Method Overview for Discovering ATE1 Substrates and their Arginylation Sites

Abstract

Arginylation is a protein modification event in which cellular machinery recognizes a conserved N-terminal or side-chain motif and post-translationally installs an arginine residue to signal a protein for degradation. This modification affects protein function, stability, and half-life and is essential to proper functions in mammalian systems. Since its discovery in the early 1960s, scientists have struggled to broadly characterize this modification in its canonical function outside of a handful of specific cases. It is known to be an essential cellular mark, as loss of the installation enzyme is embryonically lethal. However, the discovery of the substrates regulated by this mark has been slow and has required some creativity by the scientists who have chased it. Over the course of roughly six decades, the library of substrates has consistently grown through various applications. Here, we seek to summarize all approaches that have been applied to discovering and studying arginylation.

Keywords: ATE1; arginylation; cDNA screening; proteomics; radiolabeling.

Figure 1

Timeline of major advancements in arginylation discovery and method development.

Figure 2

Schemes of protein radiolabeling using heavy arginine. a) Labeling of proteins with ³H‐arginine visualized with gel electrophoresis, and confirmed the modification at the N‐terminus with Edman Degradation. b) Labeling of proteins with ¹⁴C‐arginine or ¹²C‐arginine, where the ¹⁴C‐arginine labeled protein gel is used as a guide to excise targets from the ¹²C‐arginine gel before being subjected to mass spectrometry.

Figure 3

cDNA library screening for the discovery of arginylation substrates. Sublibraries of 50 cDNAs were consecutively expressed in the presence or absence of Arg/N‐degron inhibitor to identify libraries with increasing concentration in the presence of inhibitor. Libraries with increasing protein concentration were consecutively split into smaller libraries (25 + 25, 12 + 12, 6 + 6, etc.) until a single cDNA was isolated and determined to be increasing in concentration due to being an arginylation substrate. The isolated cDNA was then confirmed to be arginylated and at the N‐terminus by the loss in signal through a single round of Edman Degradation.

Figure 4

Application of antibody enrichment for N‐terminal arginylation. Antibodies were raised against N‐terminally arginylated peptides and had confirmed specificity with dot blots that positively bound to N‐terminally arginylated peptides and did not bind to peptides of similar sequence but with no arginylation. Antibodies were then coupled with beads and applied to cell lysate to enrich arginylated proteins that were identified by LCMS analysis.

Figure 5

Schematic depicting the generation of substrates through endoproteolytic cleavage for arginylation and subsequent protein degradation. Proteins are first cleaved by a protease to reveal an N‐terminal D/E or C requiring oxidation, which can then be recognized and arginylated by ATE1 and passed through the UBR‐mediated ubiquitination and proteasomal degradation pathway.

Figure 6

Schematic of the generation of arginylation substrates from proteins containing N‐terminal methionine. A protein is cleaved by MetAP, and/or an N‐terminal asparagine or glutamine that is deamidated to ATE1 compatible aspartic and glutamic acid, respectively.

Figure 7

Application of antibody enrichment for side‐chain arginylation. Compared with the N‐terminal arginylation antibodies, here antibodies were generated against synthetic peptides containing glutamic acid residues in the middle of the polypeptide chain that were arginylated on the carboxylic acid side chain. These antibodies were validated against α‐synuclein side‐chain arginylated peptides before being applied to immunoprecipitation and mass spectrometry.

Figure 8

General overview of the bottom‐up proteomics workflow. A protein, cell, or tissue of interest is split into two aliquots, incubated with ATE1 and isotopically labeled arginine and then recombined. Following typical bottom‐up proteomics sample processing and LCMS analysis, arginylated peptides can be identified by the presence of co‐eluting doublets of isotopic peaks 10 Da apart, corresponding to the heavy and light arginine.

Figure 9

Top‐down proteomics approaches to discovery and validation of arginylated substrates. Arginylation was carried out in vivo by co‐expressing CALR and ATE1, in vitro by applying the same arginylation assay as bottom‐up proteomics, and in vitro but on‐bead during protein pull down assays, also using isotopically labeled arginine. Proteins are then quantified and sequenced with LCMS.