and are needed to build and test predictive and mechanistic models. Given the advances in high-throughput technologies and the projected increase in the availability of binding data, we found it esp. timely to evaluate the current stds. for performing and reporting binding measurements. A review of 100 studies revealed that in most cases essential controls for establishing the appropriate incubation time and concn. regime were not documented, making it impossible to det. measurement reliability. Moreover, several reported affinities could be concluded to be incorrect, thereby impacting biol. interpretations. Given these challenges, we provide a framework for a broad range of researchers to evaluate, teach about, perform, and clearly document high-quality equil. binding measurements. We apply this framework and explain underlying fundamental concepts through exptl. examples with the RNA-binding protein Puf4.
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    Wolfe, A. J.; Hsueh, Y.-C.; Blanden, A. R.; Mohammad, M. M.; Pham, B.; Thakur, A. K.; Loh, S. N.; Chen, M.; Movileanu, L. Interrogating Detergent Desolvation of Nanopore-Forming Proteins by Fluorescence Polarization Spectroscopy. Anal. Chem. 2017, 89, 8013– 8020,  DOI: 10.1021/acs.analchem.7b01339
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    Interrogating Detergent Desolvation of Nanopore-Forming Proteins by Fluorescence Polarization Spectroscopy
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    Analytical Chemistry (Washington, DC, United States) (2017), 89 (15), 8013-8020CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Understanding how membrane proteins interact with detergents is of fundamental and practical significance in structural and chem. biol. as well as in nanobiotechnol. Current methods for inspecting protein-detergent complex (PDC) interfaces require high concns. of protein and are of low throughput. Here, the authors describe a scalable, spectroscopic approach that uses nanomolar protein concns. in native solns. This approach, which is based on steady-state fluorescence polarization (FP) spectroscopy, kinetically resolves the dissocn. of detergents from membrane proteins and protein unfolding. For satisfactorily solubilizing detergents, at concns. much greater than the crit. micelle concn. (CMC), the fluorescence anisotropy was independent of detergent concn. In contrast, at detergent concns. comparable with or below the CMC, the anisotropy readout underwent a time-dependent decrease, showing a specific and sensitive protein unfolding signature. Functionally reconstituted membrane proteins into a bilayer membrane confirmed predictions made by these FP-based detns. with respect to varying refolding conditions. From a practical point of view, this 96-well anal. approach will facilitate a massively parallel assessment of the PDC interfacial interactions under a fairly broad range of micellar and environmental conditions. The authors expect that these studies will potentially accelerate research in membrane proteins pertaining to their extn., solubilization, stabilization, and crystn., as well as reconstitution into bilayer membranes.

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  1. Lauren A. Mayse, Yazheng Wang, Mohammad Ahmad, Liviu Movileanu. Real-Time Measurement of a Weak Interaction of a Transcription Factor Motif with a Protein Hub at Single-Molecule Precision. ACS Nano 2024, 18 (31) , 20468-20481. https://doi.org/10.1021/acsnano.4c04857
  2. Lauren A. Mayse, Ali Imran, Yazheng Wang, Mohammad Ahmad, Rebecca A. Oot, Stephan Wilkens, Liviu Movileanu. Evaluation of Nanopore Sensor Design Using Electrical and Optical Analyses. ACS Nano 2023, 17 (11) , 10857-10871. https://doi.org/10.1021/acsnano.3c02532
  3. Mohammad Ahmad, Ali Imran, Liviu Movileanu. Overlapping characteristics of weak interactions of two transcriptional regulators with WDR5. International Journal of Biological Macromolecules 2024, 258 , 128969. https://doi.org/10.1016/j.ijbiomac.2023.128969
  4. I. V. Astratenkova, N. D. Golberg, V. A. Rogozkin. Regulation of Gene Expression by the MYC Transcription Factor Network During Exercise. Human Physiology 2023, 49 (4) , 444-452. https://doi.org/10.1134/S036211972360011X
  5. I. V. Astratenkova, N. D. Golberg, V. A. Rogozkin. Regulation of Gene Expression by the MYC Transcription Factor Network during Exercise. Физиология человека 2023, 49 (4) , 124-132. https://doi.org/10.31857/S0131164622601014
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  • Abstract

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    Figure 1

    Figure 1. Two binding sites of WDR5 and the structure of the WDR5-MLL3Win complex. (a) Representations of the Win and WBM binding sites of WDR5. Orientations of WDR5 in the two cartoons are 180° with respect to each other. (b) Top view of the WDR5-MLL3Win complex. (c) Side view of the WDR5-MLL3Win complex. (d) Side view of the interaction sites between MLL3Win (green) and WDR5 (gray). All presented residues are within 5 Å of the other binding partner. The residues corresponding to the WDR5 mutations explored in this study are marked in dark gray. MLL3Win residues are labeled as well. Potential hydrogen bonds between the two binding partners are shown as yellow dotted lines. (e) Key residues of the WDR5 binding cavity involved in hydrogen bonding with the evolutionarily conserved Arg residue (R4710) of MLL3Win at position P0 (Table S1). The hydrogen bonds are indicated by thick dashed lines marked in yellow. The cutoff distance for identifying these hydrogen bonds was 4.0 Å. WDR5 was represented using pdb entry 4ERY. (35)

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    Figure 2

    Figure 2. Label-free optical BLI sensorgrams of the WDR5 mutant-MLL3Win interactions. (a) Locations of the surface and cavity WDR5 mutations are shown in blue using surface and cross-sectional views of WDR5, respectively. (b) BLI sensorgrams showing the association and dissociation phases. For each WDR5 mutation, sensors with immobilized MLL3Win ligand were immersed in buffers containing different WDR5 concentrations (listed on sensorgrams) to monitor association kinetics. Sensors were then transferred to buffer alone to monitor dissociation kinetics.

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    Figure 3

    Figure 3. Normalized dissociation rate constants of the WDR5 mutant-SET1Win interactions using BLI sensorgrams. The koff values for each SET1Win ligand’s interaction with mutants have been divided by the koff of that SET1Win ligand’s interaction with the native WDR5 protein. (a) MLL2Win, (b) MLL3Win, (c) MLL4Win, (d) SETd1AWin, and (e) SETd1BWin. ND stands for “Not Determined”. Using a BLI measurement, the interaction between F133L and MLL4Win was detectable, but not quantifiable.

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    Figure 4

    Figure 4. Normalized KD of the WDR5 mutant-SET1Win interactions using BLI sensorgrams. The KD values for each SET1Win ligand’s interaction with WDR5 mutants have been divided by the KD of that SET1Win ligand’s interaction with the native WDR5 protein. (a) MLL2Win, (b) MLL3Win, (c) MLL4Win, (d) SETd1AWin, and (e) SETd1BWin. ND stands for “Not Determined”. Using a BLI measurement, the interaction between F133L and MLL4Win was detectable, but not quantifiable.

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    Figure 5

    Figure 5. Normalized KD of the WDR5 mutant-SET1Win interactions using steady-state FP spectroscopy. The KD values for each SET1Win ligand’s interaction with WDR5 mutants have been divided by the KD of that SET1Win ligand’s interaction with the native WDR5 protein. (a) MLL1Win, (b) MLL2Win, (c) MLL3Win, (d) MLL4Win, (e) SETd1AWin, and (f) SETd1BWin. For vertical bars marked by “*”, the KD of those interactions could not be determined. Those values represent the lower-limit of the KD based on the highest WDR5 mutant concentrations used in this study.

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    Figure 6

    Figure 6. Quantitative comparison between affinity data resulting from BLI and FP measurements. (a) 3D graph of the ratio of the normalized KD-BLI to the normalized KD-FP. (b) Two-dimensional heat map of the ratio of the normalized KD-BLI to the normalized KD-FP. Normalized KD values are the KD measured for a specific WDR5 mutant-SET1Win interaction pair divided by the KD value corresponding to the native WDR5 protein.

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  • References

    This article references 76 other publications.

