Proteomic Identification and Analysis of Arginine-Methylated Proteins of Plasmodium falciparum at Asexual Blood Stages

Abstract

Plasmodium falciparum undergoes a tightly regulated developmental process in human erythrocytes, and recent studies suggest an important regulatory role of post-translational modifications (PTMs). As compared with Plasmodium phosphoproteome, little is known about other PTMs in the parasite. In the present study, we performed a global analysis of asexual blood stages of Plasmodium falciparum to identify arginine-methylated proteins. Using two different methyl arginine-specific antibodies, we immunoprecipitated the arginine-methylated proteins from the stage-specific parasite lysates and identified 843 putative arginine-methylated proteins by LC-MS/MS. Motif analysis of the protein sequences unveiled that the methylation sites are associated with the previously known methylation motifs such as GRx/RGx, RxG, GxxR, or WxxxR. We identified Plasmodium homologues of known arginine-methylated proteins in trypanosomes, yeast, and human. Hydrophilic interaction liquid chromatography (HILIC) was performed on the immunoprecipitates from the trophozoite stage to enrich arginine-methylated peptides. Mass spectrometry analysis of immunoprecipitated and HILIC fractions identified 55 arginine-methylated peptides having 62 methylated arginine sites. Functional classification revealed that the arginine-methylated proteins are involved in RNA metabolism, protein synthesis, intracellular protein trafficking, proteolysis, protein folding, chromatin organization, hemoglobin metabolic process, and several other functions. Summarily, the findings suggest that protein methylation of arginine residues is a widespread phenomenon in Plasmodium, and the PTM may play an important regulatory role in a diverse set of biological pathways, including host-pathogen interactions.

Photosensitized INA-Labelled protein 1 (PhIL1) is novel component of the inner membrane complex and is required for Plasmodium parasite development

“…GFP-Trap ® _A beads were allowed to bind to parasite lysate by tumbling the tube end-over-end. Proteins were eluted in 50 μl elution buffer, digested with trypsin and peptides were analysed by mass spectrometry 41 .…”

Multistage activity within a diverse set of epi-drugs against Plasmodium falciparum parasites

“…This includes Ellagic Acid, which was previously indicated as potent against asexual parasites (37). Although antioxidant pleiotropic effects of this compound cannot be ignored, the multistage activity reported here suggests a possible role of arginine methylation during these stages (38), but not in mature gametocytes.…”

Unraveling the importance of the malaria parasite helicases

“…The in silico molecular modeling and structural analysis revealed that PfU52 displayed structural similarities with its human homolog and it also exhibits some distinctive properties that can be used for drug design [65]. It has been reported in recent studies that PfU52 is methylated at arginine and lysine residues and this modification may regulate its activities [48,49].…”

“…The ATPase and DNA helicase activities of PfH45 were effectively inhibited by nogalamycin, actinomycin, daunorubicin, netropsin, ethidium bromide, and DAPI . Previous studies have reported that PfH45 is phosphorylated by protein kinase C and recent studies have shown that it is methylated at arginine residues suggesting that post‐translational modifications may regulate its activities . It will be interesting to study the effect of hippuristanol on the activity of PfH45.…”