Global Profiling and Inhibition of Protein Lipidation in Vector and Host Stages of the Sleeping Sickness Parasite Trypanosoma brucei

The enzyme N-myristoyltransferase (NMT) catalyzes the essential fatty acylation of substrate proteins with myristic acid in eukaryotes and is a validated drug target in the parasite Trypanosoma brucei, the causative agent of African trypanosomiasis (sleeping sickness). N-Myristoylation typically mediates membrane localization of proteins and is essential to the function of many. However, only a handful of proteins are experimentally validated as N-myristoylated in T. brucei. Here, we perform metabolic labeling with an alkyne-tagged myristic acid analogue, enabling the capture of lipidated proteins in insect and host life stages of T. brucei. We further compare this with a longer chain palmitate analogue to explore the chain length-specific incorporation of fatty acids into proteins. Finally, we combine the alkynyl-myristate analogue with NMT inhibitors and quantitative chemical proteomics to globally define N-myristoylated proteins in the clinically relevant bloodstream form parasites. This analysis reveals five ARF family small GTPases, calpain-like proteins, phosphatases, and many uncharacterized proteins as substrates of NMT in the parasite, providing a global view of the scope of this important protein modification and further evidence for the crucial and pleiotropic role of NMT in the cell.


Supporting Tables
Supporting tables are available as separate Excel spreadsheets. Each contains a key describing the contents of the file.            a.

b.
Pearson correlation (total data): 0.83 Figure S8. Heatmap of YnMyr intensities of all proteins showing significant differences (by ttest) between BSF (B1-B4) and PCF (P1-P6). Colour-coding globally across all rows in the matrix (note that in Fig. 4c colour-coding is within a row). FC = fold-change (Log2(BSF/PCF). pvalue = -log10(p-value t-test). MG = protein contains N-terminal glycine. One representative ID and protein name per ProteinGroup is shown (see Supp. Table S4 for complete data). Keyword based on GO annotation from TriTrypDB. Heatmap created with Gene-E (http://www.broadinstitute.org/cancer/software/GENE-E/index.html).    Figure S13. Related to Figure 7. a. Dose-response plots (treatment with 2) for protein hits (high confidence hitssee Table S6; colour-coded by clustering). b. Dose-response plots (treatment with 2) for other MG proteins not assigned as hits (grey) and for outlier non-MG protein (Tb927.8.2250, black) that is decreased only at high concentrations of inhibitors. c. Doseresponse plots for 7 proteins that show only a weak response to NMT inhibitors 1 & 2 but where the YnMyr modified peptide was identified (see Tables S3 and S6).  Tables S6 and S7. Agree high/reliable = both tools identify protein as high confidence NMT substrate; some prediction both = both tools predict some degree of N-myristoylation; disagree = one tool predicts N-myristoylation (to some degree) but the other tool does not; no myr = no myristoylation site predicted. b. Sequence logo analysis using the N-terminal 8 residues of high confidence NMT hits. Created using Weblogo (http://weblogo.berkeley.edu/).

Enzyme inhibition assays
Assessment of compound inhibition of NMT was carried out as described previously. 5 Data were fitted to a back-corrected IC50 function using GraFit 7.0 (Erithacus Software Ltd, UK).
For direct gel analysis, 4 x sample loading buffer (SLB, NuPAGE LDS sample buffer) with 2mercaptoethanol (4 % final) was added and proteins heated for 3 min at 95 °C prior to SDS-PAGE.
NaOH treatment. Samples were resuspended after CuAAC as above, then 0.2M NaOH added and samples incubated at RT for 1 hour. Samples were quenched by addition of 4 x SLB.
Pronase treatment of proteins. Following CuAAC and resuspension (1% SDS, PBS, without EDTA), samples were split in two and treated with pronase (Sigma) at 1 mg/mL or water (control) from a 10x stock and incubated at 37 °C for 4 h. 4 x SLB was added and proteins heated for 5 min at 90 °C prior to SDS-PAGE.
Pull-down for gel analysis. Protein was resuspended following CuAAC at 10 mg/mL in 2 % SDS, 10 mM EDTA in PBS, and then diluted to 1 mg/mL with PBS. DTT (from a fresh 100 × stock in water) was added to give a final concentration of 1 mM. Proteins were incubated with Dynabeads® MyOne™ Streptavidin C1 (pre-washed 3 × 0.2 % SDS in PBS) for 1.5-2 h at RT with rotation. Following removal of the supernatant, beads were washed with 3 × 1 % SDS in PBS, then boiled for 10 mins in SLB to elute bound proteins.