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      A review. The WD40 domain exhibits a β-propeller architecture, often comprising seven blades. The WD40 domain is one of the most abundant domains and also among the top interacting domains in eukaryotic genomes. In this review, we will discuss the identification, definition and architecture of the WD40 domains. WD40 domain proteins are involved in a large variety of cellular processes, in which WD40 domains function as a protein-protein or protein-DNA interaction platform. WD40 domain mediates mol. recognition events mainly through the smaller top surface, but also through the bottom surface and sides. So far, no WD40 domain has been found to display enzymic activity. We will also discuss the different binding modes exhibited by the large versatile family of WD40 domain proteins. In the last part of this review, we will discuss how post-translational modifications are recognized by WD40 domain proteins.
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      Zhang, C.; Zhang, F. The Multifunctions of WD40 Proteins in Genome Integrity and Cell Cycle Progression. J. Genomics 2015, 3, 40– 50,  DOI: 10.7150/jgen.11015
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      Eukaryotic genome encodes numerous WD40 repeat proteins, which generally function as platforms of protein-protein interactions and are involved in numerous biological process, such as signal transduction, gene transcriptional regulation, protein modifications, cytoskeleton assembly, vesicular trafficking, DNA damage and repair, cell death and cell cycle progression. Among these diverse functions, genome integrity maintenance and cell cycle progression are extremely important as deregulation of them is clinically linked to uncontrolled proliferative diseases such as cancer. Thus, we mainly summarize and discuss the recent understanding of WD40 proteins and their molecular mechanisms linked to genome stability and cell cycle progression in this review, thereby demonstrating their pervasiveness and importance in cellular networks.
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      Afanasieva, E.; Chaudhuri, I.; Martin, J.; Hertle, E.; Ursinus, A.; Alva, V.; Hartmann, M. D.; Lupas, A. N. Structural diversity of oligomeric β-propellers with different numbers of identical blades. eLife 2019, 8, e49853  DOI: 10.7554/eLife.49853
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      eLife (2019), 8 (), e49853/1-e49853/15CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)
      β-Propellers arise through the amplification of a supersecondary structure element called a blade. This process produces toroids of between four and twelve repeats, which are almost always arranged sequentially in a single polypeptide chain. We found that new propellers evolve continuously by amplification from single blades. We therefore investigated whether such nascent propellers can fold as homo-oligomers before they have been fully amplified within a single chain. One- to six-bladed building blocks derived from two seven-bladed WD40 propellers yielded stable homo-oligomers with six to nine blades, depending on the size of the building block. High resoln. structures for tetramers of two blades, trimers of three blades, and dimers of four and five blades, resp., show structurally diverse propellers and include a novel fold, highlighting the inherent flexibility of the WD40 blade. Our data support the hypothesis that subdomain-sized fragments can provide structural versatility in the evolution of new proteins.
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      Stirnimann, C. U.; Petsalaki, E.; Russell, R. B.; Müller, C. W. WD40 proteins propel cellular networks. Trends Biochem. Sci. 2010, 35, 565– 574,  DOI: 10.1016/j.tibs.2010.04.003
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      WD40 proteins propel cellular networks
      Stirnimann, Christian U.; Petsalaki, Evangelia; Russell, Robert B.; Mueller, Christoph W.
      Trends in Biochemical Sciences (2010), 35 (10), 565-574CODEN: TBSCDB; ISSN:0968-0004. (Elsevier Ltd.)
      A review. Recent findings indicate that WD40 domains play central roles in biol. processes by acting as hubs in cellular networks; however, they have been studied less intensely than other common domains, such as the kinase, PDZ or SH3 domains. As suggested by various interactome studies, they are among the most promiscuous interactors. Structural studies suggest that this property stems from their ability, as scaffolds, to interact with diverse proteins, peptides or nucleic acids using multiple surfaces or modes of interaction. A general scaffolding role is supported by the fact that no WD40 domain has been found with intrinsic enzymic activity despite often being part of large mol. machines. We discuss the WD40 domain distributions in protein networks and structures of WD40-contg. assemblies to demonstrate their versatility in mediating crit. cellular functions.
    5. 5
      Santosh Kumar, H. S.; Kumar, V.; Kumar, V.; Pattar, S.; Telkar, S. Towards the construction of an interactome for Human WD40 protein family. Bioinformation 2016, 12, 54– 61,  DOI: 10.6026/97320630012054
      5
      Towards the construction of an interactome for Human WD40 protein family
      Santosh Kumar Hulikal Shivashankara; Kumar Vadlapudi; Pattar Sharath; Telkar Sandeep
      Bioinformation (2016), 12 (2), 54-61 ISSN:0973-2063.
      WD40 proteins are involved in a variety of protein-protein interactions as part of a multi-protein assembly modulating diverse and critical cellular process. It is known that several proteins of this family have been implicated in different disorders such as developmental abnormalities and cancer. However, molecular functions of many proteins in this family are yet unknown and it is of clinical interest. Therefore, it is of interest to define, construct, understand, analyze, evaluate, redefine and refine an interactome for WD40 protein family. We used data from literature mining using Cytoscape followed by linear regression analysis between Betweenness centrality and stress scores to define a model to filter the nodes in a representative WD40 interactome construction. We identified 10 ranked nodes in this analysis and subsequent microarray data selected three of them in insulin resistance that is further demonstrated in HepG2 cell culture models. We also observed the expression of GRWD1, RBBP5 and WDR5 genes during perturbation. Thus, we report hub nodes of WD40 interactome in insulin resistance. It should be noted that the pipeline using protein interaction network help find new proteins of clinical importance.
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      Migliori, V.; Mapelli, M.; Guccione, E. On WD40 proteins: propelling our knowledge of transcriptional control?. Epigenetics 2012, 7, 815– 822,  DOI: 10.4161/epi.21140
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      On WD40 proteins: propelling our knowledge of transcriptional control?
      Migliori, Valentina; Mapelli, Marina; Guccione, Ernesto
      Epigenetics (2012), 7 (8), 815-822CODEN: EPIGB6; ISSN:1559-2294. (Landes Bioscience)
      A review. A direct effect of post-translational modifications (PTMs) on nucleosomes is the formation of a dynamic platform able to assemble the transcriptional machinery and to recruit chromatin modifiers. The histone code hypothesis suggests that histone PTMs can act as binding sites for chromatin readers and effector proteins, such as the bromodomains, that selectively interact with acetylated lysines, or the "royal family" and the PHD finger domains, which are able to recognize methylated arginines and lysines. In this review we will discuss recent data describing the function of WD40 proteins as a new class of histone readers, with particular emphasis on the ones able to recognize methylated arginine and lysine residues. We will discuss how WDR5, a classical seven-bladed WD40 propeller, is able to bind with similar affinities both the catalytic subunit of the Trithorax-like complexes, and the histone H3 tail either unmodified or sym. dimethylated on arginine 2 (H3R2me2s). Furthermore, we will speculate on how these mutually exclusive interactions of WDR5 may play a role in mediating different degrees of H3K4 methylations at both promoters and distal regulatory sites. Finally, we will summarize recent literature elucidating how other WD40 proteins such as NURF55, EED and LRWD1 recognize methylated histone tails, highlighting similarities and differences among them.
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      Jain, B. P.; Pandey, S. WD40 Repeat Proteins: Signalling Scaffold with Diverse Functions. Protein J. 2018, 37, 391– 406,  DOI: 10.1007/s10930-018-9785-7
      7
      WD40 Repeat Proteins: Signalling Scaffold with Diverse Functions
      Jain, Buddhi Prakash; Pandey, Shweta
      Protein Journal (2018), 37 (5), 391-406CODEN: PJROAH; ISSN:1572-3887. (Springer)
      A review. The WD40 domain is one of the most abundant and interacting domains in the eukaryotic genome. In proteins the WD domain folds into a β-propeller structure, providing a platform for the interaction and assembly of several proteins into a signalosome. WD40 repeats contg. proteins, in lower eukaryotes, are mainly involved in growth, cell cycle, development and virulence, while in higher organisms, they play an important role in diverse cellular functions like signal transduction, cell cycle control, intracellular transport, chromatin remodelling, cytoskeletal organization, apoptosis, development, transcriptional regulation, immune responses. To play the regulatory role in various processes, they act as a scaffold for protein-protein or protein-DNA interaction. So far, no WD40 domain has been identified with intrinsic enzymic activity. Several WD40 domain-contg. proteins have been recently characterized in prokaryotes as well. The review summarizes the vast array of functions performed by different WD40 domain contg. proteins, their domain organization and functional conservation during the course of evolution.
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      Cosgrove, M. S.; Patel, A. Mixed lineage leukemia: a structure-function perspective of the MLL1 protein. FEBS J. 2010, 277, 1832– 1842,  DOI: 10.1111/j.1742-4658.2010.07609.x
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      Mixed lineage leukemia: a structure-function perspective of the MLL1 protein
      Cosgrove, Michael S.; Patel, Anamika
      FEBS Journal (2010), 277 (8), 1832-1842CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)
      A review. Several acute lymphoblastic and myelogenous leukemias are correlated with alterations in the human mixed lineage leukemia protein-1 (MLL1) gene. MLL1 is a member of the evolutionarily conserved SET1 family of histone H3 lysine 4 (H3K4) methyltransferases, which are required for the regulation of distinct groups of developmentally regulated genes in metazoans. Despite the important biol. role of SET1 family enzymes and their involvement in human leukemias, relatively little is understood about how these enzymes work. Here, the authors review several recent structural and biochem. studies that are beginning to shed light on the mol. mechanisms for the regulation of H3K4 methylation by the human MLL1 enzyme.
    9. 9
      Li, Y.; Han, J.; Zhang, Y.; Cao, F.; Liu, Z.; Li, S.; Wu, J.; Hu, C.; Wang, Y.; Shuai, J.; Chen, J.; Cao, L.; Li, D.; Shi, P.; Tian, C.; Zhang, J.; Dou, Y.; Li, G.; Chen, Y.; Lei, M. Structural basis for activity regulation of MLL family methyltransferases. Nature 2016, 530, 447– 452,  DOI: 10.1038/nature16952
      9
      Structural basis for activity regulation of MLL family methyltransferases
      Li, Yanjing; Han, Jianming; Zhang, Yuebin; Cao, Fang; Liu, Zhijun; Li, Shuai; Wu, Jian; Hu, Chunyi; Wang, Yan; Shuai, Jin; Chen, Juan; Cao, Liaoran; Li, Dangsheng; Shi, Pan; Tian, Changlin; Zhang, Jian; Dou, Yali; Li, Guohui; Chen, Yong; Lei, Ming
      Nature (London, United Kingdom) (2016), 530 (7591), 447-452CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      The mixed lineage leukemia (MLL) family of proteins (including MLL1-MLL4, SET1A and SET1B) specifically methylate histone 3 Lys4, and have pivotal roles in the transcriptional regulation of genes involved in haematopoiesis and development. The methyltransferase activity of MLL1, by itself severely compromised, is stimulated by the three conserved factors WDR5, RBBP5 and ASH2L, which are shared by all MLL family complexes. However, the mol. mechanism of how these factors regulate the activity of MLL proteins still remains poorly understood. Here we show that a minimized human RBBP5-ASH2L heterodimer is the structural unit that interacts with and activates all MLL family histone methyltransferases. Our structural, biochem. and computational analyses reveal a two-step activation mechanism of MLL family proteins. These findings provide unprecedented insights into the common theme and functional plasticity in complex assembly and activity regulation of MLL family methyltransferases, and also suggest a universal regulation mechanism for most histone methyltransferases.
    10. 10
      Vedadi, M.; Blazer, L.; Eram, M. S.; Barsyte-Lovejoy, D.; Arrowsmith, C. H.; Hajian, T. Targeting human SET1/MLL family of proteins. Protein Sci. 2017, 26, 662– 676,  DOI: 10.1002/pro.3129
      10
      Targeting human SET1/MLL family of proteins
      Vedadi, Masoud; Blazer, Levi; Eram, Mohammad S.; Barsyte-Lovejoy, Dalia; Arrowsmith, Cheryl H.; Hajian, Taraneh
      Protein Science (2017), 26 (4), 662-676CODEN: PRCIEI; ISSN:1469-896X. (Wiley-Blackwell)
      The SET1 family of proteins, and in particular MLL1, are essential regulators of transcription and key mediators of normal development and disease. Here, we summarize the detailed characterization of the methyltransferase activity of SET1 complexes and the role of the key subunits, WDR5, RbBP5, ASH2L, and DPY30. We present new data on full kinetic characterization of human MLL1, MLL3, SET1A, and SET1B trimeric, tetrameric, and pentameric complexes to elaborate on substrate specificities and compare our findings with what has been reported before. We also review exciting recent work identifying potent inhibitors of oncogenic MLL1 function through disruption of protein-protein interactions within the MLL1 complex.
    11. 11
      Xue, H.; Yao, T.; Cao, M.; Zhu, G.; Li, Y.; Yuan, G.; Chen, Y.; Lei, M.; Huang, J. Structural basis of nucleosome recognition and modification by MLL methyltransferases. Nature 2019, 573, 445– 449,  DOI: 10.1038/s41586-019-1528-1
      11
      Structural basis of nucleosome recognition and modification by MLL methyltransferases
      Xue, Han; Yao, Tonghui; Cao, Mi; Zhu, Guanjun; Li, Yan; Yuan, Guiyong; Chen, Yong; Lei, Ming; Huang, Jing
      Nature (London, United Kingdom) (2019), 573 (7774), 445-449CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
      Methyltransferases of the mixed-lineage leukemia (MLL) family-which include MLL1, MLL2, MLL3, MLL4, SET1A and SET1B-implement methylation of histone H3 on lysine 4 (H3K4), and have crit. and distinct roles in the regulation of transcription in haematopoiesis, adipogenesis and development. The C-terminal catalytic SET (Su(var.)3-9, enhancer of zeste and trithorax) domains of MLL proteins are assocd. with a common set of regulatory factors (WDR5, RBBP5, ASH2L and DPY30) to achieve specific activities. Current knowledge of the regulation of MLL activity is limited to the catalysis of histone H3 peptides, and how H3K4 Me marks are deposited on nucleosomes is poorly understood. H3K4 methylation is stimulated by mono-ubiquitination of histone H2B on lysine 120 (H2BK120ub1), a prevalent histone H2B mark that disrupts chromatin compaction and favors open chromatin structures, but the underlying mechanism remains unknown10-12. Here we report cryo-electron microscopy structures of human MLL1 and MLL3 catalytic modules assocd. with nucleosome core particles that contain H2BK120ub1 or unmodified H2BK120. These structures demonstrate that the MLL1 and MLL3 complexes both make extensive contacts with the histone-fold and DNA regions of the nucleosome; this allows ease of access to the histone H3 tail, which is essential for the efficient methylation of H3K4. The H2B-conjugated ubiquitin binds directly to RBBP5, orienting the assocn. between MLL1 or MLL3 and the nucleosome. The MLL1 and MLL3 complexes display different structural organizations at the interface between the WDR5, RBBP5 and MLL1 (or the corresponding MLL3) subunits, which accounts for the opposite roles of WDR5 in regulating the activity of the two enzymes. These findings transform our understanding of the structural basis for the regulation of MLL activity at the nucleosome level, and highlight the pivotal role of nucleosome regulation in histone-tail modification.
    12. 12
      Jiang, H. The complex activities of the SET1/MLL complex core subunits in development and disease. Biochim. Biophys. Acta, Gene Regul. Mech. 2020, 1863, 194560,  DOI: 10.1016/j.bbagrm.2020.194560
      12
      The complex activities of the SET1/MLL complex core subunits in development and disease
      Jiang, Hao
      Biochimica et Biophysica Acta, Gene Regulatory Mechanisms (2020), 1863 (7), 194560CODEN: BBAGC6; ISSN:1874-9399. (Elsevier B.V.)
      In mammalian cells, the SET1/MLL complexes are the main writers of the H3K4 Me mark that is assocd. with active gene expression. The activities of these complexes are critically dependent on the assocn. of the catalytic subunit with their shared core subunits, WDR5, RBBP5, ASH2L, and DPY30, collectively referred as WRAD. In addn., some of these core subunits can bind to proteins other than the SET1/MLL complex components. This review starts with discussion of the mol. activities of these core subunits, with an emphasis on DPY30 in organizing the assembly of the SET1/MLL complexes with other assocd. factors. This review then focuses on the roles of the core subunits in stem cells and development, as well as in diseased cell states, mainly cancer, and ends with discussion on dissecting the responsible activities of the core subunits and how we may target them for potential disease treatment. This article is part of a Special Issue entitled: The MLL family of proteins in normal development and disease edited by Thomas A Milne.
    13. 13
      Sha, L.; Ayoub, A.; Cho, U.-S.; Dou, Y. Insights on the regulation of the MLL/SET1 family histone methyltransferases. Biochim. Biophys. Acta, Gene Regul. Mech. 2020, 1863, 194561,  DOI: 10.1016/j.bbagrm.2020.194561
      13
      Insights on the regulation of the MLL/SET1 family histone methyltransferases
      Sha, Liang; Ayoub, Alex; Cho, Uhn-Soo; Dou, Yali
      Biochimica et Biophysica Acta, Gene Regulatory Mechanisms (2020), 1863 (7), 194561CODEN: BBAGC6; ISSN:1874-9399. (Elsevier B.V.)
