Michel Pelletier, Ph.D
Associate Professor and Chair (Biology)
Office: Lennon Hall B19
The major focus of my research is to characterize and better understand lipid biosynthesis in the parasite Trypanosoma brucei, the causative agent of African sleeping sickness in human, using a dual genetic/biochemical approach. Due to the importance of lipid biosynthesis in trypanosome, it is very likely that enzymes involved in these biosynthetic pathways play an essential role and could be used as a drug target.
Human African Trypanosomiasis, also known as African sleeping sickness, is a vector-borne devastating disease caused by the parasitic protozoan Trypanosoma brucei. This parasite is transmitted between mammalian hosts by the tsetse flies of the genus Glossina. According to the World Health Organization (WHO), sleeping sickness threatens over 60 million people in 36 countries of sub-Saharan Africa. Over 70,000 deaths every year are a result of sleeping sickness and the disease is always fatal unless treated.1 During recent epidemics in several villages in the Democratic Republic of Congo, Angola, and Southern Sudan, the prevalence of the disease has reached 50%, and sleeping sickness was considered the first or second greatest cause of mortality, ahead of HIV/AIDS.1 T. brucei is also pathogenic to animals where it causes a disease known as nagana, particularly in African livestock. T. brucei is an evolutionarily ancient organism, further removed from yeast than yeast is from humans.2 The entire life cycle of T. brucei occurs extracellularly. After a mammalian host is inoculated by an infected tsetse fly, metacyclic trypomastigotes migrate from the skin tissue to the lymphatic system and pass into the bloodstream. Bloodstream form trypanosomes evade the host immune system by constantly changing the antigenic composition of the variant surface glycoprotein (VSG) coat. Inside the host, they transform into bloodstream trypomastigotes and move to the tissues and various organs. The tsetse fly becomes infected with bloodstream form trypomastigotes when taking a blood meal on an infected mammalian host. The then enter the fly midgut in which they transform into procyclic form trypomastigotes. In T. brucei, transcriptional gene regulation is essentially absent. Rather, gene expression is controlled primarily at the levels of RNA processing, stability, and translation, and likely involves a substantial number of RNA binding proteins.3-5 Besides its great health and economic importance, T. brucei represents an exceptional tool for the study of cell physiology/biology due to the possibility of carrying out RNA interference in this organism.
Protein arginine methylation is a post-translational modification resulting in the addition of methyl groups from S-adenosylmethionine (AdoMet) to the nitrogen of arginine residues in proteins.6-8 Arginine methylation is catalyzed by enzymes known as protein arginine methyltransferases (PRMTs). PRMTs have been identified in a variety of organisms including mammals, yeasts, plants, and protozoa.7, 9-11 Recent reports have established a role for arginine methylation in the control of signal transduction,12-14 RNA transport,15-16 RNA processing,17-19 protein localization,20-22 and transcription.23 However, the functional significance of many arginine methylation events is currently unknown. In mammalian cells, two major and distinct types of PRMTs have been identified. Type I enzymes catalyze the formation of both monomethylarginine (MMA) and asymmetrical dimethylarginine (ADMA), while the type II enzyme forms MMA and symmetrical dimethylarginine (SDMA). Interestingly, arginine methylation catalyzed by type I and type II often occurs within RGG, RG, or RXR motifs of RNA-binding proteins,6, 24, 25 suggesting that arginine methylation is likely to play a key role in the control of gene expression in T. brucei.
To understand the roles of arginine methylation in trypanosome gene expression, and to identify substrates and/or regulators of PRMTs in T. brucei, we began by identifying proteins interacting with the major type I PRMT, TbPRMT1, using a yeast-two-hybrid screening. This revealed several potential TbPRMT1 interacting proteins, notably a protein homologous to yeast and human lipin, a phosphatidate phosphatase involved in membrane biogenesis, energy metabolism, and adipose tissue development. The lipin family of proteins have dual cellular functions, acting as an enzyme required for triacylglycerol (TAG) and phospholipids biosynthesis, and as a transcriptional coactivator in the regulation of lipid metabolism genes (Figure 1).26 As phospholipids constitute the major constituents of biological membranes, lipin plays a pivotal role in membrane biogenesis. The first member of the family, lipin-1, was first identified through positional cloning of the mutant gene responsible for fatty liver dystrophy (fld), a genetic disease characterized by loss of body fat, fatty liver, hypertriglyceridemia, and insulin resistance.27 In mammals, 3 members of the lipin gene family have been identified. Two lipin-1 protein isoforms are generated by alternative mRNA splicing of the Lpin1 gene.28 Lipin-2 and Lipin-3 were identified through sequence similarity to lipin-1.27 While lipin-1 is mostly expressed in white and brown adipocytes, skeletal muscles, and testis,27 lipin-2 is mostly present in liver and brain, whereas lipin-3 is present in small intestine and liver, albeit at low levels.29 In mouse, two Lpin-1-related genes, Lpin2 and Lpin3, have also been identified. Orthologs have also been found in Saccharomyces cerevisiae, Schyzosaccharomyces pombe, Drosophila, Caenorhabditis elegans, Arabidopsis thaliana, and Plasmodium falciparum. Sequence alignments of the lipin-related genes revealed the presence of two highly conserved regions, designated amino-terminal and carboxy-terminal lipin (NLIP and CLIP) domains.
