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Proteome-derived peptide libraries allow detailed analysis of the substrate specificities of Nα-acetyltransferases and point to hNaa10p as the post-translational actin Nα-acetyltransferase

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Bioinformatics: from nucleotids to networks (N2N)
Abstract
The impact of N(alpha)-terminal acetylation on protein stability and protein function in general recently acquired renewed and increasing attention. Although the substrate specificity profile of the conserved enzymes responsible for N(alpha)-terminal acetylation in yeast has been well documented, the lack of higher eukaryotic models has hampered the specificity profile determination of N(alpha)-acetyltransferases (NATs) of higher eukaryotes. The fact that several types of protein N termini are acetylated by so far unknown NATs stresses the importance of developing tools for analyzing NAT specificities. Here, we report on a method that implies the use of natural, proteome-derived modified peptide libraries, which, when used in combination with two strong cation exchange separation steps, allows for the delineation of the in vitro specificity profiles of NATs. The human NatA complex, composed of the auxiliary hNaa15p (NATH/hNat1) subunit and the catalytic hNaa10p (hArd1) and hNaa50p (hNat5) subunits, cotranslationally acetylates protein N termini initiating with Ser, Ala, Thr, Val, and Gly following the removal of the initial Met. In our studies, purified hNaa50p preferred Met-Xaa starting N termini (Xaa mainly being a hydrophobic amino acid) in agreement with previous data. Surprisingly, purified hNaa10p preferred acidic N termini, representing a group of in vivo acetylated proteins for which there are currently no NAT(s) identified. The most prominent representatives of the group of acidic N termini are gamma- and beta-actin. Indeed, by using an independent quantitative assay, hNaa10p strongly acetylated peptides representing the N termini of both gamma- and beta-actin, and only to a lesser extent, its previously characterized substrate motifs. The immunoprecipitated NatA complex also acetylated the actin N termini efficiently, though displaying a strong shift in specificity toward its known Ser-starting type of substrates. Thus, complex formation of NatA might alter the substrate specificity profile as compared with its isolated catalytic subunits, and, furthermore, NatA or hNaa10p may function as a post-translational actin N(alpha)-acetyltransferase.
Keywords
TERMINAL ACETYLATION, SACCHAROMYCES-CEREVISIAE, METHIONINE AMINOPEPTIDASE, EUKARYOTIC PROTEINS, IN-VIVO, YEAST, IDENTIFICATION, COMPLEX, STABILITY, LABEL FINGERPRINTINGS SHOW

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Citation

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Chicago
Van Damme, Petra, Rune Evjenth, Havard Foyn, Kimberly Demeyer, Pieter-Jan De Bock, Johan R Lillehaug, Joël Vandekerckhove, Thomas Arnesen, and Kris Gevaert. 2011. “Proteome-derived Peptide Libraries Allow Detailed Analysis of the Substrate Specificities of Nα-acetyltransferases and Point to hNaa10p as the Post-translational Actin Nα-acetyltransferase.” Molecular & Cellular Proteomics 10 (5).
APA
Van Damme, Petra, Evjenth, R., Foyn, H., Demeyer, K., De Bock, P.-J., Lillehaug, J. R., Vandekerckhove, J., et al. (2011). Proteome-derived peptide libraries allow detailed analysis of the substrate specificities of Nα-acetyltransferases and point to hNaa10p as the post-translational actin Nα-acetyltransferase. MOLECULAR & CELLULAR PROTEOMICS, 10(5).
Vancouver
1.
Van Damme P, Evjenth R, Foyn H, Demeyer K, De Bock P-J, Lillehaug JR, et al. Proteome-derived peptide libraries allow detailed analysis of the substrate specificities of Nα-acetyltransferases and point to hNaa10p as the post-translational actin Nα-acetyltransferase. MOLECULAR & CELLULAR PROTEOMICS. 2011;10(5).
MLA
Van Damme, Petra, Rune Evjenth, Havard Foyn, et al. “Proteome-derived Peptide Libraries Allow Detailed Analysis of the Substrate Specificities of Nα-acetyltransferases and Point to hNaa10p as the Post-translational Actin Nα-acetyltransferase.” MOLECULAR & CELLULAR PROTEOMICS 10.5 (2011): n. pag. Print.
@article{1887451,
  abstract     = {The impact of N(alpha)-terminal acetylation on protein stability and protein function in general recently acquired renewed and increasing attention. Although the substrate specificity profile of the conserved enzymes responsible for N(alpha)-terminal acetylation in yeast has been well documented, the lack of higher eukaryotic models has hampered the specificity profile determination of N(alpha)-acetyltransferases (NATs) of higher eukaryotes. The fact that several types of protein N termini are acetylated by so far unknown NATs stresses the importance of developing tools for analyzing NAT specificities. Here, we report on a method that implies the use of natural, proteome-derived modified peptide libraries, which, when used in combination with two strong cation exchange separation steps, allows for the delineation of the in vitro specificity profiles of NATs. The human NatA complex, composed of the auxiliary hNaa15p (NATH/hNat1) subunit and the catalytic hNaa10p (hArd1) and hNaa50p (hNat5) subunits, cotranslationally acetylates protein N termini initiating with Ser, Ala, Thr, Val, and Gly following the removal of the initial Met. In our studies, purified hNaa50p preferred Met-Xaa starting N termini (Xaa mainly being a hydrophobic amino acid) in agreement with previous data. Surprisingly, purified hNaa10p preferred acidic N termini, representing a group of in vivo acetylated proteins for which there are currently no NAT(s) identified. The most prominent representatives of the group of acidic N termini are gamma- and beta-actin. Indeed, by using an independent quantitative assay, hNaa10p strongly acetylated peptides representing the N termini of both gamma- and beta-actin, and only to a lesser extent, its previously characterized substrate motifs. The immunoprecipitated NatA complex also acetylated the actin N termini efficiently, though displaying a strong shift in specificity toward its known Ser-starting type of substrates. Thus, complex formation of NatA might alter the substrate specificity profile as compared with its isolated catalytic subunits, and, furthermore, NatA or hNaa10p may function as a post-translational actin N(alpha)-acetyltransferase.},
  author       = {Van Damme, Petra and Evjenth, Rune and Foyn, Havard and Demeyer, Kimberly and De Bock, Pieter-Jan and Lillehaug, Johan R and Vandekerckhove, Jo{\"e}l and Arnesen, Thomas and Gevaert, Kris},
  issn         = {1535-9476},
  journal      = {MOLECULAR \& CELLULAR PROTEOMICS},
  keyword      = {TERMINAL ACETYLATION,SACCHAROMYCES-CEREVISIAE,METHIONINE AMINOPEPTIDASE,EUKARYOTIC PROTEINS,IN-VIVO,YEAST,IDENTIFICATION,COMPLEX,STABILITY,LABEL FINGERPRINTINGS SHOW},
  language     = {eng},
  number       = {5},
  pages        = {12},
  title        = {Proteome-derived peptide libraries allow detailed analysis of the substrate specificities of N\ensuremath{\alpha}-acetyltransferases and point to hNaa10p as the post-translational actin N\ensuremath{\alpha}-acetyltransferase},
  url          = {http://dx.doi.org/10.1074/mcp.M110.004580},
  volume       = {10},
  year         = {2011},
}

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