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Abstract INTRODUCTION:
Arginylation is a PTM installed by ATE1 to signal a protein for degradation by the N-degron pathway. Knockout of the ATE1 enzyme is embryonic lethal due to various cardiac defects including thinned myocardium and cardiac contractility deficiencies, however the exact mechanism of how this occurs is still unknown. Arginylation is also implicated as a regulator of aSyn folding and function, preventing aggregation as a potential mechanism in the prevention of synucleinopathies. Other roles for arginylation include protein secretion, notably serum albumin, and B-actin subcellular translocation.
This modification most commonly occurs on the N-terminus of substrates, but studies show it can be installed on aspartic and glutamic acid side chains as well. Study of arginylation is challenging due to the low abundance of the modification, aspecific antibodies, and the difficulty of distinguishing a PTM from a missed cleavage. initial qualitative proteomics studies have identified two small sets of candidates (43 and 19 proteins) potentially equipped with N-term and mid-chain arginylation, respectively. These results indicate that arginylation serves as a biological regulator of protein function and thus raises the question of how many proteins/sites are arginylated and what their functions are. We have recently developed an ABAP strategy for the discovery of arginylation sites with both bottom-up and top-down proteomics. This method takes advantage of isotopically labeled arginine to validate the N-terminal installation of the arginyl modification by ATE1 both in vitro and in vivo.
METHODS:
Peptides, peptide mixtures, proteins, cell proteomes, patient heart and brain tissue, and mouse tissues (lung, heart, and brain) were arginylated by ATE1 assay. The enzyme assay was successfully reconstituted including two key enzymes involved in the protein arginylation: RARS1 and ATE1. RARS1 charged the tRNA for arginine with either R0 or isotopically labeled R10 in solution, and ATE1 used this newly charged Arg-tRNA to label substrates. To incorporate isotopic Arg into proteomes, lysates from biological samples were used as ribosome-inactive conditions. Labeled proteomes were mixed, digested, and fractionated for proteomics analysis in data-dependent acquisition mode. For top-down proteomics, the platform was developed using calreticulin as a working standard as a known substrate of ATE1 arginylation. This protein was subjected to arginylation in vitro, in vivo, and on-bead during pull-down. Other individual protein standards were labeled with the same ATE1 assay. Labeled proteins were kept separately or in mixtures and analyzed in MRMHR mode to target specific charge states. Data was analyzed with a custom software “ArginylomePlot” for bottom-up and ProSight for top-down.
RESULTS:
In bottom-up proteomics, we first established the workflow using peptide (e.g.: standard peptide) and proteome-wide peptides (e.g.: HEK293T digest) as a proof-of-concept, then applied the technology to protein (e.g.: CALR) and whole proteomes (including iPSC cells, iPSC cardiac fibroblasts, iPSC cardiac myocytes, HEK293T, various cancer cells, patient heart, and patient brains) for arginylation discovery. As a result, a large catalog of unbiased arginylation sites (>200) has been established from various cell and tissue samples. Representative sites were validated and followed up for their biological pathways. In top-down proteomics analyses, analysis showed clear presence of two features with the 156 Da mass shift between WT and arginylated proteoforms visible following the application of the ATE1 assay. Calreticulin was readily arginylated and quantitatively determined to be an efficient target of this assay is various experiments. Further experiments validating substrates identified in bottom-up experiments demonstrated similar findings, with N-terminal arginylation reproducibly reconstituted.
DISCUSSION:
Arginylation is an essential PTM as demonstrated by the embryonic lethality of the knockout of ATE1. Given its wide role in diseases from cardiac disease, circulatory protein secretion, and aSyn folding and aggregation, understanding this PTM is crucial from a basic sciences and diagnostics perspective. Our work has generated the largest arginylation site library so far and established a series of arginylation assays (in vitro, in-bacteria, ex vivo, and in vivo) for functional validation. Arginylation profiling revealed new protein N-termini, thus opening new frontiers in protease cleavage. Further, we demonstrated the first ever experiment studying arginylation using top-down proteomics. This work could serve as the technological foundation for studying the functions of this essential PTM, and it will have a long-lasting impact on the arginylation field by opening new biochemical and biological frontiers. Further, given the observed arginylation across a diverse set of proteomes, including cancer cell lines and real patient samples, we believe there could be deep relevance to clinical pathologies that are not yet known where clinical mass spectrometry would excel. |
= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
