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Analyzing N-terminal Arginylation Through the Use of Peptide Arrays and Degradation Assays

Wadas, Brandon and Piatkov, Konstantin and Brower, Christopher and Varshavsky, Alexander (2016) Analyzing N-terminal Arginylation Through the Use of Peptide Arrays and Degradation Assays. Journal of Biological Chemistry, 291 (40). Art. No. 20976. ISSN 0021-9258. PMCID PMC5076509. doi:10.1074/jbc.M116.747956.

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Nα-terminal arginylation (Nt-arginylation) of proteins is mediated by the Ate1 arginyltransferase (R-transferase), a component of the Arg/N-end rule pathway. This proteolytic system recognizes proteins containing N terminal degradation signals called N-degrons, polyubiquitylates these proteins, and thereby causes their degradation by the proteasome. The definitively identified (canonical) residues that are Nt-arginylated by R-transferase are N-terminal Asp, Glu and (oxidized) Cys. Over the last decade, several publications suggested (i) that Ate1 can also arginylate non-canonical N-terminal residues; (ii) that Ate1 is capable of arginylating not only alpha-amino groups of N-terminal residues but also gamma-carboxyl groups of internal (non-N-terminal) Asp and Glu; and (iii) that some isoforms of Ate1 are specific for substrates bearing N-terminal Cys residues. In the present study, we employed arrays of immobilized 11-residue peptides and pulse-chase assays to examine the substrate specificity of mouse R-transferase. We show that amino acid sequences immediately downstream of canonical (Nt-arginylatable) N-terminal residue of a substrate, particularly a residue at position 2, can affect the rate of Nt-arginylation by R-transferase and thereby the rate of degradation of a substrate protein. We also show that the four major isoforms of mouse R transferase have similar Nt-arginylation specificities in vitro, contrary to the claim about specificity of some Ate1 isoforms for N terminal Cys. In addition, we found no evidence for a significant activity of the Ate1 R-transferase toward previously invoked non-canonical N-terminal or internal amino acid residues. Together, our results raise technical concerns about earlier studies that invoked non-canonical arginylation specificities of Ate1.

Item Type:Article
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URLURL TypeDescription CentralArticle
Wadas, Brandon0000-0003-2796-2384
Brower, Christopher0000-0003-0289-5541
Varshavsky, Alexander0000-0002-4011-258X
Additional Information:© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Received July 12, 2016. Revision received July 30, 2016. Revised Manuscript. Date: July 29, 2016. First Published on August 10, 2016. We thank the present and former members of the Varshavsky laboratory for helpful discussions during this study. Author contributions: B.W., K.P., C.B., and A.V. designed the experiments. B.W. performed the experiments, with participation by K.P and C.B. B.W. and A.V. wrote the paper. All authors discussed the results and commented on the manuscript. This study was supported by the U.S. National Institutes of Health grants R01-DK039520 and R01-GM031530 (to A.V.). The authors declare no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Funding AgencyGrant Number
Subject Keywords:proteasome protein degradation ubiquitin ubiquitin ligase ubiquitylation (ubiquitination) Ate1 arginyltransferase N-end rule
Issue or Number:40
PubMed Central ID:PMC5076509
Record Number:CaltechAUTHORS:20160815-140416336
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Official Citation:Analyzing N-terminal Arginylation through the Use of Peptide Arrays and Degradation Assays J. Biol. Chem. 2016 291: 20976-. doi:10.1074/jbc.M116.747956
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:69630
Deposited By: Tony Diaz
Deposited On:15 Aug 2016 21:41
Last Modified:25 Apr 2022 19:10

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