Abstract
Protein arginylation is a critical regulator of a variety of biological processes. The ability to uncover the global arginylation pattern and its associated signaling pathways would enable us to identify novel disease targets. Here, we report the development of a tool able to capture the N-terminal arginylome. This tool, termed R-catcher, is based on the ZZ domain of p62, which was previously shown to bind N-terminally arginylated proteins. Mutating the ZZ domain enhanced its binding specificity and affinity for Nt-Arg. R-catcher pulldown coupled to LC–MS/MS led to the identification of 59 known and putative arginylated proteins. Among these were a subgroup of novel ATE1-dependent arginylated ER proteins that are linked to diverse biological pathways, including cellular senescence and vesicle-mediated transport as well as diseases, such as Amyotrophic Lateral Sclerosis and Alzheimer’s disease. This study presents the first molecular tool that allows the unbiased identification of arginylated proteins, thereby unlocking the arginylome and provide a new path to disease biomarker discovery.
Highlights
The N-terminal (Nt) arginylation of proteins is typically mediated by the arginyl-tRNA-protein transferase 1 (ATE1) enzyme encoded by a single arginyltransferase gene that is highly conserved from yeast to human
The enzyme mediating this process has been identified as arginyl-tRNA-protein transferase (ATE1) whose substrates are proteins with N-terminal Asp, Glu, and oxidized Cys residue (Fig. 1a)
The generation of these residues is dependent on their cleavage by non-processive proteases, including methionine aminopeptidases (MetAP), calpains, caspases, and separases (Fig. 1a)
Summary
The N-terminal (Nt) arginylation of proteins is typically mediated by the arginyl-tRNA-protein transferase 1 (ATE1) enzyme encoded by a single arginyltransferase gene that is highly conserved from yeast to human. Mice contain four major ATE1 isoforms that exhibit similar Nt-arginylation specificity and whose expressions are tissue-specific [1, 2]. ATE1 can transfer Arg from Arg-tRNA to Nt-Asp, -Glu, and -oxidized Cys residues exposed upon the non-processive proteolytic cleavage of a protein. Regarding the oxidation of Nt-Cys, it has recently been shown to be enzymatically mediated by dioxygenases in plants and humans [3, 4]. Nt-Asp and -Glu residues can be generated through the deamidation of newly exposed Nt-Asn and -Gln, respectively (Fig. 1a). In addition to ATE1-mediated arginylation, proteolytic cleavage can directly generate neo-Nt-Arg, which results in Arg as
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