      A review. In eukaryotes, histone H3K4 methylation by the MLL/SET1 family histone methyltransferases is enriched at transcription regulatory elements including gene promoters and enhancers. The level of H3K4 methylation is highly correlated with transcription activation and is one of the most frequently used histone post-translational modifications to predict transcriptional outcome. Recently, it has been shown that rearrangement of the cellular landscape of H3K4 mono-methylation at distal enhancers precedes cell fate transition and is used for identification of novel regulatory elements for development and disease progression. Similarly, broad H3K4 tri-methylation regions have also been used to predict intrinsic tumor suppression properties of regulator regions in a variety of cellular models. Understanding the regulation for how H3K4 methylation is deposited and regulated is of paramount importance. In this review, we will discuss new findings on how the MLL/SET1 family enzymes are regulated on chromatin and their potential functional and regulatory implications. This article is part of a Special Issue entitled: The MLL family of proteins in normal development and disease edited by Thomas A Milne.
    14. 14
      Dou, Y.; Milne, T. A.; Ruthenburg, A. J.; Lee, S.; Lee, J. W.; Verdine, G. L.; Allis, C. D.; Roeder, R. G. Regulation of MLL1 H3K4 methyltransferase activity by its core components. Nat. Struct. Mol. Biol. 2006, 13, 713– 719,  DOI: 10.1038/nsmb1128
      14
      Regulation of MLL1 H3K4 methyltransferase activity by its core components
      Dou, Yali; Milne, Thomas A.; Ruthenburg, Alexander J.; Lee, Seunghee; Lee, Jae Woon; Verdine, Gregory L.; Allis, C. David; Roeder, Robert G.
      Nature Structural & Molecular Biology (2006), 13 (8), 713-719CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)
      Histone H3 Lys4 (H3K4) methylation is a prevalent mark assocd. with transcription activation. A common feature of several H3K4 methyltransferase complexes is the presence of three structural components (RbBP5, Ash2L and WDR5) and a catalytic subunit contg. a SET domain. Here we report the first biochem. reconstitution of a functional four-component mixed-lineage leukemia protein-1 (MLL1) core complex. This reconstitution, combined with in vivo assays, allows direct anal. of the contribution of each component to MLL1 enzymic activity and their roles in transcriptional regulation. Moreover, taking clues from a crystal structure anal., we demonstrate that WDR5 mediates interactions of the MLL1 catalytic unit both with the common structural platform and with the histone substrate. Mechanistic insights gained from this study can be generalized to the whole family of SET1-like histone methyltransferases in mammals.
    15. 15
      Patel, A.; Dharmarajan, V.; Vought, V. E.; Cosgrove, M. S. On the mechanism of multiple lysine methylation by the human mixed lineage leukemia protein-1 (MLL1) core complex. J. Biol. Chem. 2009, 284, 24242– 24256,  DOI: 10.1074/jbc.m109.014498
      15
      On the Mechanism of Multiple Lysine Methylation by the Human Mixed Lineage Leukemia Protein-1 (MLL1) Core Complex
      Patel, Anamika; Dharmarajan, Venkatasubramanian; Vought, Valarie E.; Cosgrove, Michael S.
      Journal of Biological Chemistry (2009), 284 (36), 24242-24256CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Transcription in eukaryotic genomes depends on enzymes that regulate the degree of histone H3 lysine 4 (H3K4) methylation. The mixed lineage leukemia protein-1 (MLL1) is a member of the SET1 family of H3K4 methyltransferases and is frequently rearranged in acute leukemias. Despite sequence comparisons that predict that SET1 family enzymes should only monomethylate their substrates, mono-, di-, and trimethylation of H3K4 has been attributed to SET1 family complexes in vivo and in vitro. To better understand this paradox, we have biochem. reconstituted and characterized a five-component 200-kDa MLL1 core complex contg. human MLL1, WDR5, RbBP5, Ash2L, and DPY-30. We demonstrate that the isolated MLL1 SET domain is a slow monomethyltransferase and that tyrosine 3942 of MLL1 prevents di- and trimethylation of H3K4. In contrast, a complex contg. the MLL1 SET domain, WDR5, RbBP5, Ash2L, and DPY-30, displays a marked ∼600-fold increase in enzymic activity but only to the di-Me form of H3K4. Single turnover kinetic expts. reveal that the reaction leading to H3K4 dimethylation involves the transient accumulation of a monomethylated species, suggesting that the MLL1 core complex uses a non-processive mechanism to catalyze multiple lysine methylation. We have also discovered that the non-SET domain components of the MLL1 core complex possess a previously unrecognized methyltransferase activity that catalyzes H3K4 dimethylation within the MLL1 core complex. Our results suggest that the mechanism of multiple lysine methylation by the MLL1 core complex involves the sequential addn. of two Me groups at two distinct active sites within the complex.
    16. 16
      Shinsky, S. A.; Monteith, K. E.; Viggiano, S.; Cosgrove, M. S. Biochemical reconstitution and phylogenetic comparison of human SET1 family core complexes involved in histone methylation. J. Biol. Chem. 2015, 290, 6361– 6375,  DOI: 10.1074/jbc.m114.627646
      16
      Biochemical Reconstitution and Phylogenetic Comparison of Human SET1 Family Core Complexes Involved in Histone Methylation
      Shinsky, Stephen A.; Monteith, Kelsey E.; Viggiano, Susan; Cosgrove, Michael S.
      Journal of Biological Chemistry (2015), 290 (10), 6361-6375CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Mixed lineage leukemia protein-1 (MLL1) is a member of the SET1 family of histone H3 lysine 4 (H3K4) methyltransferases that are required for metazoan development. MLL1 is the best characterized human SET1 family member, which includes MLL1-4 and SETd1A/B. MLL1 assembles with WDR5, RBBP5, ASH2L, DPY-30 (WRAD) to form the MLL1 core complex, which is required for H3K4 dimethylation and transcriptional activation. Because all SET1 family proteins interact with WRAD in vivo, it is hypothesized they are regulated by similar mechanisms. However, recent evidence suggests differences among family members that may reflect unique regulatory inputs in the cell. Missing is an understanding of the intrinsic enzymic activities of different SET1 family complexes under std. conditions. In this investigation, we reconstituted each human SET1 family core complex and compared subunit assembly and enzymic activities. We found that in the absence of WRAD, all but one SET domain catalyzes at least weak H3K4 monomethylation. In the presence of WRAD, all SET1 family members showed stimulated monomethyltransferase activity but differed in their di- and trimethylation activities. We found that these differences are correlated with evolutionary lineage, suggesting these enzyme complexes have evolved to accomplish unique tasks within metazoan genomes. To understand the structural basis for these differences, we employed a "phylogenetic scanning mutagenesis" assay and identified a cluster of amino acid substitutions that confer a WRAD-dependent gain-of-function dimethylation activity on complexes assembled with the MLL3 or Drosophila trithorax proteins. These results form the basis for understanding how WRAD differentially regulates SET1 family complexes in vivo.
    17. 17
      Thomas, L. R.; Wang, Q.; Grieb, B. C.; Phan, J.; Foshage, A. M.; Sun, Q.; Olejniczak, E. T.; Clark, T.; Dey, S.; Lorey, S.; Alicie, B.; Howard, G. C.; Cawthon, B.; Ess, K. C.; Eischen, C. M.; Zhao, Z.; Fesik, S. W.; Tansey, W. P. Interaction with WDR5 promotes target gene recognition and tumorigenesis by MYC. Mol. Cell. Biochem. 2015, 58, 440– 452,  DOI: 10.1016/j.molcel.2015.02.028
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    18. 18
      Thomas, L. R.; Adams, C. M.; Wang, J.; Weissmiller, A. M.; Creighton, J.; Lorey, S. L.; Liu, Q.; Fesik, S. W.; Eischen, C. M.; Tansey, W. P. Interaction of the oncoprotein transcription factor MYC with its chromatin cofactor WDR5 is essential for tumor maintenance. Proc. Natl. Acad. Sci. U.S.A. 2019, 116, 25260– 25268,  DOI: 10.1073/pnas.1910391116
      18
      Interaction of the oncoprotein transcription factor MYC with its chromatin cofactor WDR5 is essential for tumor maintenance
      Thomas, Lance R.; Adams, Clare M.; Wang, Jing; Weissmiller, April M.; Creighton, Joy; Lorey, Shelly L.; Liu, Qi; Fesik, Stephen W.; Eischen, Christine M.; Tansey, William P.
      Proceedings of the National Academy of Sciences of the United States of America (2019), 116 (50), 25260-25268CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The oncoprotein transcription factor MYC is overexpressed in the majority of cancers. Key to its oncogenic activity is the ability of MYC to regulate gene expression patterns that drive and maintain the malignant state. MYC is also considered a validated anticancer target, but efforts to pharmacol. inhibit MYC have failed. The dependence of MYC on cofactors creates opportunities for therapeutic intervention, but for any cofactor this requires structural understanding of how the cofactor interacts with MYC, knowledge of the role it plays in MYC function, and demonstration that disrupting the cofactor interaction will cause existing cancers to regress. One cofactor for which structural information is available is WDR5, which interacts with MYC to facilitate its recruitment to chromatin. To explore whether disruption of the MY-WDR5 interaction could potentially become a viable anticancer strategy, we developed a Burkitt's lymphoma system that allows replacement of wild-type MYC for mutants that are defective for WDR5 binding or all known nuclear MYC functions. Using this system, we show that WDR5 recruits MYC to chromatin to control the expression of genes linked to biomass accumulation. We further show that disrupting the MYC-WDR5 interaction within the context of an existing cancer promotes rapid and comprehensive tumor regression in vivo. These observations connect WDR5 to a core tumorigenic function of MYC and establish that, if a therapeutic window can be established, MYC-WDR5 inhibitors could be developed as anticancer agents.
    19. 19
      Thomas, L. R.; Adams, C. M.; Fesik, S. W.; Eischen, C. M.; Tansey, W. P. Targeting MYC through WDR5. Mol. Cell. Oncol. 2020, 7, 1709388,  DOI: 10.1080/23723556.2019.1709388
      19
      Targeting MYC through WDR5
      Thomas Lance R; Tansey William P; Adams Clare M; Eischen Christine M; Fesik Stephen W; Tansey William P
      Molecular & cellular oncology (2020), 7 (2), 1709388 ISSN:2372-3556.
      The oncoprotein transcription factor MYC is overexpressed in most cancers and is responsible for hundreds of thousands of cancer deaths worldwide every year. MYC is also a highly validated - but currently undruggable - anti-cancer target. We recently showed that breaking the interaction of MYC with its chromatin co-factor WD repeat-containing protein 5 (WDR5) promotes tumor regression in mouse xenografts, laying the foundation for a new strategy to inhibit MYC in the clinic.
    20. 20
      Thomas, L. R.; Foshage, A. M.; Weissmiller, A. M.; Tansey, W. P. The MYC-WDR5 Nexus and Cancer. Cancer Res. 2015, 75, 4012– 4015,  DOI: 10.1158/0008-5472.can-15-1216
      20
      The MYC-WDR5 Nexus and Cancer
      Thomas, Lance R.; Foshage, Audra M.; Weissmiller, April M.; Tansey, William P.
      Cancer Research (2015), 75 (19), 4012-4015CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)
      The MYC oncogenes encode a family of transcription factors that feature prominently in cancer. MYC proteins are overexpressed or deregulated in a majority of malignancies and drive tumorigenesis by inducing widespread transcriptional reprogramming that promotes cell proliferation, metab., and genomic instability. The ability of MYC to regulate transcription depends on its dimerization with MAX, which creates a DNA-binding domain that recognizes specific sequences in the regulatory elements of MYC target genes. Recently, we discovered that recognition of target genes by MYC also depends on its interaction with WDR5, a WD40-repeat protein that exists as part of several chromatin-regulatory complexes. Here, we discuss how interaction of MYC with WDR5 could create an avidity-based chromatin recognition mechanism that allows MYC to select its target genes in response to both genetic and epigenetic determinants. We rationalize how the MYC-WDR5 interaction provides plasticity in target gene selection by MYC and speculate on the biochem. and genomic contexts in which this interaction occurs. Finally, we discuss how properties of the MYC-WDR5 interface make it an attractive point for discovery of small-mol. inhibitors of MYC function in cancer cells.
    21. 21
      Guarnaccia, A. D.; Rose, K. L.; Wang, J.; Zhao, B.; Popay, T. M.; Wang, C. E.; Guerrazzi, K.; Hill, S.; Woodley, C. M.; Hansen, T. J.; Lorey, S. L.; Shaw, J. G.; Payne, W. G.; Weissmiller, A. M.; Olejniczak, E. T.; Fesik, S. W.; Liu, Q.; Tansey, W. P. Impact of WIN site inhibitor on the WDR5 interactome. Cell Rep. 2021, 34, 108636,  DOI: 10.1016/j.celrep.2020.108636
      21
      Impact of WIN site inhibitor on the WDR5 interactome
      Guarnaccia, Alissa D.; Rose, Kristie L.; Wang, Jing; Zhao, Bin; Popay, Tessa M.; Wang, Christina E.; Guerrazzi, Kiana; Hill, Salisha; Woodley, Chase M.; Hansen, Tyler J.; Lorey, Shelly L.; Shaw, J. Grace; Payne, William G.; Weissmiller, April M.; Olejniczak, Edward T.; Fesik, Stephen W.; Liu, Qi; Tansey, William P.
      Cell Reports (2021), 34 (3), 108636CODEN: CREED8; ISSN:2211-1247. (Cell Press)
      The chromatin-assocd. protein WDR5 is a promising pharmacol. target in cancer, with most drug discovery efforts directed against an arginine-binding cavity in WDR5 called the WIN site. Despite a clear expectation that WIN site inhibitors will alter the repertoire of WDR5 interaction partners, their impact on the WDR5 interactome remains unknown. Here, we use quant. proteomics to delineate how the WDR5 interactome is changed by WIN site inhibition. We show that the WIN site inhibitor alters the interaction of WDR5 with dozens of proteins, including those linked to phosphatidylinositol 3-kinase (PI3K) signaling. As proof of concept, we demonstrate that the master kinase PDPK1 is a bona fide high-affinity WIN site binding protein that engages WDR5 to modulate transcription of genes expressed in the G2 phase of the cell cycle. This dataset expands our understanding of WDR5 and serves as a resource for deciphering the action of WIN site inhibitors.
    22. 22
      Patel, A.; Dharmarajan, V.; Cosgrove, M. S. Structure of WDR5 bound to mixed lineage leukemia protein-1 peptide. J. Biol. Chem. 2008, 283, 32158– 32161,  DOI: 10.1074/jbc.c800164200
      22
      Structure of WDR5 Bound to Mixed Lineage Leukemia Protein-1 Peptide
      Patel, Anamika; Dharmarajan, Venkatasubramanian; Cosgrove, Michael S.
      Journal of Biological Chemistry (2008), 283 (47), 32158-32161CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      The mixed lineage leukemia protein-1 (MLL1) catalyzes histone H3 lysine 4 methylation and is regulated by interaction with WDR5 (WD-repeat protein-5), RbBP5 (retinoblastoma-binding protein-5), and the Ash2L (absent, small, homeotic disks-2-like) oncoprotein. In the accompanying investigation, we describe the identification of a conserved arginine contg. motif, called the "Win" or WDR5 interaction motif, that is essential for the assembly and H3K4 dimethylation activity of the MLL1 core complex. Here we present a 1.7-Å crystal structure of WDR5 bound to a peptide derived from the MLL1 Win motif. Our results show that Arg-3765 of MLL1 is bound in the same arginine binding pocket on WDR5 that was previously suggested to bind histone H3. Thermodn. binding expts. show that the MLL1 Win peptide is preferentially recognized by WDR5. These results are consistent with a model in which WDR5 recognizes Arg-3765 of MLL1, which is essential for the assembly and enzymic activity of the MLL1 core complex.
    23. 23
      Patel, A.; Vought, V. E.; Dharmarajan, V.; Cosgrove, M. S. A conserved arginine-containing motif crucial for the assembly and enzymatic activity of the mixed lineage leukemia protein-1 core complex. J. Biol. Chem. 2008, 283, 32162– 32175,  DOI: 10.1074/jbc.m806317200
      23
      A Conserved Arginine-containing Motif Crucial for the Assembly and Enzymatic Activity of the Mixed Lineage Leukemia Protein-1 Core Complex
      Patel, Anamika; Vought, Valarie E.; Dharmarajan, Venkatasubramanian; Cosgrove, Michael S.
      Journal of Biological Chemistry (2008), 283 (47), 32162-32175CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      The mixed lineage leukemia protein-1 (MLL1) belongs to the SET1 family of histone H3 lysine 4 methyltransferases. Recent studies indicate that the catalytic subunits of SET1 family members are regulated by interaction with a conserved core group of proteins that include the WD repeat protein-5 (WDR5), retinoblastoma-binding protein-5 (RbBP5), and the absent small homeotic-2-like protein (Ash2L). It has been suggested that WDR5 functions to bridge the interactions between the catalytic and regulatory subunits of SET1 family complexes. However, the mol. details of these interactions are unknown. To gain insight into the interactions among these proteins, we have detd. the biophys. basis for the interaction between the human WDR5 and MLL1. Our studies reveal that WDR5 preferentially recognizes a previously unidentified and conserved arginine-contg. motif, called the "Win" or WDR5 interaction motif, which is located in the N-SET region of MLL1 and other SET1 family members. Surprisingly, our structural and functional studies show that WDR5 recognizes arginine 3765 of the MLL1 Win motif using the same arginine-binding pocket on WDR5 that was previously shown to bind histone H3. We demonstrate that WDR5's recognition of arginine 3765 of MLL1 is essential for the assembly and enzymic activity of the MLL1 core complex in vitro.
    24. 24
      Song, J.-J.; Kingston, R. E. WDR5 interacts with mixed lineage leukemia (MLL) protein via the histone H3-binding pocket. J. Biol. Chem. 2008, 283, 35258– 35264,  DOI: 10.1074/jbc.m806900200
      24
      WDR5 Interacts with Mixed Lineage Leukemia (MLL) Protein via the Histone H3-binding Pocket
      Song, Ji-Joon; Kingston, Robert E.
      Journal of Biological Chemistry (2008), 283 (50), 35258-35264CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      WDR5 is a component of the mixed lineage leukemia (MLL) complex, which methylates lysine 4 of histone H3, and was identified as a methylated Lys-4 histone H3-binding protein. Here, we present a crystal structure of WDR5 bound to an MLL peptide. Surprisingly, we find that WDR5 utilizes the same pocket shown to bind histone H3 for this MLL interaction. Furthermore, the WDR5-MLL interaction is disrupted preferentially by mono- and di-methylated Lys-4 histone H3 over unmodified and tri-methylated Lys-4 histone H3. These data implicate a delicate interplay between the effector, WDR5, the catalytic subunit, MLL, and the substrate, histone H3, of the MLL complex. We suggest that the activity of the MLL complex might be regulated through this interplay.
    25. 25
      Odho, Z.; Southall, S. M.; Wilson, J. R. Characterization of a novel WDR5-binding site that recruits RbBP5 through a conserved motif to enhance methylation of histone H3 lysine 4 by mixed lineage leukemia protein-1. J. Biol. Chem. 2010, 285, 32967– 32976,  DOI: 10.1074/jbc.m110.159921
      25
      Characterization of a novel WDR5-binding site that recruits RbBP5 through a conserved motif to enhance methylation of histone H3 lysine 4 by mixed lineage leukemia protein-1
