Global Analysis of Post-Translational Side-Chain Arginylation Using Pan-Arginylation Antibodies
Ontology highlight
ABSTRACT:
SUBMITTER: MacTaggart B
PROVIDER: S-EPMC10656225 | biostudies-literature | 2023 Oct
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Arginylation is a post-translational modification mediated by the arginyltransferase ATE1, which transfers the amino acid arginine to a protein or peptide substrate from a tRNA molecule. Initially, arginylation was thought to occur only on N-terminally exposed acidic residues, and its function was thought to be limited to targeting proteins for degradation. However, more recent data has shown that ATE1 can arginylate sidechains of internal acidic residues in a protein without necessarily affecting metabolic stability. This greatly expands the potential targets and functions of arginylation, but tools for studying this process have remained limited. Here, we report the first global screen specifically for sidechain arginylation. We generate and validate pan-arginylation antibodies, which are designed to detect sidechain arginylation in any amino acid sequence context. We use these antibodies for immunoaffinity enrichment of sidechain arginylated proteins from wildtype and Ate1 knockout cell lysates. In this way, we identify a limited set of proteins that likely undergo ATE1-dependent sidechain arginylation and that are enriched in specific cellular roles, including translation, splicing, and the cytoskeleton.
2023-03-01 | MSV000091388 | MassIVE
Project description:Eukaryotic arginylation is an essential post-translational modification that modulates protein stability and regulates protein half-life. Arginylation is catalyzed by a family of enzymes known as the arginyl-tRNA transferases (ATE1s), which are conserved across the eukaryotic domain. Despite their conservation and importance, little is known regarding the structure, mechanism, and regulation of ATE1s. In this work, we show that ATE1s bind a previously undiscovered [Fe-S] cluster that is conserved across evolution. We characterize the nature of this [Fe-S] cluster and find that the presence of the [Fe-S] cluster in ATE1 is linked to its arginylation activity, both in vitro and in vivo, and the initiation of the yeast stress response. Importantly, the ATE1 [Fe-S] cluster is oxygen-sensitive, which could be a molecular mechanism of the N-degron pathway to sense oxidative stress. Taken together, our data provide the framework of a cluster-based paradigm of ATE1 regulatory control.
| S-EPMC9884297 | biostudies-literature
Project description:Ornithine decarboxylase (ODC) was purified 6500-fold from NMRI mouse kidneys under conditions designed to inhibit degradation by proteinases. The enzyme was homogeneous by SDS/polyacrylamide-gel electrophoresis, and the specific activity was among the highest reported. The yield was 70%. A monoclonal antibody against this preparation was generated and used in studies to investigate the half-life of ODC in cultured rat hepatocytes labelled with [35S]methionine. This value was 39 +/- 4 min and was unchanged when either NH4Cl (as a lysosomotropic agent) or leupeptin (as a lysosomal proteinase inhibitor) was added to the culture medium. Thus the intracellular turnover of ODC in cultured hepatocytes occurs mainly in extra-lysosomal compartments. Arginylation of rat ODC was investigated in vitro by incubation with L-[3H]arginyl-tRNA, and the incorporation of the label was compared with that of total cytosolic proteins. Arginylated ODC had a specific radioactivity 8600 times that of the bulk of cytosolic protein. Edman degradation of this ODC showed that the post-translational arginylation occurred only at the alpha-amino end of the enzyme. The inhibitor of arginyl-tRNA:protein arginyltransferase (EC 2.3.2.8), L-glutamyl-L-valyl-L-phenylalanine, increased the half-life of ODC in cultured hepatocytes from 39 min to more than 90 min. The possible significance of the preferential post-translational arginylation of ornithine decarboxylase to its rapid turnover is discussed.
| S-EPMC1131293 | biostudies-other
Project description:Arginylation is an emerging posttranslational modification mediated by Arg-tRNA-protein-transferase (ATE1). It is believed that ATE1 links Arg solely to the N terminus of proteins, requiring prior proteolysis or action by Met-aminopeptidases to expose the arginylated site. Here, we tested the possibility of Arg linkage to midchain sites within intact protein targets and found that many proteins in vivo are modified on the side chains of Asp and Glu by unconventional chemistry that targets the carboxy rather than the amino groups at the target sites. Such arginylation appears to be functionally regulated, and it can be directly mediated by ATE1, in addition to the more conventional ATE1-mediated linkage of Arg to the N-terminal alpha amino group. This midchain arginylation implies an unconventional mechanism of ATE1 action that likely facilitates its major biological role.
| S-EPMC4010198 | biostudies-literature
Project description:Posttranslational arginylation is critical for embryogenesis, cardiovascular development, and angiogenesis, but its molecular effects and the identity of proteins arginylated in vivo are largely unknown. Here we report a global analysis of this modification on the protein level and identification of 43 proteins arginylated in vivo on highly specific sites. Our data demonstrate that unlike previously believed, arginylation can occur on any N-terminally exposed residue likely defined by a structural recognition motif on the protein surface, and that it preferentially affects a number of physiological systems, including cytoskeleton and primary metabolic pathways. The results of our study suggest that protein arginylation is a general mechanism for regulation of protein structure and function and outline the potential role of protein arginylation in cell metabolism and embryonic development.
