Global proteomic analysis in trypanosomes reveals unique proteins and conserved cellular processes impacted by arginine methylation

Abstract

Arginine methylation is a common posttranslational modification with reported functions in transcription, RNA processing and translation, and DNA repair. Trypanosomes encode five protein arginine methyltransferases, suggesting that arginine methylation exerts widespread impacts on the biology of these organisms. Here, we performed a global proteomic analysis of T. brucei to identify arginine methylated proteins and their sites of modification. Using an approach entailing two-dimensional chromatographic separation, and alternating electron transfer dissociation and collision induced dissociation, we identified 1332 methylarginines in 676 proteins. The resulting data set represents the largest compilation of arginine methylated proteins in any organism to date. Functional classification revealed numerous arginine methylated proteins involved in flagellar function, RNA metabolism, DNA replication and repair, and intracellular protein trafficking. Thus, arginine methylation has the potential to impact aspects of T. brucei gene expression, cell biology, and pathogenesis. Interestingly, pathways with known methylated proteins in higher eukaryotes were identified in this study, but often different components of the pathway were methylated in trypanosomes. Methylarginines were often identified in glycine rich contexts, although exceptions to this rule were detected. Collectively, these data inform on a multitude of aspects of trypanosome biology and serve as a guide for the identification of homologous arginine methylated proteins in higher eukaryotes.

Arginine methylation of the cellular nucleic acid binding protein does not affect its subcellular localization but impedes RNA binding

“…Such as we have shown that the modification is involved in the regulation of subcellular localization or shuttling of SERBP1 [26]. From a proteomic study in trypanosomes, a subset of arginine methylated protein are involved in intracellular trafficking [31], suggesting that the cellular methylation conditions might regulate the protein transport machinery in some way. We detected predominant nuclear distributions of the endogenous or transfected CNBP proteins whether HeLa cells were under normal methylation or hypomethylation conditions.…”

Large Scale Mass Spectrometry-based Identifications of Enzyme-mediated Protein Methylation Are Subject to High False Discovery Rates

“…together with manual data curation or with orthogonal methylpeptide validation), remains a highly popular method of filtering methyl-PSMs (see Table I). When applied as a standalone technique, the most common application of the approach involves obtaining methyl-PSM thresholding criteria based on global FDR estimates (4,5,8,9); two recent studies have, however, made use of separate methyl-PSM FDR esti-…”

“…Despite the prospective issues associated with sequence database search-derived methylpeptide identifications, the use of orthogonal methylpeptide validation in large scale methylation site discovery studies remains sporadic. Although several such studies have employed heavy-methyl SILAC (6,7,14,16) or other closely related methylpeptide-specific labeling techniques (12,13) to validate methylation sites, others have chosen to bypass orthogonal validation and to instead predominantly rely on the target-decoy approach to provide estimates for high stringency methylpeptide filtering criteria (4,5,8,9,15) or to inform manual data curation (see Table I) (10,11). This irregular use of orthogonal methylpeptide validation is a reflection of the fact that in-depth studies into methylpeptide FDRs have yet to be performed.…”

The Major Protein Arginine Methyltransferase in Trypanosoma brucei Functions as an Enzyme-Prozyme Complex

“…Posttranslational modification of proteins is one such mechanism that allows rapid responses to internal and external cues (8). Our proteome-wide studies revealed that about 15% of the proteome of T. brucei insect vector procyclic forms (PFs) 2 harbors arginine methyl marks (9,10). 3 Thus, arginine methylation is poised to play a crucial role in regulating T. brucei biology.…”