      Odho, Zain; Southall, Stacey M.; Wilson, Jon R.
      Journal of Biological Chemistry (2010), 285 (43), 32967-32976CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Histone modification is well established as a fundamental mechanism driving the regulation of transcription, replication, and DNA repair through the control of chromatin structure. Likewise, it is apparent that incorrect targeting of histone modifications contributes to misregulated gene expression and hence to developmental disorders and diseases of genomic instability such as cancer. The KMT2 family of SET domain methyltransferases, typified by mixed lineage leukemia protein-1 (MLL1), is responsible for histone H3 lysine 4 methylation, a marker of active genes. To ensure that this modification is correctly targeted, a multiprotein complex assocs. with the methyltransferase and directs activity. We have identified a novel interaction site on the core complex protein WD repeat protein-5 (WDR5), and we mapped the complementary site on its partner retinoblastoma-binding protein-5 (RbBP5). We have characterized this interaction by x-ray crystallog. and show how it is fundamental to the assembly of the complex and to the regulation of methyltransferase activity. We show which region of RbBP5 contributes directly to mixed lineage leukemia activation, and we combine our structural and biochem. data to produce a model to show how WDR5 and RbBP5 act cooperatively to stimulate activity.
    26. 26
      Chacón Simon, S.; Wang, F.; Thomas, L. R.; Phan, J.; Zhao, B.; Olejniczak, E. T.; Macdonald, J. D.; Shaw, J. G.; Schlund, C.; Payne, W.; Creighton, J.; Stauffer, S. R.; Waterson, A. G.; Tansey, W. P.; Fesik, S. W. Discovery of WD Repeat-Containing Protein 5 (WDR5)-MYC Inhibitors Using Fragment-Based Methods and Structure-Based Design. J. Med. Chem. 2020, 63, 4315– 4333,  DOI: 10.1021/acs.jmedchem.0c00224
      26
      Discovery of WD Repeat-Containing Protein 5 (WDR5)-MYC Inhibitors Using Fragment-Based Methods and Structure-Based Design
      Chacon Simon, Selena; Wang, Feng; Thomas, Lance R.; Phan, Jason; Zhao, Bin; Olejniczak, Edward T.; MacDonald, Jonathan D.; Shaw, J. Grace; Schlund, Caden; Payne, William; Creighton, Joy; Stauffer, Shaun R.; Waterson, Alex G.; Tansey, William P.; Fesik, Stephen W.
      Journal of Medicinal Chemistry (2020), 63 (8), 4315-4333CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      The frequent deregulation of MYC and its elevated expression via multiple mechanisms drives cells to a tumorigenic state. Indeed, MYC is overexpressed in up to ~ 50% of human cancers and is considered a highly validated anticancer target. Recently, we discovered that WD repeat-contg. protein 5 (WDR5) binds to MYC and is a crit. cofactor required for the recruitment of MYC to its target genes and reported the first small mol. inhibitors of the WDR5-MYC interaction using structure-based design. These compds. display high binding affinity, but have poor physicochem. properties and are hence not suitable for in vivo studies. Herein, we conducted an NMR-based fragment screening to identify addnl. chem. matter and, using a structure-based approach, we merged a fragment hit with the previously reported sulfonamide series. Compds. in this series can disrupt the WDR5-MYC interaction in cells, and as a consequence, we obsd. a redn. of MYC localization to chromatin.
    27. 27
      Gao, J.; Chang, M. T.; Johnsen, H. C.; Gao, S. P.; Sylvester, B. E.; Sumer, S. O.; Zhang, H.; Solit, D. B.; Taylor, B. S.; Schultz, N.; Sander, C. 3D clusters of somatic mutations in cancer reveal numerous rare mutations as functional targets. Genome Med. 2017, 9, 4,  DOI: 10.1186/s13073-016-0393-x
      27
      3D clusters of somatic mutations in cancer reveal numerous rare mutations as functional targets
      Gao, Jianjiong; Chang, Matthew T.; Johnsen, Hannah C.; Gao, Sizhi Paul; Sylvester, Brooke E.; Sumer, Selcuk Onur; Zhang, Hongxin; Solit, David B.; Taylor, Barry S.; Schultz, Nikolaus; Sander, Chris
      Genome Medicine (2017), 9 (), 4/1-4/13CODEN: GMEECG; ISSN:1756-994X. (BioMed Central Ltd.)
      Many mutations in cancer are of unknown functional significance. Std. methods use statistically significant recurrence of mutations in tumor samples as an indicator of functional impact. We extend such analyses into the long tail of rare mutations by considering recurrence of mutations in clusters of spatially close residues in protein structures. Analyzing 10,000 tumor exomes, we identify more than 3000 rarely mutated residues in proteins as potentially functional and exptl. validate several in RAC1 and MAP2K1. These potential driver mutations (web resources: 3dhotspots.org and cBioPortal.org) can extend the scope of genomically informed clin. trials and of personalized choice of therapy.
    28. 28
      Acuner, S. E.; Sumbul, F.; Torun, H.; Haliloglu, T. Oncogenic mutations on Rac1 affect global intrinsic dynamics underlying GTP and PAK1 binding. Biophys. J. 2021, 120, 866– 876,  DOI: 10.1016/j.bpj.2021.01.016
      28
      Oncogenic mutations on Rac1 affect global intrinsic dynamics underlying GTP and PAK1 binding
      Acuner, Saliha Ece; Sumbul, Fidan; Torun, Hamdi; Haliloglu, Turkan
      Biophysical Journal (2021), 120 (5), 866-876CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
      Rac1 is a small member of the Rho GTPase family. One of the most important downstream effectors of Rac1 is a serine/threonine kinase, p21-activated kinase 1 (PAK1). Mutational activation of PAK1 by Rac1 has oncogenic signaling effects. Here, although we focus on Rac1-PAK1 interaction by at.-force-microscopy-based single-mol. force spectroscopy expts., we explore the effect of active mutations on the intrinsic dynamics and binding interactions of Rac1 by Gaussian network model anal. and mol. dynamics simulations. We observe that Rac1 oncogenic mutations are at the hinges of three global modes of motion, suggesting the mech. changes as potential markers of oncogenicity. Indeed, the dissocn. of wild-type Rac1-PAK1 complex shows two distinct unbinding dynamic states that are reduced to one with constitutively active Q61L and oncogenic Y72C mutant Rac1, as revealed by single-mol. force spectroscopy expts. Q61L and Y72C mutations change the mechanics of the Rac1-PAK1 complex by increasing the elasticity of the protein and slowing down the transition to the unbound state. On the other hand, Rac1's intrinsic dynamics reveal more flexible GTP and PAK1-binding residues on switches I and II with Q61L, Y72C, oncogenic P29S and Q61R, and neg. T17N mutations. The cooperativity in the fluctuations of GTP-binding sites around the p-loop and switch I decreases in all mutants, mostly in Q61L, whereas some PAK1-binding residues display enhanced coupling with GTP-binding sites in Q61L and Y72C and within each other in P29S. The predicted binding free energies of the modeled Rac1-PAK1 complexes show that the change in the dynamic behavior likely means a more favorable PAK1 interaction. Overall, these findings suggest that the active mutations affect intrinsic functional dynamic events and alter the mechanics underlying the binding of Rac1 to GTP and upstream and downstream partners including PAK1.
    29. 29
      Forbes, S. A.; Beare, D.; Bindal, N.; Bamford, S.; Ward, S.; Cole, C. G.; Jia, M.; Kok, C.; Boutselakis, H.; De, T.; Sondka, Z.; Ponting, L.; Stefancsik, R.; Harsha, B.; Tate, J.; Dawson, E.; Thompson, S.; Jubb, H.; Campbell, P. J. COSMIC: High-Resolution Cancer Genetics Using the Catalogue of Somatic Mutations in Cancer. Curr. Protoc. Hum. Genet. 2016, 91, 10– 37,  DOI: 10.1002/cphg.21
      29
      COSMIC: High-Resolution Cancer Genetics Using the Catalogue of Somatic Mutations in Cancer
      Forbes S A; Beare D; Bindal N; Bamford S; Ward S; Cole C G; Jia M; Kok C; Boutselakis H; De T; Sondka Z; Ponting L; Stefancsik R; Harsha B; Tate J; Dawson E; Thompson S; Jubb H; Campbell P J
      Current protocols in human genetics (2016), 91 (), 10.11.1-10.11.37 ISSN:.
      COSMIC (http://cancer.sanger.ac.uk) is an expert-curated database of somatic mutations in human cancer. Broad and comprehensive in scope, recent releases in 2016 describe over 4 million coding mutations across all human cancer disease types. Mutations are annotated across the entire genome, but expert curation is focused on over 400 key cancer genes. Now encompassing the majority of molecular mutation mechanisms in oncogenetics, COSMIC additionally describes 10 million non-coding mutations, 1 million copy-number aberrations, 9 million gene-expression variants, and almost 8 million differentially methylated CpGs. This information combines a consistent interpretation of the data from the major cancer genome consortia and cancer genome literature with exhaustive hand curation of over 22,000 gene-specific literature publications. This unit describes the graphical Web site in detail; alternative protocols overview other ways the entire database can be accessed, analyzed, and downloaded. © 2016 by John Wiley & Sons, Inc.
    30. 30
      Tate, J. G.; Bamford, S.; Jubb, H. C.; Sondka, Z.; Beare, D. M.; Bindal, N.; Boutselakis, H.; Cole, C. G.; Creatore, C.; Dawson, E.; Fish, P.; Harsha, B.; Hathaway, C.; Jupe, S. C.; Kok, C. Y.; Noble, K.; Ponting, L.; Ramshaw, C. C.; Rye, C. E.; Speedy, H. E.; Stefancsik, R.; Thompson, S. L.; Wang, S.; Ward, S.; Campbell, P. J.; Forbes, S. A. COSMIC: the Catalogue Of Somatic Mutations In Cancer. Nucleic Acids Res. 2019, 47, D941– d947,  DOI: 10.1093/nar/gky1015
      30
      COSMIC: the Catalogue Of Somatic Mutations In Cancer
      Tate, John G.; Bamford, Sally; Jubb, Harry C.; Sondka, Zbyslaw; Beare, David M.; Bindal, Nidhi; Boutselakis, Harry; Cole, Charlotte G.; Creatore, Celestino; Dawson, Elisabeth; Fish, Peter; Harsha, Bhavana; Hathaway, Charlie; Jupe, Steve C.; Kok, Chai Yin; Noble, Kate; Ponting, Laura; Ramshaw, Christopher C.; Rye, Claire E.; Speedy, Helen E.; Stefancsik, Ray; Thompson, Sam L.; Wang, Shicai; Ward, Sari; Campbell, Peter J.; Forbes, Simon A.
      Nucleic Acids Research (2019), 47 (D1), D941-D947CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
      COSMIC, the Catalog Of Somatic Mutations In Cancer (https://cancer.sanger.ac.uk) is the most detailed and comprehensive resource for exploring the effect of somatic mutations in human cancer. The latest release, COSMIC v86 (August 2018), includes almost 6 million coding mutations across 1.4 million tumor samples, curated from over 26,000 publications. In addn. to coding mutations, COSMIC covers all the genetic mechanisms by which somatic mutations promote cancer, including non-coding mutations, gene fusions, copy-no. variants and drug resistance mutations. COSMIC is primarily handcurated, ensuring quality, accuracy and descriptive data capture. Building on our manual curation processes, we are introducing new initiatives that allow us to prioritize key genes and diseases, and to react more quickly and comprehensively to new findings in the literature. Alongside improvements to the public website and data-download systems, new functionality in COSMIC-3D allows exploration of mutations within three-dimensional protein structures, their protein structural and functional impacts, and implications for druggability. In parallel with COSMIC's deep and broad variant coverage, the Cancer Gene Census (CGC) describes a curated catalog of genes driving every form of human cancer. Currently describing 719 genes, the CGC has recently introduced functional descriptions of how each gene drives disease, summarized into the 10 cancer Hallmarks.
    31. 31
      Torkamani, A.; Schork, N. J. Prediction of cancer driver mutations in protein kinases. Cancer Res. 2008, 68, 1675– 1682,  DOI: 10.1158/0008-5472.can-07-5283
      31
      Prediction of Cancer Driver Mutations in Protein Kinases
      Torkamani, Ali; Schork, Nicholas J.
      Cancer Research (2008), 68 (6), 1675-1682CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)
      A large no. of somatic mutations accumulate during the process of tumorigenesis. A subset of these mutations contribute to tumor progression (known as "driver" mutations) whereas the majority of these mutations are effectively neutral (known as "passenger" mutations). The ability to differentiate between drivers and passengers will be crit. to the success of upcoming large-scale cancer DNA resequencing projects. Here we show a method capable of discriminating between drivers and passengers in the most frequently cancer-assocd. protein family, protein kinases. We apply this method to multiple cancer data sets, validating its accuracy by showing that it is capable of identifying known drivers, has excellent agreement with previous statistical ests. of the frequency of drivers, and provides strong evidence that predicted drivers are under pos. selection by various sequence and structural analyses. Furthermore, we identify particular positions in protein kinases that seem to play a role in oncogenesis. Finally, we provide a ranked list of candidate driver mutations.
    32. 32
      Ryslik, G. A.; Cheng, Y.; Modis, Y.; Zhao, H. Leveraging protein quaternary structure to identify oncogenic driver mutations. BMC Bioinf. 2016, 17, 137,  DOI: 10.1186/s12859-016-0963-3
      32
      Leveraging protein quaternary structure to identify oncogenic driver mutations
      Ryslik, Gregory A.; Cheng, Yuwei; Modis, Yorgo; Zhao, Hongyu
      BMC Bioinformatics (2016), 17 (), 137/1-137/13CODEN: BBMIC4; ISSN:1471-2105. (BioMed Central Ltd.)
      Background: Identifying key "driver" mutations which are responsible for tumorigenesis is crit. in the development of new oncol. drugs. Due to multiple pharmacol. successes in treating cancers that are caused by such driver mutations, a large body of methods have been developed to differentiate these mutations from the benign "passenger" mutations which occur in the tumor but do not further progress the disease. Under the hypothesis that driver mutations tend to cluster in key regions of the protein, the development of algorithms that identify these clusters has become a crit. area of research. Results: We have developed a novel methodol., QuartPAC (Quaternary Protein Amino acid Clustering), that identifies non-random mutational clustering while utilizing the protein quaternary structure in 3D space. By integrating the spatial information in the Protein Data Bank (PDB) and the mutational data in the Catalog of Somatic Mutations in Cancer (COSMIC), QuartPAC is able to identify clusters which are otherwise missed in a variety of proteins. The R package is available on Bioconductor at: http://bioconductor.jp/packages/3.1/bioc/html/QuartPAC.html. Conclusion:QuartPAC provides a unique tool to identify mutational clustering while accounting for the complete folded protein quaternary structure.
    33. 33
      Kumar, S.; Clarke, D.; Gerstein, M. B. Leveraging protein dynamics to identify cancer mutational hotspots using 3D structures. Proc. Natl. Acad. Sci. U.S.A. 2019, 116, 18962– 18970,  DOI: 10.1073/pnas.1901156116
      33
      Leveraging protein dynamics to identify cancer mutational hotspots using 3D structures
      Kumar, Sushant; Clarke, Declan; Gerstein, Mark B.
      Proceedings of the National Academy of Sciences of the United States of America (2019), 116 (38), 18962-18970CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Large-scale exome sequencing of tumors has enabled the identification of cancer drivers using recurrence-based approaches. Some of these methods also employ 3D protein structures to identify mutational hotspots in cancer-assocd. genes. In detg. such mutational clusters in structures, existing approaches overlook protein dynamics, despite its essential role in protein function. We present a framework to identify cancer driver genes using a dynamics-based search of mutational hotspot communities. Mutations are mapped to protein structures, which are partitioned into distinct residue communities. These communities are identified in a framework where residue-residue contact edges are weighted by correlated motions (as inferred by dynamics-based models). We then search for signals of pos. selection among these residue communities to identify putative driver genes, while applying our method to the TCGA (The Cancer Genome Atlas) PanCancer Atlas missense mutation catalog. Overall, we predict 1 or more mutational hotspots within the resolved structures of proteins encoded by 434 genes. These genes were enriched among biol. processes assocd. with tumor progression. Addnl., a comparison between our approach and existing cancer hotspot detection methods using structural data suggests that including protein dynamics significantly increases the sensitivity of driver detection.
    34. 34
      Kamburov, A.; Lawrence, M. S.; Polak, P.; Leshchiner, I.; Lage, K.; Golub, T. R.; Lander, E. S.; Getz, G. Comprehensive assessment of cancer missense mutation clustering in protein structures. Proc. Natl. Acad. Sci. U.S.A. 2015, 112, E5486– E5495,  DOI: 10.1073/pnas.1516373112
      34
      Comprehensive assessment of cancer missense mutation clustering in protein structures
      Kamburov, Atanas; Lawrence, Michael S.; Polak, Paz; Leshchiner, Ignaty; Lage, Kasper; Golub, Todd R.; Lander, Eric S.; Getz, Gad
      Proceedings of the National Academy of Sciences of the United States of America (2015), 112 (40), E5486-E5495CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Large-scale tumor sequencing projects enabled the identification of many new cancer gene candidates through computational approaches. Here, we describe a general method to detect cancer genes based on significant 3D clustering of mutations relative to the structure of the encoded protein products. The approach can also be used to search for proteins with an enrichment of mutations at binding interfaces with a protein, nucleic acid, or small mol. partner. We applied this approach to systematically analyze the PanCancer compendium of somatic mutations from 4,742 tumors relative to all known 3D structures of human proteins in the Protein Data Bank. We detected significant 3D clustering of missense mutations in several previously known oncoproteins including HRAS, EGFR, and PIK3CA. Although clustering of missense mutations is often regarded as a hallmark of oncoproteins, we obsd. that a no. of tumor suppressors, including FBXW7, VHL, and STK11, also showed such clustering. Beside these known cases, we also identified significant 3D clustering of missense mutations in NUF2, which encodes a component of the kinetochore, that could affect chromosome segregation and lead to aneuploidy. Anal. of interaction interfaces revealed enrichment of mutations in the interfaces between FBXW7-CCNE1, HRAS-RASA1, CUL4B-CAND1, OGT-HCFC1, PPP2R1A-PPP2R5C/PPP2R2A, DICER1-Mg2+, MAX-DNA, SRSF2-RNA, and others. Together, our results indicate that systematic consideration of 3D structure can assist in the identification of cancer genes and in the understanding of the functional role of their mutations.
    35. 35
      Dharmarajan, V.; Lee, J.-H.; Patel, A.; Skalnik, D. G.; Cosgrove, M. S. Structural basis for WDR5 interaction (Win) motif recognition in human SET1 family histone methyltransferases. J. Biol. Chem. 2012, 287, 27275– 27289,  DOI: 10.1074/jbc.m112.364125