| S-EPMC1988855 | biostudies-literature
Project description:Autoantibodies are a hallmark of autoimmune diseases such as lupus and have the potential to be used as biomarkers for diverse diseases, including immunodeficiency, infectious disease, and cancer. More precise detection of antibodies to specific targets is needed to improve diagnosis of such diseases. Here, we report the development of reusable peptide microarrays, based on giant magnetoresistive (GMR) nanosensors optimized for sensitively detecting magnetic nanoparticle labels, for the detection of antibodies with a resolution of a single post-translationally modified amino acid. We have also developed a chemical regeneration scheme to perform multiplex assays with a high level of reproducibility, resulting in greatly reduced experimental costs. In addition, we show that peptides synthesized directly on the nanosensors are approximately two times more sensitive than directly spotted peptides. Reusable peptide nanosensor microarrays enable precise detection of autoantibodies with high resolution and sensitivity and show promise for investigating antibody-mediated immune responses to autoantigens, vaccines, and pathogen-derived antigens as well as other fundamental peptide-protein interactions.
| S-EPMC5367622 | biostudies-literature
Project description:Arginylation is a protein post-translational modification catalyzed by arginyl-tRNA transferases (ATE1s), which are critical enzymes conserved across all eukaryotes. Arginylation is a key step in the Arg N-degron pathway, a hierarchical cellular signaling pathway that links the ubiquitin-dependent degradation of a protein to the identity of its N-terminal amino acid side chain. The fidelity of ATE1-catalyzed arginylation is imperative, as this post-translational modification regulates several essential biological processes such as cardiovascular maturation, chromosomal segregation, and even the stress response. While the process of ATE1-catalyzed arginylation has been studied in detail at the cellular level, much remains unknown about the structure of this important enzyme, its mechanism of action, and its regulation. In this work, we detail the current state of knowledge on ATE1-catalyzed arginylation, and we discuss both ongoing and future directions that will reveal the structural and mechanistic details of this essential eukaryotic cellular regulator.
| S-EPMC7749041 | biostudies-literature
Project description:A new global post-translational modification (PTM) discovery strategy, G-PTM-D, is described. A proteomics database containing UniProt-curated PTM information is supplemented with potential new modification types and sites discovered from a first-round search of mass spectrometry data with ultrawide precursor mass tolerance. A second-round search employing the supplemented database conducted with standard narrow mass tolerances yields deep coverage and a rich variety of peptide modifications with high confidence in complex unenriched samples. The G-PTM-D strategy represents a major advance to the previously reported G-PTM strategy and provides a powerful new capability to the proteomics research community.
| S-EPMC5387672 | biostudies-literature
Project description:A variety of post-translational modifications (PTMs) are believed to regulate the behavior and function of α-synuclein (αS), an intrinsically disordered protein that mediates synaptic vesicle trafficking. Fibrils of αS are implicated in neurodegenerative disorders such as Parkinson's disease. In this study, we used chemical synthesis and biophysical techniques to characterize the neuroprotective effects of glutamate arginylation, a hitherto little characterized PTM in αS. We developed semisynthetic routes combining peptide synthesis, unnatural amino acid mutagenesis, and native chemical ligation (NCL) to site-specifically introduce the PTM of interest along with fluorescent probes into αS. We synthesized the arginylated glutamate as a protected amino acid, as well as a novel ligation handle for NCL, in order to generate full-length αS modified at various individual sites or a combination of sites. We assayed the lipid-vesicle binding affinities of arginylated αS using fluorescence correlation spectroscopy (FCS) and found that arginylated αS has the same vesicle affinity compared to control protein, suggesting that this PTM does not alter the native function of αS. On the other hand, we studied the aggregation kinetics of modified αS and found that arginylation at E83, but not E46, slows aggregation and decreases the percentage incorporation of monomer into fibrils in a dose-dependent manner. Arginylation at both sites also resulted in deceleration of fibril formation. Our study represents the first synthetic strategy for incorporating glutamate arginylation into proteins and provides insight into the neuroprotective effect of this unusual PTM.
| S-EPMC8042788 | biostudies-literature
Project description:Post-translational modifications (PTMs) play key roles in regulating cell signaling and physiology in both normal and cancer cells. Advances in mass spectrometry enable high-throughput, accurate, and sensitive measurement of PTM levels to better understand their role, prevalence, and crosstalk. Here, we analyze the largest collection of proteogenomics data from 1,110 patients with PTM profiles across 11 cancer types (10 from the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium [CPTAC]). Our study reveals pan-cancer patterns of changes in protein acetylation and phosphorylation involved in hallmark cancer processes. These patterns revealed subsets of tumors, from different cancer types, including those with dysregulated DNA repair driven by phosphorylation, altered metabolic regulation associated with immune response driven by acetylation, affected kinase specificity by crosstalk between acetylation and phosphorylation, and modified histone regulation. Overall, this resource highlights the rich biology governed by PTMs and exposes potential new therapeutic avenues.
| S-EPMC10680287 | biostudies-literature