Browsing Biology Master’s Theses by Author "Bumstead, Andrew R."
Arginine Methylation and Enzymatic Activity of TbLpn, a Lipin Homologue from T. BruceiPelletier, Michel; Ortega, Bernardo; Shen, Rongkun; Bumstead, Andrew R.; The College at Brockport (2016-07-21)Trypanosoma brucei is a flagellated protozoan parasite responsible for African Trypanosomiasis. T. brucei is a deadly immune-evasive parasite which circulates the bloodstream of mammalian hosts and requires the Tsetse fly as a transmission vector. The parasite is capable of antigenic variation which allows it to change its glycoprotein coat in order to escape immune detection. Of great concern is the parasite’s growing resistance to various chemotherapeutic treatments which may allow for reemergence. Protein-arginine methyltransferases (PRMTs) are a group of proteins responsible for transferring methyl groups to arginine residues within proteins. Protein methylation causes epigenetic modification of histones or changes in protein-protein interactions which, in turn, leads to the regulation of a variety of biological functions including, but not limited to: transcriptional/translational activation or repression, signal transduction, protein localization, and cell differentiation. Several PRMTs have been discovered in T. brucei including TbPRMT1, TbPRMT3, TbPRMT5, TbPRMT6 and TbPRMT7. TbLpn, a lipin family protein, was discovered based on its protein interactions with PRMTs. Lipins act as Mg2+-dependent phosphatidate phosphatases (PAPs) which catalyze the dephosphorylation of phosphatidic acid (PA) to diacylglycerol (DAG). DAG is a powerful cell signaling molecule which can then be channeled into the synthesis of triacylglycerol (TAG) as well as the phospholipids phosphatidylcholine (PC) and phosphotidlyethanolamine (PE) via the Kennedy pathway. PE and PC are both core constituents of the protozoan cell membrane, and PE in particular is necessary for synthesis of the glycosylphosphatidylinositol (GPI) anchor. Importantly, T. brucei synthesizes its phospholipids de novo, ensuring that these phospholipids are produced by the Kennedy pathway and not by host scavenging. To observe any methylation interaction between TbPRMTs and TbLpn, a methylation assay was conducted using Adomet as a methyl source. The results show that TbPRMT1, TbPRMT5, and TbPRMT7 all successfully methylated TbLpn independently in vitro. TbPRMT3 and TbPRMT6 were incapable of methylating TbLpn in vitro. Furthermore a phosphatidic acid phosphatase assay was conducted to observe how effective TbLpn functions as an enzyme when methylated by TbPRMTs. This assay determined the enzymatic activity of TbLpn based on the release of organic phosphate (Pi) released during the dephosphorylation of PA to DAG. Results from the phosphatidic phosphatase assay show that the enzymatic activity of TbLpn increases greatly following methylation by TbPRMT5 or TbPRMT7, but not TbPRMT1, over control TbLpn.