      35
      Structural Basis for WDR5 Interaction (Win) Motif Recognition in Human SET1 Family Histone Methyltransferases
      Dharmarajan, Venkatasubramanian; Lee, Jeong-Heon; Patel, Anamika; Skalnik, David G.; Cosgrove, Michael S.
      Journal of Biological Chemistry (2012), 287 (33), 27275-27289CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Translocations and amplifications of the mixed lineage leukemia-1 (MLL1) gene are assocd. with aggressive myeloid and lymphocytic leukemias in humans. MLL1 is a member of the SET1 family of histone H3 lysine 4 (H3K4) methyltransferases, which are required for transcription of genes involved in hematopoiesis and development. MLL1 assocs. with a subcomplex contg. WDR5, RbBP5, Ash2L, and DPY-30 (WRAD), which together form the MLL1 core complex that is required for sequential mono- and dimethylation of H3K4. We previously demonstrated that WDR5 binds the conserved WDR5 interaction (Win) motif of MLL1 in vitro, an interaction that is required for the H3K4 dimethylation activity of the MLL1 core complex. In this investigation, we demonstrate that arginine 3765 of the MLL1 Win motif is required to co-immunoppt. WRAD from mammalian cells, suggesting that the WDR5-Win motif interaction is important for the assembly of the MLL1 core complex in vivo. We also demonstrate that peptides that mimic SET1 family Win motif sequences inhibit H3K4 dimethylation by the MLL1 core complex with varying degrees of efficiency. To understand the structural basis for these differences, we detd. structures of WDR5 bound to six different naturally occurring Win motif sequences at resolns. ranging from 1.9 to 1.2 Å. Our results reveal that binding energy differences result from interactions between non-conserved residues C-terminal to the Win motif and to a lesser extent from subtle variation of residues within the Win motif. These results highlight a new class of methylation inhibitors that may be useful for the treatment of MLL1-related malignancies.
    36. 36
      Zhang, P.; Lee, H.; Brunzelle, J. S.; Couture, J.-F. The plasticity of WDR5 peptide-binding cleft enables the binding of the SET1 family of histone methyltransferases. Nucleic Acids Res. 2012, 40, 4237– 4246,  DOI: 10.1093/nar/gkr1235
      36
      The plasticity of WDR5 peptide-binding cleft enables the binding of the SET1 family of histone methyltransferases
      Zhang, Pamela; Lee, Hwabin; Brunzelle, Joseph S.; Couture, Jean-Francois
      Nucleic Acids Research (2012), 40 (9), 4237-4246CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      In mammals, the SET1 family of lysine methyltransferases (KMTs), which includes MLL1-5, SET1A and SET1B, catalyzes the methylation of lysine-4 (Lys-4) on histone H3. Recent reports have demonstrated that a three-subunit complex composed of WD-repeat protein-5 (WDR5), retinoblastoma-binding protein-5 (RbBP5) and absent, small, homeotic disks-2-like (ASH2L) stimulates the methyltransferase activity of MLL1. On the basis of studies showing that this stimulation is in part controlled by an interaction between WDR5 and a small region located in close proximity of the MLL1 catalytic domain [referred to as the WDR5-interacting motif (Win)], it has been suggested that WDR5 might play an analogous role in scaffolding the other SET1 complexes. We herein provide biochem. and structural evidence showing that WDR5 binds the Win motifs of MLL2-4, SET1A and SET1B. Comparative anal. of WDR5-Win complexes reveals that binding of the Win motifs is achieved by the plasticity of WDR5 peptidyl-arginine-binding cleft allowing the C-terminal ends of the Win motifs to be maintained in structurally divergent conformations. Consistently, enzymic assays reveal that WDR5 plays an important role in the optimal stimulation of MLL2-4, SET1A and SET1B methyltransferase activity by the RbBP5-ASH2L heterodimer. Overall, our findings illustrate the function of WDR5 in scaffolding the SET1 family of KMTs and further emphasize on the important role of WDR5 in regulating global histone H3 Lys-4 methylation.
    37. 37
      Muntean, A. G.; Hess, J. L. The pathogenesis of mixed-lineage leukemia. Annu. Rev. Pathol.: Mech. Dis. 2012, 7, 283– 301,  DOI: 10.1146/annurev-pathol-011811-132434
      37
      The pathogenesis of mixed-lineage leukemia
      Muntean, Andrew G.; Hess, Jay L.
      Annual Review of Pathology: Mechanisms of Disease (2012), 7 (), 283-301CODEN: ARPMCU; ISSN:1553-4006. (Annual Reviews Inc.)
      A review. Aggressive leukemias arise in both children and adults as a result of rearrangements to the mixed-lineage leukemia gene (MLL) located on chromosome 11q23. MLL encodes a large histone methyltransferase that directly binds DNA and pos. regulates gene transcription, including homeobox (HOX) genes. MLL is involved in chromosomal translocations, partial tandem duplications, and amplifications, all of which result in hematopoietic malignancies due to sustained HOX expression and stalled differentiation. MLL lesions are assocd. with both acute myeloid leukemia and acute lymphoid leukemia and are usually assocd. with a relatively poor prognosis despite improved treatment options such as allogeneic hematopoietic stem cell transplantation, which underscores the need for new treatment regimens. Recent advances have begun to reveal the mol. mechanisms that drive MLL-assocd. leukemias, which, in turn, have provided opportunities for therapeutic development. Here, we discuss the etiol. of MLL leukemias and potential directions for future therapy.
    38. 38
      Chen, X.; Xie, W.; Gu, P.; Cai, Q.; Wang, B.; Xie, Y.; Dong, W.; He, W.; Zhong, G.; Lin, T.; Huang, J. Upregulated WDR5 promotes proliferation, self-renewal and chemoresistance in bladder cancer via mediating H3K4 trimethylation. Sci. Rep. 2015, 5, 8293,  DOI: 10.1038/srep08293
      38
      Upregulated WDR5 promotes proliferation, self-renewal and chemoresistance in bladder cancer via mediating H3K4 trimethylation
      Chen, Xu; Xie, Weibin; Gu, Peng; Cai, Qingqing; Wang, Bo; Xie, Yun; Dong, Wen; He, Wang; Zhong, Guangzheng; Lin, Tianxin; Huang, Jian
      Scientific Reports (2015), 5 (), 8293CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
      WD repeat domain 5 (WDR5) plays an important role in various biol. functions through the epigenetic regulation of gene transcription; however, its role in bladder cancer remains largely unknown. Our study investigated the role of WDR5 in bladder cancer and demonstrated that WDR5 was upregulated in bladder cancer tissues, and elevated WDR5 protein levels pos. correlated with advanced tumor stage and poor survival. Through gain or loss of function, we demonstrated that WDR5 promoted proliferation, self-renewal and chemoresistance to cisplatin in bladder cancer cells in vitro, and tumor growth in vivo. Mechanistically, WDR5 regulated various functions in bladder cancer by mediating the transcription of cyclin B1, cyclin E1, cyclin E2, UHMK1, MCL1, BIRC3 and Nanog by histone H3 lysine 4 trimethylation. Therefore, we have discovered that WDR5 plays an important role in bladder cancer suggesting that WDR5 is a potential biomarker and a promising target in the treatment of bladder cancer.
    39. 39
      Chen, X.; Gu, P.; Li, K.; Xie, W.; Chen, C.; Lin, T.; Huang, J. Gene expression profiling of WDR5 regulated genes in bladder cancer. Genomics Data 2015, 5, 27– 29,  DOI: 10.1016/j.gdata.2015.05.003
      39
      Gene expression profiling of WDR5 regulated genes in bladder cancer
      Chen Xu; Gu Peng; Lin Tianxin; Li Kuiqing; Xie Weibin; Chen Changhao; Huang Jian
      Genomics data (2015), 5 (), 27-9 ISSN:2213-5960.
      WD repeat domain 5 (WDR5) plays an important role in various biological functions through the epigenetic regulation of gene transcription (Wysocka et al., 2005 [1]; Sandstrom et al., 2014[2]; Ang et al., 2011[3]). Recently, our study found that WDR5 was upregulated in bladder cancer tissues, promoted bladder cancer cell proliferation, self-renewal and chemoresistance to cisplatin in bladder cancer cells in vitro, and tumor growth in vivo (Chen et al., 2015). To gain a molecular understanding of the role of WDR5 in promoting bladder cancer, we performed a genome-wide analysis on WDR5 knockdown by microarray gene expression profiling. Here we provide detailed experimental methods and analysis for the microarray data, which have been deposited into Gene Expression Omnibus (GEO): GSE59132.
    40. 40
      Ge, Z.; Song, E. J.; Kawasawa, Y. I.; Li, J.; Dovat, S.; Song, C. WDR5 high expression and its effect on tumorigenesis in leukemia. Oncotarget 2016, 7, 37740– 37754,  DOI: 10.18632/oncotarget.9312
      40
      WDR5 high expression and its effect on tumorigenesis in leukemia
      Ge Zheng; Ge Zheng; Song Evelyn J; Dovat Sinisa; Song Chunhua; Ge Zheng; Li Jianyong; Kawasawa Yuka Imamura
      Oncotarget (2016), 7 (25), 37740-37754 ISSN:.
      WD repeat domain 5 (WDR5) plays an important role in various biological functions through the epigenetic regulation of gene transcription. However, the oncogenic effect of WDR5 in leukemia remains largely unknown. Here, we found WDR5 expression is increased in leukemia patients. High expression of WDR5 is associated with high risk leukemia; Patients with WDR5 and MLL1 high expression have poor complete remission rate. We further identified the global genomic binding of WDR5 in leukemic cells and found the genomic co-localization of WDR5 binding with H3K4me3 enrichment. Moreover, WDR5 knockdown by shRNA suppresses cell proliferation, induces apoptosis, inhibits the expression of WDR5 targets, and blocks the H3K4me3 enrichment on the promoter of its targets. We also observed the positive correlation of WDR5 expression with these targets in the cohort study of leukemia patients. Our data reveal that WDR5 may have oncogenic effect and WDR5-mediated H3K4 methylation plays an important role in leukemogenesis.
    41. 41
      Sun, W.; Guo, F.; Liu, M. Up-regulated WDR5 promotes gastric cancer formation by induced cyclin D1 expression. J. Cell. Biochem. 2018, 119, 3304– 3316,  DOI: 10.1002/jcb.26491
      41
      Up-regulated WDR5 promotes gastric cancer formation by induced cyclin D1 expression
      Sun, Wei; Guo, Fuchao; Liu, Mingkai
      Journal of Cellular Biochemistry (2018), 119 (4), 3304-3316CODEN: JCEBD5; ISSN:0730-2312. (Wiley-Blackwell)
      Gastric cancer (GC) is the fourth common cancer and second leading cause of cancer-related mortality in the world. WD repeat domain 5 (WDR5) has been identified that its functions as an important role in various biol. functions through the epigenetic regulation of gene transcription. However, the oncogenic effect of WDR5 in gastric cancer remains largely unknown. In this study, we investigated the role of WDR5 in gastric cancer genesis. We found that WDR5 expression is increased in gastric cancer patients. Through survival anal., we found that high expression of WDR5 is assocd. with high risk gastric cancer; patients who with WDR5 high expression have poor survival rate compared with those who with WDR5 low expression. To make further investigation, we identified that WDR5 is targeted for cell cycle arrest by the Cyclin D1 in a process that is regulated by H3K4me3. Moreover, over-expression of WDR5 promotes cell proliferation, induces S/G2/M arrest in cell cycle, and promotes the expression of WDR5 targets, as well as that of H3K4me3 on the promoter of its targets. Inversely, WDR5 knockdown by shRNA inhibits cell proliferation, reverses S/G2/M arrest in cell cycle, and suppresses the expression of WDR5 targets, as well as that of H3K4me3. We also obsd. the pos. correlation of WDR5 expression with its target in the cohort study of gastric patients. Taken together, our data reveal that WDR5 may have oncogenic effect and WDR5-mediated H3K4 methylation plays an important role in gastric cancer.
    42. 42
      Wang, F.; Zhang, J.; Ke, X.; Peng, W.; Zhao, G.; Peng, S.; Xu, J.; Xu, B.; Cui, H. WDR5-Myc axis promotes the progression of glioblastoma and neuroblastoma by transcriptional activating CARM1. Biochem. Biophys. Res. Commun. 2020, 523, 699– 706,  DOI: 10.1016/j.bbrc.2019.12.101
      42
      WDR5-Myc axis promotes the progression of glioblastoma and neuroblastoma by transcriptional activating CARM1
      Wang, Feng; Zhang, Jiayi; Ke, Xiaoxue; Peng, Wen; Zhao, Gaichao; Peng, Shihan; Xu, Jie; Xu, Bo; Cui, Hongjuan
      Biochemical and Biophysical Research Communications (2020), 523 (3), 699-706CODEN: BBRCA9; ISSN:0006-291X. (Elsevier B.V.)
      The WD repeat domain 5 (WDR5), also known as SWD3 and BIG-3, is often overexpressed in cancers, however its mol. function in cancer remains to be elucidated. In this study, we found that WDR5 promoted the proliferation and self-renewal of glioblastoma and neuroblastoma cells. The data from databases and Western blot assay showed that CARM1 is a downstream gene of WDR5-Myc axis. In addn., we obsd. that WDR5 promoted the binding of Myc to CARM1 promoter by interacting with Myc and inducing histone 3 lysine 4 trimethylation (H3K4me3). Dual luciferase reporter system indicated that Myc binds to the upstream region (-520 to -515) before transcription start site (TSS) of CARM1 promoter. These findings suggest a novel regulatory model for the proliferation and tumorigenesis of glioblastoma and neuroblastoma by WDR5-Myc axis.
    43. 43
      Bryan, A. F.; Wang, J.; Howard, G. C.; Guarnaccia, A. D.; Woodley, C. M.; Aho, E. R.; Rellinger, E. J.; Matlock, B. K.; Flaherty, D. K.; Lorey, S. L.; Chung, D. H.; Fesik, S. W.; Liu, Q.; Weissmiller, A. M.; Tansey, W. P. WDR5 is a conserved regulator of protein synthesis gene expression. Nucleic Acids Res. 2020, 48, 2924– 2941,  DOI: 10.1093/nar/gkaa051
      43
      WDR5 is a conserved regulator of protein synthesis gene expression
      Bryan, Audra F.; Wang, Jing; Howard, Gregory C.; Guarnaccia, Alissa D.; Woodley, Chase M.; Aho, Erin R.; Rellinger, Eric J.; Matlock, Brittany K.; Flaherty, David K.; Lorey, Shelly L.; Chung, Dai H.; Fesik, Stephen W.; Liu, Qi; Weissmiller, April M.; Tansey, William P.
      Nucleic Acids Research (2020), 48 (6), 2924-2941CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
      WDR5 is a highly-conserved nuclear protein that performs multiple scaffolding functions in the context of chromatin. WDR5 is also a promising target for pharmacol. inhibition in cancer, with small mol. inhibitors of an arginine-binding pocket of WDR5 (the 'WIN' site) showing efficacy against a range of cancer cell lines in vitro. Efforts to understand WDR5, or establish the mechanism of action of WIN site inhibitors, however, are stymied by its many functions in the nucleus, and a lack of knowledge of the conserved gene networks--if any--that are under its control. Here, we have performed comparative genomic analyses to identify the conserved sites of WDR5 binding to chromatin, and the conserved genes regulated by WDR5, across a diverse panel of cancer cell lines. We show that a specific cohort of protein synthesis genes (PSGs) are invariantly bound by WDR5, demonstrate that the WIN site anchors WDR5 to chromatin at these sites, and establish that PSGs are bona fide, acute, and persistent targets of WIN site blockade. Together, these data reveal that WDR5 plays a predominant transcriptional role in biomass accumulation and provide further evidence that WIN site inhibitors act to repress gene networks linked to protein synthesis homeostasis. In Saccharomyces cerevisiae, ribosomal protein gene (RPG) promoters display binding sites for either Rap1 or Abf1 transcription factors. Unlike Rap1-assocd. promoters, the small cohort of Abf1-dependent RPGs (Abf1-RPGs) has not been extensively investigated. We show that RPL3, RPL4B, RPP1A, RPS22B and RPS28A/B share a common promoter architecture, with an Abf1 site upstream of a conserved element matching the sequence recognized by Fhl1, a transcription factor which together with Ifh1 orchestrates Rap1-assocd. RPG regulation. Abf1 and Fhl1 promoter assocn. was confirmed by ChIP and/or gel retardation assays. Mutational anal. revealed a more severe requirement of Abf1 than Fhl1 binding sites for RPG transcription. In the case of RPS22B an unusual Tbf1 binding site promoted both RPS22B and intron-hosted SNR44 expression. Abf1-RPG down-regulation upon TOR pathway inhibition was much attenuated at defective mutant promoters unable to bind Abf1. TORC1 inactivation caused the expected redn. of Ifh1 occupancy at RPS22B and RPL3 promoters, but unexpectedly it entailed largely increased Abf1 assocn. with Abf1-RPG promoters. We present evidence that Abf1 recruitment upon nutritional stress, also obsd. for representative ribosome biogenesis genes, favors RPG transcriptional rescue upon nutrient replenishment, thus pointing to nutrient-regulated Abf1 dynamics at promoters as a novel mechanism in ribosome biogenesis control.
    44. 44
      Imran, A.; Moyer, B. S.; Canning, A. J.; Kalina, D.; Duncan, T. M.; Moody, K. J.; Wolfe, A. J.; Cosgrove, M. S.; Movileanu, L. Kinetics of the multitasking high-affinity Win binding site of WDR5 in restricted and unrestricted conditions. Biochem. J. 2021, 478, 2145– 2161,  DOI: 10.1042/bcj20210253
      44
      Kinetics of the multitasking high-affinity Win binding site of WDR5 in restricted and unrestricted conditions
      Imran, Ali; Moyer, Brandon S.; Canning, Ashley J.; Kalina, Dan; Duncan, Thomas M.; Moody, Kelsey J.; Wolfe, Aaron J.; Cosgrove, Michael S.; Movileanu, Liviu
      Biochemical Journal (2021), 478 (11), 2145-2161CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
      Recent advances in quant. proteomics show that WD40 proteins play a pivotal role in numerous cellular networks. Yet, they have been fairly unexplored and their phys. assocns. with other proteins are ambiguous. A quant. understanding of these interactions has wide-ranging significance. WD40 repeat protein 5 (WDR5) interacts with all members of human SET1/MLL methyltransferases, which regulate methylation of the histone 3 lysine 4 (H3K4). Here, using real-time binding measurements in a high-throughput setting, we identified the kinetic fingerprint of transient assocns. between WDR5 and 14-residue WDR5 interaction (Win) motif peptides of each SET1 protein (SET1Win). Our results reveal that the high-affinity WDR5-SET1Win interactions feature slow assocn. kinetics. This finding is likely due to the requirement of SET1Win to insert into the narrow WDR5 cavity, also named the Win binding site. Furthermore, our explorations indicate fairly slow dissocn. kinetics. This conclusion is in accordance with the primary role of WDR5 in maintaining the functional integrity of a large multisubunit complex, which regulates the histone methylation. Because the Win binding site is considered a key therapeutic target, the immediate outcomes of this study could form the basis for accelerated developments in medical biotechnol.
    45. 45
      Mayse, L. A.; Imran, A.; Larimi, M. G.; Cosgrove, M. S.; Wolfe, A. J.; Movileanu, L. Disentangling the recognition complexity of a protein hub using a nanopore. Nat. Commun. 2022, 13, 978,  DOI: 10.1038/s41467-022-28465-8
      45
      Disentangling the recognition complexity of a protein hub using a nanopore
