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N-terminal acetylome analyses reveals the substrate specificity profile of Naa50 (Nat5) and implicate NATs in influencing initiator Methionine processing
Co-translational N-terminal (Nt-) acetylation of nascent polypeptides is catalyzed by N-terminal acetyltransferases (NATs). The very N-terminal amino acid sequence is the major factor determining whether or not a given protein is Nt-acetylated. In humans, six different NATs, denoted NatA-NatF, are identified. In the current study we used N-terminal COFRADIC analysis to define the in vivo substrate specificity of Naa50 (Nat5)/NatE as this has long remained elusive. Three yeast strains were generated; a control strain endogenously expressing yNaa50, a deletion strain depleted of yNaa50 and a strain deleted of yNaa50 but ectopically expressing human Naa50. When comparing the Nt-acetylation status of different N-termini in the control strain with the deletion strain, a reduction in Nt-acetylation for several yeast proteins was observed. To our surprise, these substrates were not of the predicted NatE-type substrates, but rather canonical NatA substrates. Further, ectopic expression of hNaa50 mainly resulted in the Nt-acetylation of a selected class of otherwise Nt-free yeast N-termini besides increasing the degree of Nt-acetylation of several other yeast proteins, and as such (partially) complementing those N-termini displaying reduced Nt-acetylation upon yNAA50-deletion. The preferred substrates of hNaa50 were predominantly Met-starting N-termini including Met-Lys-, Met-Val-, Met-Ala-, Met-Tyr-, Met-Phe-, Met-Leu-, Met-Ser- and Met-Thr, and highly overlapped with the previously identified human Naa60/NatF substrate specificity profile. Identification of several hNaa50 substrates with a small amino acid in the second position also revealed a potential interplay between the NATs and methionine aminopeptidases (MetAPs). The initiator Methionine (iMet) is normally cleaved off by MetAPs when the second amino acid is small, but our in vitro data suggest that in contrast to a free iMet, an Nt-acetylated iMet is not hydrolyzed by MetAPs. Thus, Naa50-mediated Nt-acetylation may potentially act as a mechanism to retain the iMet of proteins with a small amino acid at the second position that normally would be hydrolyzed.
Sample Processing Protocol
Yeast strains were cultivated in 300 ml synthetic Dextrose containing medium lacking uracil to an O.D600 of 3.5. Yeast cells were harvested by centrifugation and re-suspended in 1 ml buffer (50 mM Tris-HCl, pH 7.4, 12 mM EDTA, 140 mM Na2HPO4, 250 mM NaCl, 1 tablet complete protease inhibitor (Roche)/100 ml). To lyse the cells, glass beads were added and the lysate was vortexed vigorously for 30 seconds and then put on ice. This step was repeated 6 times. The lysate was cleared by centrifugation at 10,000 x g for 15 minutes at 4°C. Solid guanidinium hydrochloride was added to a final concentration of 4 M in order to inactivate proteases and denature all proteins before proteins reduction by the addition of TCEP (1 mM final concentration) and alkylated by IAA (2 mM final concentration). The reaction was carried out at 30°C for 60 minutes while protected from light. The protein samples were desalted in 1.4 M Gu-HCl, 50 mM Sodium Phosphate, pH 8.0, and free amines were acetylated with 2C13Trideutero-N-Hydroxysuccinimide (2C13D3-NHS) for 2 hours at 30°C protected from light. Partial acetylation of serine and threonine was reversed by adding hydroxylamine (5x in excess of 2C13D3-NHS) and quenching of 2C13D3-NHS was performed by adding glycine following incubation for 10 minutes at room temperature. Protein samples were desalted in 10 mM NH4HCO3, pH 8.0 and the volume was reduced to 0.75 ml by vacuum drying. The protein mixture was boiled for 5 minutes followed by 5 minutes on ice and subsequently digested with trypsin at 37°C for 16 hours. Following digestion, the samples were dried completely and subjected to COFRADIC analyses. Subsequent steps of the N-terminal COFRADIC protocol were performed as described previously . Significant variations in Nt-acetylation were set to 10% for partially acetylated N-termini in the control strain as compared to the deletion strain or hNaa50 overexpression strain. Reference: Staes, A., Van Damme, P. et al., Improved recovery of proteome-informative, protein N-terminal peptides by combined fractional diagonal chromatography (COFRADIC). Proteomics 2008, 8, 1362-1370.