Gel and Western blot analysis
Samples were separated by SDS-PAGE and the gel soaked in fixing solution (10 % AcOH, 40 % MeOH), then rinsed in water for in-gel fluorescent imaging: gels were scanned with Cy3 filters to detect the TAMRA fluorophore using an Ettan DIGE scanner, GE Healthcare. Molecular weight markers (Precision Plus All Blue Standards, Bio-Rad) were detected with Cy5 filters. For base treatment of gels: the gel was first fixed by treatment with gel soaking solution (10 % AcOH, 40 % MeOH) for 20 min, then washed briefly with water. A solution of 0.5 M NaOH in 50 % MeOH was added and the gel agitated gently for 1 hr at RT. The gel was washed briefly in water and then treated again with gel soaking solution for 20 min. The gel was washed again briefly with water before reimaging. ImageJ 7 was used for quantification of fluorescent bands. A thin rectangle was dropped down the length of the lane and the 'gel analyzer' function used to plot the profile of intensity down the lane (with averaging across horizontally). The signal was measured by integrating the area under each band of interest and normalizing relative to no inhibition (YnMyr only). Any background (from Myr treated sample) was subtracted. Total protein loading was checked by Coomassie. TC50 was calculated by fitting data to a back-corrected IC50 function using GraFit 7.0 (Erithacus Software Ltd, UK).
For Western blot detection of biotinylated proteins, proteins were transferred from gels to a PVDF membrane (Immobilon-PSQ, Millipore) using a semi-dry Western blot system (Invitrogen). Tris-glycine transfer buffer (NuPAGE, Invitrogen) was used to soak the blotting paper (2.5 mm paper, Invitrogen) and membrane prior to transfer. PVDF membrane was soaked first in MeOH (~30 sec), then water (2 min) before soaking in transfer buffer (at least 5 min). Transfer took 27 min at 20 V. Membranes were blocked for 1 hr RT with BSA (3 % w/v in 10 mL TBST) and then washed with TBST (3 x 10 mL for 5 min). Membranes were then incubated with NeutrAvidin-HRP (1:10000, Invitrogen) in TBST for one hour and washed again with TBST (3 x 10 mL for 10 min). Detection was carried out using Luminata Crescendo Western HRP substrate (Millipore) according to the manufactures instructions and on a Fujifilm LAS 3000 imager.