      Mayse, Lauren Ashley; Imran, Ali; Larimi, Motahareh Ghahari; Cosgrove, Michael S.; Wolfe, Aaron James; Movileanu, Liviu
      Nature Communications (2022), 13 (1), 978CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)
      WD40 repeat proteins are frequently involved in processing cell signaling and scaffolding large multi-subunit machineries. Despite their significance in physiol. and disease-like conditions, their reversible interactions with other proteins remain modestly examd. Here, we show the development and validation of a protein nanopore for the detection and quantification of WD40 repeat protein 5 (WDR5), a chromatin-assocd. hub involved in epigenetic regulation of histone methylation. Our nanopore sensor is equipped with a 14-residue Win motif of mixed lineage leukemia 4 methyltransferase (MLL4Win), a WDR5 ligand. Our approach reveals a broad dynamic range of MLL4Win-WDR5 interactions and three distant subpopulations of binding events, representing three modes of protein recognition. The three binding events are confirmed as specific interactions using a weakly binding WDR5 deriv. and various environmental contexts. These outcomes demonstrate the substantial sensitivity of our nanopore sensor, which can be utilized in protein analytics.
    46. 46
      Karatas, H.; Townsend, E. C.; Cao, F.; Chen, Y.; Bernard, D.; Liu, L.; Lei, M.; Dou, Y.; Wang, S. High-affinity, small-molecule peptidomimetic inhibitors of MLL1/WDR5 protein-protein interaction. J. Am. Chem. Soc. 2013, 135, 669– 682,  DOI: 10.1021/ja306028q
      46
      High-Affinity, Small-Molecule Peptidomimetic Inhibitors of MLL1/WDR5 Protein-Protein Interaction
      Karatas, Hacer; Townsend, Elizabeth C.; Cao, Fang; Chen, Yong; Bernard, Denzil; Liu, Liu; Lei, Ming; Dou, Yali; Wang, Shaomeng
      Journal of the American Chemical Society (2013), 135 (2), 669-682CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Mixed lineage leukemia 1 (MLL1) is a histone H3 lysine 4 (H3K4) methyltransferase, and targeting the MLL1 enzymic activity has been proposed as a novel therapeutic strategy for the treatment of acute leukemia harboring MLL1 fusion proteins. The MLL1/WDR5 protein-protein interaction is essential for MLL1 enzymic activity. In the present study, we designed a large no. of peptidomimetics to target the MLL1/WDR5 interaction based upon -CO-ARA-NH-, the min. binding motif derived from MLL1. Our study led to the design of high-affinity peptidomimetics, which bind to WDR5 with Ki < 1 nM and function as potent antagonists of MLL1 activity in a fully reconstituted in vitro H3K4 methyltransferase assay. Detn. of co-crystal structures of two potent peptidomimetics in complex with WDR5 establishes their structural basis for high-affinity binding to WDR5. Evaluation of one such peptidomimetic, MM-102, in bone marrow cells transduced with MLL1-AF9 fusion construct shows that the compd. effectively decreases the expression of HoxA9 and Meis-1, two crit. MLL1 target genes in MLL1 fusion protein mediated leukemogenesis. MM-102 also specifically inhibits cell growth and induces apoptosis in leukemia cells harboring MLL1 fusion proteins. Our study provides the first proof-of-concept for the design of small-mol. inhibitors of the WDR5/MLL1 protein-protein interaction as a novel therapeutic approach for acute leukemia harboring MLL1 fusion proteins.
    47. 47
      Cao, F.; Townsend, E. C.; Karatas, H.; Xu, J.; Li, L.; Lee, S.; Liu, L.; Chen, Y.; Ouillette, P.; Zhu, J.; Hess, J. L.; Atadja, P.; Lei, M.; Qin, Z. S.; Malek, S.; Wang, S.; Dou, Y. Targeting MLL1 H3K4 methyltransferase activity in mixed-lineage leukemia. Mol. Cell 2014, 53, 247– 261,  DOI: 10.1016/j.molcel.2013.12.001
      47
      Targeting MLL1 H3K4 Methyltransferase Activity in Mixed-Lineage Leukemia
      Cao, Fang; Townsend, Elizabeth C.; Karatas, Hacer; Xu, Jing; Li, Li; Lee, Shirley; Liu, Liu; Chen, Yong; Ouillette, Peter; Zhu, Jidong; Hess, Jay L.; Atadja, Peter; Lei, Ming; Qin, Zhaohui S.; Malek, Sami; Wang, Shaomeng; Dou, Yali
      Molecular Cell (2014), 53 (2), 247-261CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)
      Here we report a comprehensive characterization of our recently developed inhibitor MM-401 that targets the MLL1 H3K4 methyltransferase activity. MM-401 is able to specifically inhibit MLL1 activity by blocking MLL1-WDR5 interaction and thus the complex assembly. This targeting strategy does not affect other mixed-lineage leukemia (MLL) family histone methyltransferases (HMTs), revealing a unique regulatory feature for the MLL1 complex. Using MM-401 and its enantiomer control MM-NC-401, we show that inhibiting MLL1 methyltransferase activity specifically blocks proliferation of MLL cells by inducing cell-cycle arrest, apoptosis, and myeloid differentiation without general toxicity to normal bone marrow cells or non-MLL cells. More importantly, transcriptome analyses show that MM-401 induces changes in gene expression similar to those of MLL1 deletion, supporting a predominant role of MLL1 activity in regulating MLL1-dependent leukemia transcription program. We envision broad applications for MM-401 in basic and translational research.
    48. 48
      Alicea-Velázquez, N. L.; Shinsky, S. A.; Loh, D. M.; Lee, J. H.; Skalnik, D. G.; Cosgrove, M. S. Targeted Disruption of the Interaction between WD-40 Repeat Protein 5 (WDR5) and Mixed Lineage Leukemia (MLL)/SET1 Family Proteins Specifically Inhibits MLL1 and SETd1A Methyltransferase Complexes. J. Biol. Chem. 2016, 291, 22357– 22372,  DOI: 10.1074/jbc.M116.752626
      48
      Targeted Disruption of the Interaction between WD-40 Repeat Protein 5 (WDR5) and Mixed Lineage Leukemia (MLL)/SET1 Family Proteins Specifically Inhibits MLL1 and SETd1A Methyltransferase Complexes
      Alicea-Velazquez, Nilda L.; Shinsky, Stephen A.; Loh, Daniel M.; Lee, Jeong-Heon; Skalnik, David G.; Cosgrove, Michael S.
      Journal of Biological Chemistry (2016), 291 (43), 22357-22372CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      MLL1 belongs to the SET1 family of histone H3 lysine 4 (H3K4) methyltransferases, composed of MLL1-4 and SETd1A/B. MLL1 translocations are present in acute leukemias, and mutations in several family members are assocd. with cancer and developmental disorders. MLL1 assocs. with a subcomplex contg. WDR5, RbBP5, ASH2L, and DPY-30 (WRAD), forming the MLL1 core complex required for H3K4 mono- and dimethylation and transcriptional activation. Core complex assembly requires interaction of WDR5 with the MLL1 Win (WDR5 interaction) motif, which is conserved across the SET1 family. Agents that mimic the SET1 family Win motif inhibit the MLL1 core complex and have become an attractive approach for targeting MLL1 in cancers. Like MLL1, other SET1 family members interact with WRAD, but the roles of the Win motif in complex assembly and enzymic activity remain unexplored. Here, we show that the Win motif is necessary for interaction of WDR5 with all members of the human SET1 family. Mutation of the Win motif-WDR5 interface severely disrupts assembly and activity of MLL1 and SETd1A complexes but only modestly disrupts MLL2/4 and SETd1B complexes without significantly altering enzymic activity in vitro. Notably, in the absence of WDR5, MLL3 interacts with RAD and shows enhanced activity. To further probe the role of the Win motif-WDR5 interaction, we designed a peptidomimetic that binds WDR5 (Kd ∼3 nm) and selectively inhibits activity of MLL1 and SETd1A core complexes within the SET1 family. Our results reveal that SET1 family complexes with the weakest Win motif-WDR5 interaction are more susceptible to Win motif-based inhibitors.
    49. 49
      Karatas, H.; Li, Y.; Liu, L.; Ji, J.; Lee, S.; Chen, Y.; Yang, J.; Huang, L.; Bernard, D.; Xu, J.; Townsend, E. C.; Cao, F.; Ran, X.; Li, X.; Wen, B.; Sun, D.; Stuckey, J. A.; Lei, M.; Dou, Y.; Wang, S. Discovery of a Highly Potent, Cell-Permeable Macrocyclic Peptidomimetic (MM-589) Targeting the WD Repeat Domain 5 Protein (WDR5)-Mixed Lineage Leukemia (MLL) Protein-Protein Interaction. J. Med. Chem. 2017, 60, 4818– 4839,  DOI: 10.1021/acs.jmedchem.6b01796
      49
      Discovery of a Highly Potent, Cell-Permeable Macrocyclic Peptidomimetic (MM-589) Targeting the WD Repeat Domain 5 Protein (WDR5)-Mixed Lineage Leukemia (MLL) Protein-Protein Interaction
      Karatas, Hacer; Li, Yangbing; Liu, Liu; Ji, Jiao; Lee, Shirley; Chen, Yong; Yang, Jiuling; Huang, Liyue; Bernard, Denzil; Xu, Jing; Townsend, Elizabeth C.; Cao, Fang; Ran, Xu; Li, Xiaoqin; Wen, Bo; Sun, Duxin; Stuckey, Jeanne A.; Lei, Ming; Dou, Yali; Wang, Shaomeng
      Journal of Medicinal Chemistry (2017), 60 (12), 4818-4839CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      We report herein the design, synthesis and evaluation of macrocyclic peptidomimetics that bind to WDR5 and block the WDR5-MLL protein-protein interaction. Compd. (I) (R1 = Me; n = 1) (MM-589) binds to WDR5 with an IC50 value of 0.90 nM (Ki value <1 nM), and inhibits the MLL H3K4 methyltransferase (HMT) activity with an IC50 value of 12.7 nM. Compd. I (R1 = Me; n = 1) potently and selectively inhibits cell growth in human leukemia cell lines harboring MLL translocations, and is >40 times better than the previously reported compd. MM-401. Co-crystal structures of I (R1 = H, n = 5) and (R1 = Me; n = 1) complexed with WDR5 provide structural basis for their high affinity binding to WDR5. Addnl., we have developed and optimized a new, AlphaLISA-based MLL HMT functional assay to facilitate the functional evaluation of these designed compds. Compd. I (R1 = Me; n = 1) represents the most potent inhibitor of the WDR5-MLL interaction reported to date and further optimization of I (R1 = Me; n = 1) may yield a new therapy for acute leukemia.
    50. 50
      Schapira, M.; Tyers, M.; Torrent, M.; Arrowsmith, C. H. WD40 repeat domain proteins: a novel target class?. Nat. Rev. Drug Discovery 2017, 16, 773– 786,  DOI: 10.1038/nrd.2017.179
      50
      WD40 repeat domain proteins: a novel target class?
      Schapira, Matthieu; Tyers, Mike; Torrent, Maricel; Arrowsmith, Cheryl H.
      Nature Reviews Drug Discovery (2017), 16 (11), 773-786CODEN: NRDDAG; ISSN:1474-1776. (Nature Research)
      Antagonism of protein-protein interactions (PPIs) with small mols. is becoming more feasible as a therapeutic approach. Successful PPI inhibitors tend to target proteins contg. deep peptide-binding grooves or pockets rather than the more common large, flat protein interaction surfaces. Here, we review one of the most abundant PPI domains in the human proteome, the WD40 repeat (WDR) domain, which has a central peptide-binding pocket and is a member of the β-propeller domain-contg. protein family. Recently, two WDR domain-contg. proteins, WDR5 and EED, as well as other β-propeller domains have been successfully targeted by potent, specific, cell-active, drug-like chem. probes. Could WDR domains be a novel target class for drug discovery. Although the research is at an early stage and therefore not clin. validated, cautious optimism is justified, as WDR domain-contg. proteins are involved in multiple disease-assocd. pathways. The druggability and structural diversity of WDR domain binding pockets suggest that understanding how to target this prevalent domain class will open up areas of disease biol. that have so far resisted drug discovery efforts.
    51. 51
      Aho, E. R.; Weissmiller, A. M.; Fesik, S. W.; Tansey, W. P. Targeting WDR5: A WINning Anti-Cancer Strategy?. Epigenet. Insights 2019, 12, 2516865719865282,  DOI: 10.1177/2516865719865282
      There is no corresponding record for this reference.
    52. 52
      Aho, E. R.; Wang, J.; Gogliotti, R. D.; Howard, G. C.; Phan, J.; Acharya, P.; Macdonald, J. D.; Cheng, K.; Lorey, S. L.; Lu, B.; Wenzel, S.; Foshage, A. M.; Alvarado, J.; Wang, F.; Shaw, J. G.; Zhao, B.; Weissmiller, A. M.; Thomas, L. R.; Vakoc, C. R.; Hall, M. D.; Hiebert, S. W.; Liu, Q.; Stauffer, S. R.; Fesik, S. W.; Tansey, W. P. Displacement of WDR5 from Chromatin by a WIN Site Inhibitor with Picomolar Affinity. Cell Rep. 2019, 26, 2916– 2928.e13,  DOI: 10.1016/j.celrep.2019.02.047
      52
      Displacement of WDR5 from Chromatin by a WIN Site Inhibitor with Picomolar Affinity
      Aho, Erin R.; Wang, Jing; Gogliotti, Rocco D.; Howard, Gregory C.; Phan, Jason; Acharya, Pankaj; Macdonald, Jonathan D.; Cheng, Ken; Lorey, Shelly L.; Lu, Bin; Wenzel, Sabine; Foshage, Audra M.; Alvarado, Joseph; Wang, Feng; Shaw, J. Grace; Zhao, Bin; Weissmiller, April M.; Thomas, Lance R.; Vakoc, Christopher R.; Hall, Matthew D.; Hiebert, Scott W.; Liu, Qi; Stauffer, Shaun R.; Fesik, Stephen W.; Tansey, William P.
      Cell Reports (2019), 26 (11), 2916-2928.e13CODEN: CREED8; ISSN:2211-1247. (Cell Press)
      The chromatin-assocd. protein WDR5 is a promising target for pharmacol. inhibition in cancer. Drug discovery efforts center on the blockade of the "WIN site" of WDR5, a well-defined pocket that is amenable to small mol. inhibition. Various cancer contexts have been proposed to be targets for WIN site inhibitors, but a lack of understanding of WDR5 target genes and of the primary effects of WIN site inhibitors hampers their utility. Here, by the discovery of potent WIN site inhibitors, we demonstrate that the WIN site links WDR5 to chromatin at a small cohort of loci, including a specific subset of ribosome protein genes. WIN site inhibitors rapidly displace WDR5 from chromatin and decrease the expression of assocd. genes, causing translational inhibition, nucleolar stress, and p53 induction. Our studies define a mode by which WDR5 engages chromatin and forecast that WIN site blockade could have utility against multiple cancer types.
    53. 53
      Dennis, M. L.; Morrow, B. J.; Dolezal, O.; Cuzzupe, A. N.; Stupple, A. E.; Newman, J.; Bentley, J.; Hattarki, M.; Nuttall, S. D.; Foitzik, R. C.; Street, I. P.; Stupple, P. A.; Monahan, B. J.; Peat, T. S. Fragment screening for a protein-protein interaction inhibitor to WDR5. Struct. Dyn. 2019, 6, 064701,  DOI: 10.1063/1.5122849
      53
      Fragment screening for a protein-protein interaction inhibitor to WDR5
      Dennis, Matthew L.; Morrow, Benjamin J.; Dolezal, Olan; Cuzzupe, Anthony N.; Stupple, Alexandra E.; Newman, Janet; Bentley, John; Hattarki, Meghan; Nuttall, Stewart D.; Foitzik, Richard C.; Street, Ian P.; Stupple, Paul A.; Monahan, Brendon J.; Peat, Thomas. S.
      Structural Dynamics (2019), 6 (6), 064701/1-064701/20CODEN: SDTYAE; ISSN:2329-7778. (American Institute of Physics)
      In this study, a fragment screen with Surface Plasmon Resonance (SPR) was used to identify a highly ligand-efficient imidazole-contg. compd. that is bound in the WIN site. The subsequent medicinal chem. campaign-guided by a suite of high-resoln. cocrystal structures with WDR5-progressed the initial hit to a low micromolar binder. One outcome from this study is a moiety that substitutes well for the side chain of arginine; a tripeptide contg. one such substitution was resolved in a high resoln. structure (1.5 Å) with a binding mode analogous to the native tripeptide. SPR furthermore indicates a similar residence time (kd = ∼0.06 s-1) for these two analogs. This novel scaffold therefore represents a possible means to overcome the potential permeability issues of WDR5 ligands that possess highly basic groups like guanidine. The series reported here furthers the understanding of the WDR5 WIN site and functions as a starting point for the development of more potent WDR5 inhibitors that may serve as cancer therapeutics. (c) 2019 American Institute of Physics.
    54. 54
      Bolshan, Y.; Getlik, M.; Kuznetsova, E.; Wasney, G. A.; Hajian, T.; Poda, G.; Nguyen, K. T.; Wu, H.; Dombrovski, L.; Dong, A.; Senisterra, G.; Schapira, M.; Arrowsmith, C. H.; Brown, P. J.; Al-Awar, R.; Vedadi, M.; Smil, D. Synthesis, Optimization, and Evaluation of Novel Small Molecules as Antagonists of WDR5-MLL Interaction. ACS Med. Chem. Lett. 2013, 4, 353– 357,  DOI: 10.1021/ml300467n
      54
      Synthesis, Optimization, and Evaluation of Novel Small Molecules as Antagonists of WDR5-MLL Interaction
      Bolshan, Yuri; Getlik, Matthaus; Kuznetsova, Ekaterina; Wasney, Gregory A.; Hajian, Taraneh; Poda, Gennadiy; Nguyen, Kong T.; Wu, Hong; Dombrovski, Ludmila; Dong, Aiping; Senisterra, Guillermo; Schapira, Matthieu; Arrowsmith, Cheryl H.; Brown, Peter J.; Al-awar, Rima; Vedadi, Masoud; Smil, David
      ACS Medicinal Chemistry Letters (2013), 4 (3), 353-357CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)
      The WD40-repeat protein WDR5 plays a crit. role in maintaining the integrity of MLL complexes and fully activating their methyltransferase function. MLL complexes, the trithorax-like family of SET1 methyltransferases, catalyze trimethylation of lysine 4 on histone 3, and they have been widely implicated in various cancers. Antagonism of WDR5 and MLL subunit interaction by small mols. has recently been presented as a practical way to inhibit activity of the MLL1 complex, and N-(2-(4-methylpiperazin-1-yl)-5-substituted-phenyl) benzamides were reported as potent and selective antagonists of such an interaction. Here, we describe the protein crystal structure guided optimization of prototypic compd. I (Kdis = 7 μM), leading to identification of more potent antagonist II (Kdis = 0.3 μM).
    55. 55
      Getlik, M.; Smil, D.; Zepeda-Velázquez, C.; Bolshan, Y.; Poda, G.; Wu, H.; Dong, A.; Kuznetsova, E.; Marcellus, R.; Senisterra, G.; Dombrovski, L.; Hajian, T.; Kiyota, T.; Schapira, M.; Arrowsmith, C. H.; Brown, P. J.; Vedadi, M.; Al-Awar, R. Structure-Based Optimization of a Small Molecule Antagonist of the Interaction Between WD Repeat-Containing Protein 5 (WDR5) and Mixed-Lineage Leukemia 1 (MLL1). J. Med. Chem. 2016, 59, 2478– 2496,  DOI: 10.1021/acs.jmedchem.5b01630
      55
      Structure-Based Optimization of a Small Molecule Antagonist of the Interaction Between WD Repeat-Containing Protein 5 (WDR5) and Mixed-Lineage Leukemia 1 (MLL1)
      Getlik, Matthaus; Smil, David; Zepeda-Velazquez, Carlos; Bolshan, Yuri; Poda, Gennady; Wu, Hong; Dong, Aiping; Kuznetsova, Ekaterina; Marcellus, Richard; Senisterra, Guillermo; Dombrovski, Ludmila; Hajian, Taraneh; Kiyota, Taira; Schapira, Matthieu; Arrowsmith, Cheryl H.; Brown, Peter J.; Vedadi, Masoud; Al-awar, Rima
      Journal of Medicinal Chemistry (2016), 59 (6), 2478-2496CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      WD repeat-contg. protein 5 (WDR5) is an important component of the multiprotein complex essential for activating mixed-lineage leukemia 1 (MLL1). Rearrangement of the MLL1 gene is assocd. with onset and progression of acute myeloid and lymphoblastic leukemias, and targeting the WDR5-MLL1 interaction may result in new cancer therapeutics. Our previous work showed that binding of small mol. ligands to WDR5 can modulate its interaction with MLL1, suppressing MLL1 methyltransferase activity. Initial structure-activity relationship studies identified N-(2-(4-methylpiperazin-1-yl)-5-substituted-phenyl) benzamides as potent and selective antagonists of this protein-protein interaction. Guided by crystal structure data and supported by in silico library design, we optimized the scaffold by varying the C-1 benzamide and C-5 substituents. This allowed us to develop the first highly potent (Kdisp < 100 nM) small mol. antagonists of the WDR5-MLL1 interaction and demonstrate that N-(4-(4-methylpiperazin-1-yl)-3'-(morpholinomethyl)-[1,1'-biphenyl]-3-yl)-6-oxo-4-(trifluoromethyl)-1,6-dihydropyridine-3-carboxamide 16d (OICR-9429) is a potent and selective chem. probe suitable to help dissect the biol. role of WDR5.
