Hazbun, T. | Search bioRxiv

bioRxiv2021

Discovering the N-terminal Methylome by Repurposing of Proteomic Datasets

Protein -N-methylation is an underexplored post-translational modification involving the covalent addition of methyl groups to the free -amino group at protein N-termini. To systematically explore the extent of -N-terminal methylation in yeast and humans, we reanalyzed publicly accessible proteomic datasets to identify N-terminal peptides contributing to the -N-terminal methylome. This repurposing approach found evidence of -N-methylation of established and novel protein substrates with canonical N-terminal motifs of established -N-terminal methyltransferases, including human NTMT1/2 and yeast Tae1. NTMT1/2 are implicated in cancer and aging processes but have unclear and context-dependent roles. Moreover, -N-methylation of non-canonical sequences was surprisingly prevalent, suggesting unappreciated and cryptic methylation events. Analysis of the amino acid frequencies of -N-methylated peptides revealed a [S]1-[S/A/Q]2 pattern in yeast and [A/N/G]1-[A/S/V]2-[A/G]3 in humans, which differs from the canonical motif. We delineated the distribution of the two types of prevalent N-terminal modifications, acetylation, and methylation, on amino acids at the 1st position. We tested three potentially methylated proteins and confirmed the -N-terminal methylation of Hsp31 by additional proteomic analysis and immunoblotting. The other two proteins, Vma1 and Ssa3, were found to be predominantly acetylated, indicating proteomic searching for -N-terminal methylation requires careful consideration of mass spectra. This study demonstrates the feasibility of reprocessing proteomic data for global -N-terminal methylome investigations. The raw MS data that supports the findings of this study were deposited with PRIDE identifier: PXD022833. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/439552v3_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@124e5b9org.highwire.dtl.DTLVardef@1660eb7org.highwire.dtl.DTLVardef@15027aaorg.highwire.dtl.DTLVardef@15c0da6_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract (For TOC only). C_FIG

bioinformatics↗

bioRxiv2020

Specific OCRL1 patient mutations differentially impact Lowe Syndrome cellular phenotypes

Lowe Syndrome (LS) is a lethal genetic disorder caused by mutations in the OCRL1 gene which encodes the lipid 5 phosphatase Ocrl1. Patients exhibit a characteristic triad of symptoms including eyes, brain and kidneys abnormalities with renal failure as the most common cause of premature death. Over 200 OCRL1 mutations have been identified in LS, but their specific impact on cellular processes is unknown. Despite observations of heterogeneity in patient symptom severity, there is little understanding of the correlation between genotype and its impact on phenotype. Here, we show that different mutations had diverse effects on protein localization and on triggering LS cellular phenotypes. In addition, some mutations affecting specific domains imparted unique characteristics to the resulting mutated protein. We also propose that certain mutations conformationally affect the 5-phosphatase domain of the protein, resulting in loss of enzymatic activity and causing common and specific phenotypes. This study is the first to show the differential effect of patient 5-phosphatase mutations on cellular phenotypes and introduces a conformational disease component in LS. This work provides a framework that can help stratify patients as well as to produce a more accurate prognosis depending on the nature and location of the mutation within the OCRL1 gene.

cell biology↗