Data Processing Protocol
LC-MS/MS analysis and data processing: The obtained peptide mixtures were introduced into an LC-MS/MS system, the Ultimate 3000 (Dionex, Amsterdam, The Netherlands) in-line connected to an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Samples were first loaded on a trapping column (made in-house, 100 µm internal diameter (I.D.) x 20 mm, 5 µm beads C18 Reprosil-HD, Dr. Maisch). After back-flushing from the trapping column, the sample was loaded on a reverse-phase column (made in-house, 75 m I.D. x 150 mm, 5 µm beads C18 Reprosil-HD, Dr. Maisch). Peptides were loaded with solvent A (0.1% trifluoroacetic acid, 2% acetonitrile), and were separated with a linear gradient from 2% solvent A’ (0.05% formic acid) to 55% solvent B’ (0.05% formic acid and 80% acetonitrile) at a flow rate of 300 nL/min followed by a wash reaching 100% solvent B’. The mass spectrometer was operated in data-dependent mode, automatically switching between MS and MS/MS acquisition for the six most abundant peaks in a given MS spectrum. Full scan MS spectra were acquired in the Orbitrap at a target value of 1E6 with a resolution of 60,000. The six most intense ions were then isolated for fragmentation in the linear ion trap, with a dynamic exclusion of 60 s. Peptides were fragmented after filling the ion trap at a target value of 1E4 ion counts. From the MS/MS data in each LC run, Mascot Generic Files were created using the Mascot Distiller software (version 2.3.01, Matrix Science). While generating these peak lists, grouping of spectra was allowed with a maximum intermediate retention time of 30 s and a maximum intermediate scan count of 5 was used where possible. Grouping was done with 0.005 Da precursor tolerance. A peak list was only generated when the MS/MS spectrum contained more than 10 peaks. There was no de-isotoping and the relative signal to noise limit was set at 2. These peak lists were then searched with the Mascot search engine (Matrix Science) using the Mascot Daemon interface (version 2.3, Matrix Science). Spectra were searched against the baker’s yeast (S. cerevisiae) Swiss-Prot database. 13C2D3-acetylation of lysine side-chains, carbamidomethylation of cysteine and methionine oxidation to methionine-sulfoxide were set as fixed modifications for the N-terminal COFRADIC analyses. Variable modifications were 13C2D3-acetylation and acetylation of protein N-termini. Pyroglutamate formation of N-terminal glutamine was additionally set as a variable modification. Mass tolerance on precursor ions was set to 10 ppm (with Mascot’s C13 option set to 1) and on fragment ions to 0.5 Da. Endoproteinase semi-Arg-C/P (Arg-C specificity with arginine-proline cleavage allowed) was set as enzyme allowing no missed cleavages. The peptide charge was set to 1+, 2+, 3+ and instrument setting was put to ESI-TRAP. Only peptides that were ranked one and scored above the threshold score, set at 99% confidence, were withheld. Quantification of the degree of Nt-Acetylation was performed as described previously (1). All data management was done in ms_lims (2). References: 1. Van Damme, P., Hole, K., Pimenta-Marques, A., Helsens, K., Vandekerckhove, J., Martinho, R. G., Gevaert, K., and Arnesen, T. (2011) NatF contributes to an evolutionary shift in protein N-terminal acetylation and is important for normal chromosome segregation. PLoS genetics 7, e1002169. 2. Helsens, K., Colaert, N., Barsnes, H., Muth, T., Flikka, K., Staes, A., Timmerman, E., Wortelkamp, S., Sickmann, A., Vandekerckhove, J., Gevaert, K., and Martens, L. (2010) ms_lims, a simple yet powerful open source laboratory information management system for MS-driven proteomics. Proteomics 10, 1261-1264.
Van Damme P, Hole K, Gevaert K, Arnesen T. N-terminal acetylome analysis reveals the specificity of Naa50 (Nat5) and suggests a kinetic competition between N-terminal acetyltransferases and methionine aminopeptidases. Proteomics. 2015 Apr 17 PubMed: 25886145
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