Proteomics experiments
Pull-down and preparation of peptides for MS. 0.25-0.5 mg lysate was prepared for proteomic analysis. Proteins were precipitated with acetone (4 volumes added and sample left at -20 °C for at least 1 h) or chloroform/methanol and resuspended at 1 mg/mL in 1% SDS in PBS. Chloroform/methanol precipitation: 4 volumes of MeOH were added, followed by 1 volume of CHCl3 and 3 volumes of H2O, with vortexing between each addition. Samples were vortexed vigorously and centrifuged at 17,000 ×g for 5 min at RT. The top aqueous-MeOH layer was removed and 4 volumes of MeOH added. Samples were gently mixed and centrifuged again. The supernatant was removed and the pellet washed 2x with MeOH.
Proteins were captured by CuAAC as before with the following modifications: CuAAC reaction was carried out for 2 hours and, for some samples, with AzRB or AzRTB in place of AzTB. When AzRB/RTB was used, proteins were precipitated following CuAAC via a modified chloroform/methanol precipitation procedure: 4 volumes of MeOH, 1 vol. CHCl3, 3 vol. H2O were added to the sample, which was centrifuged at 17,000 ×g for 5 min to pellet proteins at the interface. Both layers were then removed simultaneously, the pellet resuspended in 0.2 % SDS/PBS to the original volume and the precipitation procedure repeated. The pellet was then washed 2x with MeOH. Proteins were finally resuspended at 10 mg/mL in 2 % SDS, 10 mM EDTA in PBS, and then diluted to 1 mg/mL with PBS. DTT (from a fresh 100 × stock in water) was added to give a final concentration of 1 mM.
For base-treatment (inhibition samples), proteins were resuspended in 50 µL 2 % SDS, 10 mM EDTA in PBS; to this was added 50 µL PBS and 25 µL 2M NaOH. Samples were shaken for 1 hr RT then 25 µL 2M HCl added to neutralise; next 350 µL PBS was added and 5 µL 0.1M DTT (giving 1 mM DTT final concentration). Samples were centrifuged at 17000xg, 10 min RT to pellet any undissolved protein.
Affinity enrichment were performed for all samples as before with the following modifications: NeutrAvidin agarose resin (Thermo Scientific, pre-washed 3 × 0.2 % SDS in PBS; typically 50 μL of bead slurry was used for 0.5 mg of lysate) was used in place of the magnetic Dynabeads. Beads were stringently washed following pull-down: 3 × 1 % SDS in PBS, 3 × 4M Urea in 50 mM PBS, 5 × AMBIC (50 mM ammonium bicarbonate). For a 50 μL bed of beads resuspended in 50 μL AMBIC, samples were reduced (5 μL of 100 mM DTT in 50 mM AMBIC) at 60 °C for 30 minutes and allowed to cool to room temperature. The beads were washed with 2 x AMBIC. Cysteines were alkylated (5 μL of 100 mM iodoacetamide in AMBIC) at room temperature for 30 min in the dark. The beads were washed with 2 x AMBIC. Trypsin (1 μg Sequencing Grade Modified Trypsin (Promega) dissolved at 0.2 μg/μL in AMBIC per mg starting lysate) was added to the beads and samples were placed on a shaker and digested overnight at 37 °C. The samples were centrifuged and the supernatant was transferred into clean tubes. The beads were washed twice with 0.1% aqueous formic acid, and these washes were combined with the first supernatant. The solutions were stage-tipped according to a published protocol 8 . Elution from the sorbent (SDC-XC from 3M) with 70 % acetonitrile in water was followed by speed-vacassisted solvent removal, reconstitution of peptides in 0.5 % TFA, 2 % acetonitrile in water, and transferred into LC-MS sample vials.

LC-MS/MS analysis.
The analysis was performed using an Acclaim PepMap RSLC column 50 cm × 75 μm inner diameter (Thermo Fisher Scientific) using a 2 h acetonitrile gradient in 0.1 % aqueous formic acid at a flow rate of 250 nL/min. Easy nLC-1000 was coupled to a Q Exactive mass spectrometer via an easy-spray source (all Thermo Fisher Scientific). The Q Exactive was operated in data-dependent mode with survey scans acquired at a resolution of 75,000 at m/z 200 (transient time 256 ms). Up to 10 of the most abundant isotope patterns with charge +2 or higher from the survey scan were selected with an isolation window of 3.0 m/z and fragmented by HCD with normalized collision energies of 25. The maximum ion injection times for the survey scan and the MS/MS scans (acquired with a resolution of 17 500 at m/z 200) were 20 and 120 ms, respectively. The ion target value for MS was set to 10 6 and for MS/MS to 10 5 , and the intensity threshold was set to 8.3 × 10 2 .
Bioinformatics and comparisons with literature datasets. Prediction of myristoylation was performed using the Myristoylator (http://web.expasy.org/myristoylator/) 4a  For analysis of hits during differentiation of T. brucei from BSF to PCF, the dataset of Dejung et al. was used. 2 Briefly, the LFQ intensity data (ProteinGroups file) was downloaded from PRIDE (PXD003319), intensities logarithmized (base 2) and missing intensities imputed with random numbers from a normal distribution, whose mean and standard deviation were chosen to simulate low abundance values close to noise level (impute criteria: width 0.1 and down shift 2.1; imputation for each sample individually). Intensities were averaged (mean) across replicates and cross-referenced with the current dataset to generate profile plots of select proteins during the differentiation process (Fig. S7).