    56. 56
      Schapira, M.; Arrowsmith, C. H. Methyltransferase inhibitors for modulation of the epigenome and beyond. Curr. Opin. Chem. Biol. 2016, 33, 81– 87,  DOI: 10.1016/j.cbpa.2016.05.030
      56
      Methyltransferase inhibitors for modulation of the epigenome and beyond
      Schapira, Matthieu; Arrowsmith, Cheryl H.
      Current Opinion in Chemical Biology (2016), 33 (), 81-87CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)
      Over the past two years tremendous progress has been made in the discovery of new inhibitors of protein lysine and arginine methyltransferases, establishing this class of epigenetic enzymes, along with DNA methyltransferases, as druggable protein families. New inhibitors of protein methyltransferases have been described with a variety of mechanisms of action including cofactor competitive, substrate competitive, allosteric inhibitors and disruptors of protein-protein interactions. Inhibitors have been used extensively in oncol. studies, and inhibitors of EZH2, and DOT1L are currently in clin. trials. Finally, advances in understanding the clin. mechanism of action of 5-azacytidine and related DNA hypomethylation agents were reported, revealing a likely role for a cell autonomous innate immune response.
    57. 57
      Gupta, A.; Xu, J.; Lee, S.; Tsai, S. T.; Zhou, B.; Kurosawa, K.; Werner, M. S.; Koide, A.; Ruthenburg, A. J.; Dou, Y.; Koide, S. Facile target validation in an animal model with intracellularly expressed monobodies. Nat. Chem. Biol. 2018, 14, 895– 900,  DOI: 10.1038/s41589-018-0099-z
      57
      Facile target validation in an animal model with intracellularly expressed monobodies
      Gupta, Ankit; Xu, Jing; Lee, Shirley; Tsai, Steven T.; Zhou, Bo; Kurosawa, Kohei; Werner, Michael S.; Koide, Akiko; Ruthenburg, Alexander J.; Dou, Yali; Koide, Shohei
      Nature Chemical Biology (2018), 14 (9), 895-900CODEN: NCBABT; ISSN:1552-4450. (Nature Research)
      Rapidly detg. the biol. effect of perturbing a site within a potential drug target could guide drug discovery efforts, but it remains challenging. Here, the authors describe a facile target validation approach that exploits monobodies, small synthetic binding proteins that can be fully functionally expressed in cells. The authors developed a potent and selective monobody to WDR5, a core component of the mixed lineage leukemia (MLL) methyltransferase complex. The monobody bound to the MLL interaction site of WDR5, the same binding site for small-mol. inhibitors whose efficacy has been demonstrated in cells but not in animals. As a genetically encoded reagent, the monobody inhibited proliferation of an MLL-AF9 cell line in vitro, suppressed its leukemogenesis and conferred a survival benefit in an in vivo mouse leukemia model. The capacity of this approach to readily bridge biochem., structural, cellular characterization and tests in animal models may accelerate discovery and validation of druggable sites.
    58. 58
      Concepcion, J.; Witte, K.; Wartchow, C.; Choo, S.; Yao, D.; Persson, H.; Wei, J.; Li, P.; Heidecker, B.; Ma, W.; Varma, R.; Zhao, L.-S.; Perillat, D.; Carricato, G.; Recknor, M.; Du, K.; Ho, H.; Ellis, T.; Gamez, J.; Howes, M.; Phi-Wilson, J.; Lockard, S.; Zuk, R.; Tan, H. Label-free detection of biomolecular interactions using BioLayer interferometry for kinetic characterization. Comb. Chem. High Throughput Screen. 2009, 12, 791– 800,  DOI: 10.2174/138620709789104915
      58
      Label-free detection of biomolecular interactions using biolayer interferometry for kinetic characterization
      Concepcion, Joy; Witte, Krista; Wartchow, Charles; Choo, Sae; Yao, Danfeng; Persson, Henrik; Wei, Jing; Li, Pu; Heidecker, Bettina; Ma, Weilei; Varma, Ram; Zhao, Lian-She; Perillat, Donald; Carricato, Greg; Recknor, Michael; Du, Kevin; Ho, Huddee; Ellis, Tim; Gamez, Juan; Howes, Michael; Phi-Wilson, Janette; Lockard, Scott; Zuk, Robert; Tan, Hong
      Combinatorial Chemistry & High Throughput Screening (2009), 12 (8), 791-800CODEN: CCHSFU; ISSN:1386-2073. (Bentham Science Publishers Ltd.)
      A review. The anal. of biomol. interactions is key in the drug development process. Label-free biosensor methods provide information on binding, kinetics, concn., and the affinity of an interaction. These techniques provide real-time monitoring of interactions between an immobilized ligand (such as a receptor) to an analyte in soln. without the use of labels. Advances in biosensor design and detection using BioLayer Interferometry (BLI) provide a simple platform that enables label-free monitoring of biomol. interactions without the use of flow cells. We review the applications of BLI in a wide variety of research and development environments for quantifying antibodies and proteins and measuring kinetics parameters.
    59. 59
      Weeramange, C. J.; Fairlamb, M. S.; Singh, D.; Fenton, A. W.; Swint-Kruse, L. The strengths and limitations of using biolayer interferometry to monitor equilibrium titrations of biomolecules. Protein Sci 2020, 29, 1018– 1034,  DOI: 10.1002/pro.3827
      59
      The strengths and limitations of using biolayer interferometry to monitor equilibrium titrations of biomolecules
      Weeramange, Chamitha J.; Fairlamb, Max S.; Singh, Dipika; Fenton, Aron W.; Swint-Kruse, Liskin
      Protein Science (2020), 29 (4), 1018-1034CODEN: PRCIEI; ISSN:1469-896X. (Wiley-Blackwell)
      Every method used to quantify biomol. interactions has its own strengths and limitations. To quantify protein-DNA binding affinities, nitrocellulose filter binding assays with 32P-labeled DNA quantify Kd values from 10-12 to 10-8 M but have several tech. limitations. Here, we considered the suitability of biolayer interferometry (BLI), which monitors assocn. and dissocn. of a sol. macromol. to an immobilized species; the ratio koff/kon dets. Kd. However, for lactose repressor protein (LacI) and an engineered repressor protein ("LLhF") binding immobilized DNA, complicated kinetic curves precluded this anal. Thus, we detd. whether the amplitude of the BLI signal at equil. related linearly to the fraction of protein bound to DNA. A key question was the effective concn. of immobilized DNA. Equil. titrn. expts. with DNA concns. below Kd (equil. binding regime) must be analyzed differently than those with DNA near or above Kd (stoichiometric binding regime). For ForteBio streptavidin tips, the most frequent effective DNA concn. was ∼2 x 10-9 M. Although variation occurred among different lots of sensor tips, binding events with Kd ≥ 10-8 M should reliably be in the equil. binding regime. We also obsd. effects from multi-valent interactions: Tetrameric LacI bound two immobilized DNAs whereas dimeric LLhF did not. We next used BLI to quantify the amt. of inducer sugars required to allosterically diminish protein-DNA binding and to assess the affinity of fructose-1-kinase for the DNA-LLhF complex. Overall, when exptl. design corresponded with appropriate data interpretation, BLI was convenient and reliable for monitoring equil. titrns. and thereby quantifying a variety of binding interactions.
    60. 60
      Imran, A.; Moyer, B. S.; Wolfe, A. J.; Cosgrove, M. S.; Makarov, D. E.; Movileanu, L. Interplay of Affinity and Surface Tethering in Protein Recognition. J. Phys. Chem. Lett. 2022, 13, 4021– 4028,  DOI: 10.1021/acs.jpclett.2c00621
      60
      Interplay of Affinity and Surface Tethering in Protein Recognition
      Imran, Ali; Moyer, Brandon S.; Wolfe, Aaron J.; Cosgrove, Michael S.; Makarov, Dmitrii E.; Movileanu, Liviu
      Journal of Physical Chemistry Letters (2022), 13 (18), 4021-4028CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
      Surface-tethered ligand-receptor complexes are key components in biol. signaling and adhesion. They also find increasing utility in single-mol. assays and biotechnol. applications. Here, we study the real-time binding kinetics between various surface-immobilized peptide ligands and their unrestrained receptors. A long peptide tether increases the assocn. of ligand-receptor complexes, exptl. proving the fly casting mechanism where the disorder accelerates protein recognition. On the other hand, a short peptide tether enhances the complex dissocn. Notably, the rate consts. measured for the same receptor, but under different spatial constraints, are strongly correlated to one another. Furthermore, this correlation can be used to predict how surface tethering on a ligand-receptor complex alters its binding kinetics. Our results have immediate implications in the broad areas of biomol. recognition, intrinsically disordered proteins, and biosensor technol.
    61. 61
      Wolfe, A. J.; Si, W.; Zhang, Z.; Blanden, A. R.; Hsueh, Y.-C.; Gugel, J. F.; Pham, B.; Chen, M.; Loh, S. N.; Rozovsky, S.; Aksimentiev, A.; Movileanu, L. Quantification of membrane protein-detergent complex interactions. J. Phys. Chem. B 2017, 121, 10228– 10241,  DOI: 10.1021/acs.jpcb.7b08045
      61
      Quantification of Membrane Protein-Detergent Complex Interactions
      Wolfe, Aaron J.; Si, Wei; Zhang, Zhengqi; Blanden, Adam R.; Hsueh, Yi-Ching; Gugel, Jack F.; Pham, Bach; Chen, Min; Loh, Stewart N.; Rozovsky, Sharon; Aksimentiev, Aleksei; Movileanu, Liviu
      Journal of Physical Chemistry B (2017), 121 (44), 10228-10241CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
      Although fundamentally significant in structural, chem., and membrane biol., the interfacial protein-detergent complex (PDC) interactions have been modestly examd. because of the complicated behavior of both detergents and membrane proteins in aq. phase. Membrane proteins are prone to unproductive aggregation resulting from poor detergent solvation, but the participating forces in this phenomenon remain ambiguous. Here, using rational membrane protein design, targeted chem. modification, and steady-state fluorescence polarization spectroscopy, the detergent desolvation of membrane proteins can be quant. evaluated. Depleting the detergent in the sample well produced a two-state transition of membrane proteins between a fully detergent-solvated state and a detergent-desolvated state, the nature of which depended on the interfacial PDC interactions. Using a panel of six membrane proteins of varying hydrophobic topog., structural fingerprint, and charge distribution on the solvent-accessible surface, the authors provide direct exptl. evidence for the contributions of the electrostatic and hydrophobic interactions to the protein solvation properties. Moreover, all-atom mol. dynamics simulations report the major contribution of the hydrophobic forces exerted at the PDC interface. This semiquant. approach might be extended in the future to include studies of the interfacial PDC interactions of other challenging membrane protein systems of unknown structure. This would have practical importance in protein extn., solubilization, stabilization, and crystn.
    62. 62
      Wolfe, A. J.; Gugel, J. F.; Chen, M.; Movileanu, L. Kinetics of Membrane Protein-Detergent Interactions Depend on Protein Electrostatics. J. Phys. Chem. B 2018, 122, 9471– 9481,  DOI: 10.1021/acs.jpcb.8b07889
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      Kinetics of Membrane Protein-Detergent Interactions Depend on Protein Electrostatics
      Wolfe, Aaron J.; Gugel, Jack F.; Chen, Min; Movileanu, Liviu
      Journal of Physical Chemistry B (2018), 122 (41), 9471-9481CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
      Interactions of a membrane protein with a detergent micelle represent a fundamental process with practical implications in structural and chem. biol. Quant. assessment of the kinetics of protein-detergent complex (PDC) interactions has always been challenged by complicated behavior of both membrane proteins and solubilizing detergents in aq. phase. Here, we show the kinetic reads of the desorption of maltoside-contg. detergents from β-barrel membrane proteins. Using steady-state fluorescence polarization (FP) anisotropy measurements, we recorded real-time, specific signatures of the PDC interactions. The results of these measurements were used to infer the model-dependent rate consts. of assocn. and dissocn. of the proteomicelles. Remarkably, the kinetics of the PDC interactions depend on the overall protein charge despite the nonionic nature of the detergent monomers. In the future, this approach might be employed for high-throughput screening of kinetic fingerprints of different membrane proteins stabilized in micelles that contain mixts. of various detergents.
    63. 63
      Forbes, S. A.; Beare, D.; Boutselakis, H.; Bamford, S.; Bindal, N.; Tate, J.; Cole, C. G.; Ward, S.; Dawson, E.; Ponting, L.; Stefancsik, R.; Harsha, B.; Kok, C. Y.; Jia, M.; Jubb, H.; Sondka, Z.; Thompson, S.; De, T.; Campbell, P. J. COSMIC: somatic cancer genetics at high-resolution. Nucleic Acids Res. 2017, 45, D777– d783,  DOI: 10.1093/nar/gkw1121
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      COSMIC: somatic cancer genetics at high-resolution
      Forbes, Simon A.; Beare, David; Boutselakis, Harry; Bamford, Sally; Bindal, Nidhi; Tate, John; Cole, Charlotte G.; Ward, Sari; Dawson, Elisabeth; Ponting, Laura; Stefancsik, Raymund; Harsha, Bhavana; Kok, Chai Yin; Jia, Mingming; Jubb, Harry; Sondka, Zbyslaw; Thompson, Sam; De, Tisham; Campbell, Peter J.
      Nucleic Acids Research (2017), 45 (D1), D777-D783CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
      COSMIC, the Catalog of Somatic Mutations in Cancer (http://cancer.sanger.ac.uk) is a high-resoln. resource for exploring targets and trends in the genetics of human cancer. Currently the broadest database of mutations in cancer, the information in COSMIC is curated by expert scientists, primarily by scrutinizing large nos. of scientific publications. Over 4 million coding mutations are described in v78 (Sept. 2016), combining genome-wide sequencing results from 28 366 tumors with complete manual curation of 23 489 individual publications focused on 186 key genes and 286 key fusion pairs across all cancers. Mol. profiling of large tumor nos. has also allowed the annotation of more than 13 million non-coding mutations, 18 029 gene fusions, 187 429 genome rearrangements, 1 271 436 abnormal copy no. segments, 9 175 462 abnormal expression variants and 7 879 142 differentially methylated CpG dinucleotides. COSMIC now details the genetics of drug resistance, novel somatic gene mutations which allow a tumor to evade therapeutic cancer drugs. Focusing initially on highly characterized drugs and genes, COSMIC v78 contains wide resistance mutation profiles across 20 drugs, detailing the recurrence of 301 unique resistance alleles across 1934 drug-resistant tumors. All information from the COSMIC database is available freely on the COSMIC website.
    64. 64
      Kumar, S.; Nussinov, R. Relationship between ion pair geometries and electrostatic strengths in proteins. Biophys. J. 2002, 83, 1595– 1612,  DOI: 10.1016/s0006-3495(02)73929-5
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      Relationship between ion pair geometries and electrostatic strengths in proteins
      Kumar, Sandeep; Nussinov, Ruth
      Biophysical Journal (2002), 83 (3), 1595-1612CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
      The electrostatic free energy contribution of an ion pair in a protein depends on two factors, geometrical orientation of the side-chain charged groups with respect to each other and the structural context of the ion pair in the protein. Conformers in NMR ensembles enable studies of the relationship between geometry and electrostatic strengths of ion pairs, because the protein structural contexts are highly similar across different conformers. We have studied this relationship using a dataset of 22 unique ion pairs in 14 NMR conformer ensembles for 11 nonhomologous proteins. In different NMR conformers, the ion pairs are classified as salt bridges, nitrogen-oxygen (N-O) bridges and longer-range ion pairs on the basis of geometrical criteria. In salt bridges, centroids of the side-chain charged groups and at least a pair of side-chain nitrogen and oxygen atoms of the ion-pairing residues are within a 4 Å distance. In N-O bridges, at least a pair of the side-chain nitrogen and oxygen atoms of the ion-pairing residues are within 4 Å distance, but the distance between the side-chain charged group centroids is greater than 4 Å. In the longer-range ion pairs, the side-chain charged group centroids as well as the side-chain nitrogen and oxygen atoms are more than 4 Å apart. Continuum electrostatic calcns. indicate that most of the ion pairs have stabilizing electrostatic contributions when their side-chain charged group centroids are within 5 Å distance. Hence, most (∼92%) of the salt bridges and a majority (68%) of the N-O bridges are stabilizing. Most (∼89%) of the destabilizing ion pairs are the longer-range ion pairs. In the NMR conformer ensembles, the electrostatic interaction between side-chain charged groups of the ion-pairing residues is the strongest for salt bridges, considerably weaker for N-O bridges, and the weakest for longer-range ion pairs. These results suggest empirical rules for stabilizing electrostatic interactions in proteins.
    65. 65
      Nogal, B.; Bowman, C. A.; Ward, A. B. Time-course, negative-stain electron microscopy-based analysis for investigating protein-protein interactions at the single-molecule level. J. Biol. Chem. 2017, 292, 19400– 19410,  DOI: 10.1074/jbc.m117.808352
      65
      Time-course, negative-stain electron microscopy-based analysis for investigating protein-protein interactions at the single-molecule level
      Nogal, Bartek; Bowman, Charles A.; Ward, Andrew B.
      Journal of Biological Chemistry (2017), 292 (47), 19400-19410CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Several biophys. approaches are available to study protein-protein interactions. Most approaches are conducted in bulk soln. and are therefore limited to an av. measurement of the ensemble of mol. interactions. Here, single-particle EM can enrich the understanding of protein-protein interactions at the single-mol. level and potentially capture states that are unobservable with ensemble methods because they are below the limit of detection or not conducted on an appropriate time scale. Using the HIV-1 envelope glycoprotein (Env) and its interaction with receptor CD4-binding site neutralizing antibodies as a model system, the authors both corroborate ensemble kinetics-derived parameters and demonstrate how time-course EM can further dissect stoichiometric states of complexes that are not readily observable with other methods. Visualization of the kinetics and stoichiometry of Env-antibody complexes demonstrated the applicability of this approach to qual. and semi-quant. differentiate two highly similar neutralizing antibodies. Furthermore, implementation of machine-learning techniques for sorting class avs. of these complexes into discrete subclasses of particles helped reduce human bias. The authors' data provide proof of concept that single-particle EM can be used to generate a "visual" kinetic profile that should be amenable to studying many other protein-protein interactions, is relatively simple and complementary to well-established biophys. approaches. Moreover, the authors' method provides crit. insights into broadly neutralizing antibody recognition of Env, which may inform vaccine immunogen design and immunotherapeutic development.