Two distinct functions of lipin-1 have recently been identified. First, lipin-1 acts as a Mg2+-dependent phosphatidate phosphatase (PAP1) involved in TAG and phospholipids biosynthesis. Lipin-1 catalyzes the dephosphorylation of phosphatidic acid (PA) to diacylglycerol (DAG) which, in turn, is used for the synthesis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), as well as TAG Figure 2). In eukaryotic cells, PC and PE are the most abundant phospholipids, usually accounting for 30-50% and 20-40%, respectively, of total phospholipids.30-31 In the presence of high levels of fatty acids within the cells, lipin translocates from the cytosol to the endoplasmic reticulum, where it converts phosphatidic acid to diacylglycerol. Lipin-1-deficient cells are immature adipocytes unable to store lipid or express mature adipocyte markers.32 This results in a loss of body weight, peripheral neuropathy due to myelin degradation, Schwann cell differentiation defects and proliferation, and nerve conduction velocity (Figure 3).33-35
The yeast ortholog of lipin, PAH1, was later identified in Saccharomyces cerevisiae, and was shown to possess the same Mg2+-dependent PAP1 activity. The catalytic activity was mapped to the DxDxT motif present in the CLIP domain of PAH1.36 and was later shown to be present in all lipin-related proteins. As for their mammalian counterparts, pah1ƒ¢ mutants contain elevated levels of PA and low levels of DAG and TAG. The levels of phosphatidylinositol (PI), PC, and PE, are also affected in these mutants.36, 37. In addition, PAH1-deficient mutants exhibit aberrant expansion of the nuclear/ER membrane due to elevated PA levels and derepression of phospholipids synthesis genes, attributed to increased concentration of PA at the nuclear/ER membrane .37
The T. brucei lipin homologue was identified by yeast-two-hybrid as a TbPRMT1-interacting protein. The gene, located on chromosome VII (locus Tb927.7.5450), encodes a predicted 806 amino acid protein, which we termed TbLpn. The predicted protein has a molecular weight of 86.7 kDa and a pI of 4.9, and displays, at the amino acid level, between 15 and 25% identity to the lipin family of protein. Most importantly, the two conserved regions NLIP and CLIP, characteristic of the lipin family of protein, are also present in TbLpn (Figure 4). In addition, careful examination of the predicted amino acid sequence revealed the presence of the DxDxT motif, namely DVDGT, located from amino acid 445 to 449. This clearly suggests that TbLpn possesses PAP1 activity and represent a functional homologue of lipin proteins
As for other eukaryotes, PC and PE constitute the majority of phospholipids in trypanosomes.38 Of great importance is the fact that, as opposed to other parasitic organisms, trypanosomes synthesize phospholipids de novo. This makes the trypanosome phospholipids biosynthesis machinery a very attractive target for new drug design. Although the pathways for phospholipids biosynthesis have not been very well characterized, recent data have helped to better understand how trypanosomes are able to assemble phospholipids. For example, the steps involved in the biosynthesis of glycosylphosphatidylinositol (GPI), a process essential for T. brucei bloodstream form survival, have been well studied. This synthesis differs in certain aspects from the pathway in mammalian cells and yeast, making phospholipids synthesis even more important as a drug target. Furthermore, the pathways for the synthesis of PI and PE have recently been determined.39, 40 We recently carried out down-regulation of TbLpn expression in procyclic form T. brucei by RNA interference, and found that TbLpn is essential for cell growth (Figure 5).
The major focus ofmy research is to identify the function(s) of Tblpn and its importance in trypanosome metabolism and infectivity using a dual genetic/biochemical approach. Functions of Tblpn in vivo will be assessed by disrupting its expression by RNA interference and determining the effect of the disruption on cellular growth, cellular phospholipid content, phosphatidic acid phosphatase activity, cell morphology, and expression of lipid biosynthetic genes, just to name a few.