Raw MassSpec Data for TopDown Analysis of CALR Arginylation (Main)
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ABSTRACT: Raw files of data used for main figures in manuscript "Top-down Proteomics for the Characterization and Quantification of Calreticulin Arginylation".
Project description:Raw files of data used for Supplemental figures and tables in manuscript "Top-down Proteomics for the Characterization and Quantification of Calreticulin Arginylation".
Project description:Raw files of data used for Supplemental figures and tables in manuscript "Top-down Proteomics for the Characterization and Quantification of Calreticulin Arginylation".
Project description:Arginyltransferase ATE1 mediates posttranslational arginylation that plays key roles in mammalian embryogenesis, cell migration, and normal brain function. The molecular mechanisms of arginylation remain elusive. ATE1 utilizes arginyl-tRNAArg as the donor of Arg, putting this reaction into a direct competition with the protein synthesis machinery. Here, we addressed these questions of ATE1- arginyl-tRNAArg specificity as a potential mechanism enabling this competition in vivo. Using in vitro arginylation assays and ATE1 knockout models, we find that while arginylation is specific to tRNAArg, it is able to utilize short tRNAArg derivatives that bear structural resemblance to tRNA-derived fragments (tRF), a new class of small regulatory non-coding RNAs with poorly characterized but critical functions in vivo. Arginyl-tRFArg can be generated in vitro directly from pre-charged -tRNAArg, and ATE1 is able to utilize these arginyl-tRFArg fragments with similar efficiency as arginyl-tRNAArg. Lack of arginylation in ATE1 knockout cells leads to a decrease in tRFArg generation and a significant increase in the ratio of tRNAArg to tRFArg compared to wild type, suggesting a functional link between tRFArg and arginylation in vivo. We propose that generation of physiologically important tRFs can play a critical role as a switch between protein translation and arginylation in vivo.
Project description:Arginyltransferase ATE1 mediates posttranslational arginylation that plays key roles in mammalian embryogenesis, cell migration, and normal brain function. The molecular mechanisms of arginylation remain elusive. ATE1 utilizes arginyl-tRNAArg as the donor of Arg, putting this reaction into a direct competition with the protein synthesis machinery. Here, we addressed these questions of ATE1- arginyl-tRNAArg specificity as a potential mechanism enabling this competition in vivo. Using in vitro arginylation assays and ATE1 knockout models, we find that while arginylation is specific to tRNAArg, it is able to utilize short tRNAArg derivatives that bear structural resemblance to tRNA-derived fragments (tRF), a new class of small regulatory non-coding RNAs with poorly characterized but critical functions in vivo. Arginyl-tRFArg can be generated in vitro directly from pre-charged -tRNAArg, and ATE1 is able to utilize these arginyl-tRFArg fragments with similar efficiency as arginyl-tRNAArg. Lack of arginylation in ATE1 knockout cells leads to a decrease in tRFArg generation and a significant increase in the ratio of tRNAArg to tRFArg compared to wild type, suggesting a functional link between tRFArg and arginylation in vivo. We propose that generation of physiologically important tRFs can play a critical role as a switch between protein translation and arginylation in vivo.
Project description:Targeted proteomics of Nt-arginylation on HeLa proteome under the stress of unfolded protein response. UPR was derived by combinational treatment of MG132 and thapsigargin. PRM method was selected to undertake targeted MS2 analysis. Data was prepared to have duplicates.
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.