    66. 66
      Chen, C.; Liu, Y.; Rappaport, A. R.; Kitzing, T.; Schultz, N.; Zhao, Z.; Shroff, A. S.; Dickins, R. A.; Vakoc, C. R.; Bradner, J. E.; Stock, W.; LeBeau, M. M.; Shannon, K. M.; Kogan, S.; Zuber, J.; Lowe, S. W. MLL3 is a haploinsufficient 7q tumor suppressor in acute myeloid leukemia. Cancer Cell 2014, 25, 652– 665,  DOI: 10.1016/j.ccr.2014.03.016
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      MLL3 Is a Haploinsufficient 7q Tumor Suppressor in Acute Myeloid Leukemia
      Chen, Chong; Liu, Yu; Rappaport, Amy R.; Kitzing, Thomas; Schultz, Nikolaus; Zhao, Zhen; Shroff, Aditya S.; Dickins, Ross A.; Vakoc, Christopher R.; Bradner, James E.; Stock, Wendy; LeBeau, Michelle M.; Shannon, Kevin M.; Kogan, Scott; Zuber, Johannes; Lowe, Scott W.
      Cancer Cell (2014), 25 (5), 652-665CODEN: CCAECI; ISSN:1535-6108. (Elsevier Inc.)
      Recurring deletions of chromosome 7 and 7q [-7/del(7q)] occur in myelodysplastic syndromes and acute myeloid leukemia (AML) and are assocd. with poor prognosis. However, the identity of functionally relevant tumor suppressors on 7q remains unclear. Using RNAi and CRISPR/Cas9 approaches, we show that an ∼50% redn. in gene dosage of the mixed lineage leukemia 3 (MLL3) gene, located on 7q36.1, cooperates with other events occurring in -7/del(7q) AMLs to promote leukemogenesis. Mll3 suppression impairs the differentiation of HSPC. Interestingly, Mll3-suppressed leukemias, like human -7/del(7q) AMLs, are refractory to conventional chemotherapy but sensitive to the BET inhibitor JQ1. Thus, our mouse model functionally validates MLL3 as a haploinsufficient 7q tumor suppressor and suggests a therapeutic option for this aggressive disease.
    67. 67
      Wu, H.-T.; Liu, Y.-E.; Hsu, K.-W.; Wang, Y.-F.; Chan, Y.-C.; Chen, Y.; Chen, D.-R. MLL3 Induced by Luteolin Causes Apoptosis in Tamoxifen-Resistant Breast Cancer Cells through H3K4 Monomethylation and Suppression of the PI3K/AKT/mTOR Pathway. Am. J. Chin. Med. 2020, 48, 1221– 1241,  DOI: 10.1142/s0192415x20500603
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      MLL3 Induced by Luteolin Causes Apoptosis in Tamoxifen-Resistant Breast Cancer Cells through H3K4 Monomethylation and Suppression of the PI3K/AKT/mTOR Pathway
      Wu, Han-Tsang; Liu, Yi-En; Hsu, Kai-Wen; Wang, Yu-Fen; Chan, Ya-Chi; Chen, Yeh; Chen, Dar-Ren
      American Journal of Chinese Medicine (2020), 48 (5), 1221-1241CODEN: AJCMBA; ISSN:0192-415X. (World Scientific Publishing Co. Pte. Ltd.)
      Tamoxifen is one of the most common hormone therapy drug for estrogen receptor pos. breast cancer. Tumor cells with drug resistance often cause recurrence and metastasis in cancer patients. Luteolin is a natural compd. found from various types of vegetables and exhibit anticancer activity in different cancers. This study demonstrated that luteolin inhibits the proliferation and induces apoptosis of tamoxifen-resistant ER-pos. breast cancer cells. Luteolin also causes cell cycle arrest at the G2/M phase and decreases mitochondrial membrane potential. Besides, luteolin reduces the levels of activated PI3K/AKT/mTOR signaling pathway. The combination treatment of luteolin and PI3K, AKT, or mTOR inhibitors synergistically increases apoptosis in tamoxifen-resistant ER-pos. breast cancer cells. Ras gene family (K-Ras, H-Ras, and N-Ras), an activator of PI3K, was transcriptionally repressed by luteolin via induction of tumor suppressor mixed-lineage leukemia 3 expression. MLL3 increases the level of monomethylation of Histone 3 Lysine 4 on the enhancer and promoter region of Ras genes, thus causes repression of Ras expressions. Our finding implies that luteolin was a promising natural agent against tamoxifen resistance of breast cancer.
    68. 68
      Rampias, T.; Karagiannis, D.; Avgeris, M.; Polyzos, A.; Kokkalis, A.; Kanaki, Z.; Kousidou, E.; Tzetis, M.; Kanavakis, E.; Stravodimos, K.; Manola, K. N.; Pantelias, G. E.; Scorilas, A.; Klinakis, A. The lysine-specific methyltransferase KMT2C/MLL3 regulates DNA repair components in cancer. EMBO Rep. 2019, 20, e46821  DOI: 10.15252/embr.201846821
      There is no corresponding record for this reference.
    69. 69
      Wong, S. H. K.; Goode, D. L.; Iwasaki, M.; Wei, M. C.; Kuo, H.-P.; Zhu, L.; Schneidawind, D.; Duque-Afonso, J.; Weng, Z.; Cleary, M. L. The H3K4-Methyl Epigenome Regulates Leukemia Stem Cell Oncogenic Potential. Cancer Cell 2015, 28, 198– 209,  DOI: 10.1016/j.ccell.2015.06.003
      69
      The H3K4-Methyl Epigenome Regulates Leukemia Stem Cell Oncogenic Potential
      Wong, Stephen H. K.; Goode, David L.; Iwasaki, Masayuki; Wei, Michael C.; Kuo, Hsu-Ping; Zhu, Li; Schneidawind, Dominik; Duque-Afonso, Jesus; Weng, Ziming; Cleary, Michael L.
      Cancer Cell (2015), 28 (2), 198-209CODEN: CCAECI; ISSN:1535-6108. (Elsevier Inc.)
      The genetic programs that maintain leukemia stem cell (LSC) self-renewal and oncogenic potential have been well defined; however, the comprehensive epigenetic landscape that sustains LSC cellular identity and functionality is less well established. We report that LSCs in MLL-assocd. leukemia reside in an epigenetic state of relative genome-wide high-level H3K4me3 and low-level H3K79me2. LSC differentiation is assocd. with reversal of these broad epigenetic profiles, with concomitant downregulation of crucial MLL target genes and the LSC maintenance transcriptional program that is driven by the loss of H3K4me3, but not H3K79me2. The H3K4-specific demethylase KDM5B neg. regulates leukemogenesis in murine and human MLL-rearranged AML cells, demonstrating a crucial role for the H3K4 global methylome in detg. LSC fate.
    70. 70
      Shinsky, S. A.; Cosgrove, M. S. Unique Role of the WD-40 Repeat Protein 5 (WDR5) Subunit within the Mixed Lineage Leukemia 3 (MLL3) Histone Methyltransferase Complex. J. Biol. Chem. 2015, 290, 25819– 25833,  DOI: 10.1074/jbc.m115.684142
      70
      Unique role of the WD-40 repeat protein 5 (WDR5) subunit within the mixed lineage leukemia 3 (MLL3) histone methyltransferase complex
      Shinsky, Stephen A.; Cosgrove, Michael S.
      Journal of Biological Chemistry (2015), 290 (43), 25819-25833CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      The MLL3 (mixed lineage leukemia 3) protein is a member of the human SET1 family of histone H3 lysine 4 methyltransferases and contains the conserved WDR5 interaction (Win) motif and the catalytic suppressor of variegation, enhancer of zeste, trithorax (SET) domain. The human SET1 family includes MLL1-4 and SETd1A/B, which all interact with a conserved subcomplex contg. WDR5, RbBP5, Ash2L, and DPY-30 (WRAD) to form the minimal core complex required for full methyltransferase activity. However, recent evidence suggests that the WDR5 subunit may not be utilized in an identical manner within all SET1 family core complexes. Although the roles of WDR5 within the MLL1 core complex have been extensively studied, not much is known about the roles of WDR5 in other SET1 family core complexes. In this investigation, we set out to characterize the roles of the WDR5 subunit in the MLL3 core complex. We found that unlike MLL1, the MLL3 SET domain assembles with the RbBP5/Ash2L heterodimer independently of the Win motif-WDR5 interaction. Furthermore, we obsd. that WDR5 inhibits the monomethylation activity of the MLL3 core complex, which is dependent on the Win motif. We also found evidence suggesting that the WRAD subcomplex catalyzes weak H3K4 monomethylation within the context of the MLL3 core complex. Furthermore, soln. structures of the MLL3 core complex assembled with and without WDR5 by small angle x-ray scattering show similar overall topologies. Together, this work demonstrates a unique role for WDR5 in modulating the enzymic activity of the MLL3 core complex.
    71. 71
      Wysocka, J.; Swigut, T.; Milne, T. A.; Dou, Y.; Zhang, X.; Burlingame, A. L.; Roeder, R. G.; Brivanlou, A. H.; Allis, C. D. WDR5 associates with histone H3 methylated at K4 and is essential for H3 K4 methylation and vertebrate development. Cell 2005, 121, 859– 872,  DOI: 10.1016/j.cell.2005.03.036
      71
      WDR5 associates with histone H3 methylated at K4 and is essential for H3 K4 methylation and vertebrate development
      Wysocka, Joanna; Swigut, Tomek; Milne, Thomas A.; Dou, Yali; Zhang, Xin; Burlingame, Alma L.; Roeder, Robert G.; Brivanlou, Ali H.; Allis, C. David
      Cell (Cambridge, MA, United States) (2005), 121 (6), 859-872CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      Histone H3 lysine 4 (K4) methylation has been linked to the transcriptional activation in a variety of eukaryotic species. Here authors show that a common component of MLL1, MLL2, and hSet1 H3 K4 methyltransferase complexes, the WD40-repeat protein WDR5, directly assocs. with histone H3 di- and trimethylated at K4 and with H3-K4-dimethylated nucleosomes. WDR5 is required for binding of the methyltransferase complex to the K4-dimethylated H3 tail as well as for global H3 K4 trimethylation and HOX gene activation in human cells. WDR5 is essential for vertebrate development, in that WDR5-depleted X. laevis tadpoles exhibit a variety of developmental defects and abnormal spatial Hox gene expression. These results are the first demonstration that a WD40-repeat protein acts as a module for recognition of a specific histone modification and suggest a mechanism for reading and writing an epigenetic mark for gene activation.
    72. 72
      Itan, Y.; Casanova, J.-L. Can the impact of human genetic variations be predicted?. Proc. Natl. Acad. Sci. U.S.A. 2015, 112, 11426– 11427,  DOI: 10.1073/pnas.1515057112
      72
      Can the impact of human genetic variations be predicted?
      Itan, Yuval; Casanova, Jean-Laurent
      Proceedings of the National Academy of Sciences of the United States of America (2015), 112 (37), 11426-11427CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      There is no expanded citation for this reference.
    73. 73
      Ali, A.; Veeranki, S. N.; Tyagi, S. A SET-domain-independent role of WRAD complex in cell-cycle regulatory function of mixed lineage leukemia. Nucleic Acids Res. 2014, 42, 7611– 7624,  DOI: 10.1093/nar/gku458
      73
      A SET-domain-independent role of WRAD complex in cell-cycle regulatory function of mixed lineage leukemia
      Ali, Aamir; Veeranki, Sailaja Naga; Tyagi, Shweta
      Nucleic Acids Research (2014), 42 (12), 7611-7624CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      MLL, the trithorax ortholog, is a well-characterized histone 3 lysine 4 methyltransferase that is crucial for proper regulation of the Hox genes during embryonic development. Chromosomal translocations, disrupting the Mll gene, lead to aggressive leukemia with poor prognosis. However, the functions of MLL in cellular processes like cell-cycle regulation are not well studied. Here we show that the MLL has a regulatory role during multiple phases of the cell cycle. RNAi-mediated knockdown reveals that MLL regulates S-phase progression and, proper segregation and cytokinesis during M phase. Using deletions and mutations, we narrow the cell-cycle regulatory role to the C subunit of MLL. Our anal. reveals that the transactivation domain and not the SET domain is important for the S-phase function of MLL. Surprisingly, disruption of MLL-WRAD interaction is sufficient to disrupt proper mitotic progression. These mitotic functions of WRAD are independent of SET domain of MLL and, therefore, define a new role of WRAD in subset of MLL functions. Finally, we address the overlapping and unique roles of the different SET family members in the cell cycle.
    74. 74
      Movileanu, L.; Cheley, S.; Howorka, S.; Braha, O.; Bayley, H. Location of a Constriction in the Lumen of a Transmembrane Pore by Targeted Covalent Attachment of Polymer Molecules. J. Gen. Physiol. 2001, 117, 239– 252,  DOI: 10.1085/jgp.117.3.239
      74
      Location of a constriction in the lumen of a transmembrane pore by targeted covalent attachment of polymer molecules
      Movileanu, Liviu; Cheley, Stephen; Howorka, Stefan; Braha, Orit; Bayley, Hagan
      Journal of General Physiology (2001), 117 (3), 239-251CODEN: JGPLAD; ISSN:0022-1295. (Rockefeller University Press)
      Few methods exist for obtaining the internal dimensions of transmembrane pores for which 3-D structures are lacking or for showing that structures detd. by crystallog. reflect the internal dimensions of pores in lipid bilayers. Several approaches, involving polymer penetration and transport, have revealed limiting diams. for various pores. But, in general, these approaches do not indicate the locations of constrictions in the channel lumen. Here, the authors combine cysteine mutagenesis and chem. modification with sulfhydryl-reactive polymers to locate the constriction in the lumen of the staphylococcal α-hemolysin pore, a model protein of known structure. The rates of reaction of each of four polymeric regents (MePEG-OPSS) of different masses towards individual single cysteine mutants, comprising a set with cysteines distributed over the length of the lumen of the pore, were detd. by macroscopic current recording. The rates for the three larger polymers (1.8, 2.5, and 5.0 kDa) were normalized with respect to the rates of reaction with a 1.0 kDa polymer for each of the seven positions in the lumen. The rate of reaction of the 5.0 kDa polymer dropped dramatically at the centrally located Cys-111 residue and positions distal to Cys-111, whether the reagent was applied from the trans or the cis side of the bilayer. This semi-quant. anal. sufficed to demonstrate that a constriction is located at the midpoint of the pore lumen, as predicted by the crystal structure, and although the constriction allows a 2.5 kDa polymer to pass, transport of a 5.0 kDa mol. is greatly restricted. In addn., PEG chains gave greater redns. in pore conductance when covalently attached to the narrower regions of the lumen, permitting further definition of the interior of the pore. The procedures described here should be applicable to other pores and to related structures such as the vestibules of ion channels.
    75. 75
      Jarmoskaite, I.; AlSadhan, I.; Vaidyanathan, P. P.; Herschlag, D. How to measure and evaluate binding affinities. eLife 2020, 9, e57264  DOI: 10.7554/eLife.57264
      75
      How to measure and evaluate binding affinities
      Jarmoskaite, Inga; Alsadhan, Ishraq; Vaidyanathan, Pavanapuresan P.; Herschlag, Daniel
      eLife (2020), 9 (), e57264CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)
      Quant. measurements of biomol. assocns. are central to biol. understanding and are needed to build and test predictive and mechanistic models. Given the advances in high-throughput technologies and the projected increase in the availability of binding data, we found it esp. timely to evaluate the current stds. for performing and reporting binding measurements. A review of 100 studies revealed that in most cases essential controls for establishing the appropriate incubation time and concn. regime were not documented, making it impossible to det. measurement reliability. Moreover, several reported affinities could be concluded to be incorrect, thereby impacting biol. interpretations. Given these challenges, we provide a framework for a broad range of researchers to evaluate, teach about, perform, and clearly document high-quality equil. binding measurements. We apply this framework and explain underlying fundamental concepts through exptl. examples with the RNA-binding protein Puf4.
    76. 76
      Wolfe, A. J.; Hsueh, Y.-C.; Blanden, A. R.; Mohammad, M. M.; Pham, B.; Thakur, A. K.; Loh, S. N.; Chen, M.; Movileanu, L. Interrogating Detergent Desolvation of Nanopore-Forming Proteins by Fluorescence Polarization Spectroscopy. Anal. Chem. 2017, 89, 8013– 8020,  DOI: 10.1021/acs.analchem.7b01339
      76
      Interrogating Detergent Desolvation of Nanopore-Forming Proteins by Fluorescence Polarization Spectroscopy
      Wolfe, Aaron J.; Hsueh, Yi-Ching; Blanden, Adam R.; Mohammad, Mohammad M.; Pham, Bach; Thakur, Avinash K.; Loh, Stewart N.; Chen, Min; Movileanu, Liviu
      Analytical Chemistry (Washington, DC, United States) (2017), 89 (15), 8013-8020CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Understanding how membrane proteins interact with detergents is of fundamental and practical significance in structural and chem. biol. as well as in nanobiotechnol. Current methods for inspecting protein-detergent complex (PDC) interfaces require high concns. of protein and are of low throughput. Here, the authors describe a scalable, spectroscopic approach that uses nanomolar protein concns. in native solns. This approach, which is based on steady-state fluorescence polarization (FP) spectroscopy, kinetically resolves the dissocn. of detergents from membrane proteins and protein unfolding. For satisfactorily solubilizing detergents, at concns. much greater than the crit. micelle concn. (CMC), the fluorescence anisotropy was independent of detergent concn. In contrast, at detergent concns. comparable with or below the CMC, the anisotropy readout underwent a time-dependent decrease, showing a specific and sensitive protein unfolding signature. Functionally reconstituted membrane proteins into a bilayer membrane confirmed predictions made by these FP-based detns. with respect to varying refolding conditions. From a practical point of view, this 96-well anal. approach will facilitate a massively parallel assessment of the PDC interfacial interactions under a fairly broad range of micellar and environmental conditions. The authors expect that these studies will potentially accelerate research in membrane proteins pertaining to their extn., solubilization, stabilization, and crystn., as well as reconstitution into bilayer membranes.

  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschembio.2c00273.

    • Sequence of SET1Win peptide ligands; location of somatic cancer mutations from tumor samples with N < 500; results of mutation clustering for different mutation subsets; list of tumor locations associated with missense WDR5 mutations within and around the Win binding site; list of noncovalent bonds at the WDR5-SET1Win interface; location of surface WDR5 mutations within the A and B pockets; BLI measurements of very weak interactions; kinetic rate constants of association and dissociation of WDR5 mutants with SET1Win ligands; equilibrium dissociation constants of WDR5 mutants with SET1Win ligands; structural information on the effect of the S175L mutation; steady-state FP spectroscopy curves for the interactions of WDR5 mutants with SET1win ligands; equilibrium dissociation constants of WDR5 mutants with SET1Win ligands using steady-state FP spectroscopy; quantitative comparisons of affinity data acquired with BLI and FP; and supporting references (PDF)


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