Functional Characterization of Trypanosoma Brucei Protein Arginine Methyltransferase 1

Abstract

Trypanosoma brucei is an early divergent parasitic protozoan that causes sleeping sickness in humans and Nagana in cattle in sub-Saharan Africa. The disease is almost always fatal when untreated. While treatments exist, they are expensive, hard to administer and an emerging resistance has been documented. Therefore, development of new drugs is highly desirable. One avenue of drug discovery is to target essential processes that contain parasite-specific components with rational drug design techniques. In depth understanding of these processes is vital for precise targeting of the inhibitors. T. brucei differs from other eukaryotes in the manner by which it controls gene expression. The genes are transcribed in large, functionally unrelated, polycistronic units which are then processed into individual mRNAs by trans-splicing coupled with polyadenylation. Thus, the vast majority of protein expression control occurs post-transcriptionally, mostly through the function of mRNA binding proteins that alter mRNA stability or translation rates. RNA binding protein function is then modified by post-translational modifications. Arginine methylation, a modification catalyzed by protein arginine methyltransferases (PRMTs), was shown to primarily target RNA binding proteins in humans as well as in T. brucei. This prompted us to more closely investigate the role of PRMTs in T. brucei biology.In this thesis, I examine the structure and function of TbPRMT1, a major T. brucei PRMT. From our previous work we knew that TbPRMT1 is responsible for the majority of asymmetric dimethylarginine formation in vivo; however, the enzyme did not have robust activity in vitro. The data presented in Chapter 2 of this thesis show that TbPRMT1 is an obligate PRMT heterotetramer formed by a catalytic subunit we call enzyme (TbPRMT1ENZ or ENZ) and an allosteric activator called prozyme (TbPRMT1PRO or PRO). The majority of PRMTs function as homodimers, and no PRMT to date has been shown to form obligate heteromer with another PRMT. Therefore, TbPRMT1 structure is completely novel.Chapter 3 of this thesis focuses on the in vivo role of TbPRMT1. Studies in an animal model show that TbPRMT1 contributes to T. brucei virulence through an unknown mechanism. I determined protein steady state levels in cells lacking TbPRMT1 and observed changes in energy metabolism, namely a decrease in glycolytic enzyme abundance and an increase in the levels of components of the proline degradation pathway. These changes resemble the metabolic remodeling T. brucei undergoes through its life cycle progression. Potential TbPRMT1 substrates were identified among proteins that change mRNA association in TbPRMT1 depleted cells and among TbPRMT1 interacting proteins. Many proteins involved in T. brucei starvation stress response were found interacting with TbPRMT1, which prompted us to examine the ability of TbPRMT1ENZ RNAi cell line to respond to stress. My results show that lack of TbPRMT1 strongly hinders the ability of T. brucei to form cytoplasmic mRNA granules in starvation conditions. Finally, I show that TbPRMT1 has an ability to bind nucleic acids in vitro and in vivo, a feature completely novel to PRMT enzymes. And which may factor into its ability to modulate RNA biology.