Determination of the roles of the Trypanosoma brucei lipin homologue TbLpn

Abstract

African Sleeping Sickness is caused by Trypanosoma brucei. T.b is a parasitic protozoan that affects humans and other animals alike in sub-Saharan Africa. Epidemics have occurred throughout the 20th century, including Uganda and Kenya in 1900 and Alego and Kenya from 1976 to 1983. Due to its antigenic variation, which helps it evade the immune system, there have only been five drugs approved for the treatment of this disease and they tend to be toxic. Therefore, in order to better understand how T.b functions, a lipin homologue termed TbLpn was examined. Lipins help catalyze the formation of diacylglycerol (DAG), which is used to form triacylglycerol (TAG). TAG is then used to form phosphatidylethanolamine (PE) and phosphatidylcholine (PC), which are major lipids that constitute the membrane of T.b. In order to better understand how TbLpn functions, we checked if it was phosphorylated in vivo by protein methyl transferases termed (TbPRMT1 and TbPRMT7) in procyclic T.b forms. TbLpn was immunoprecipitated from whole cell extracts using anti-TbLpn. Then, TbLpn treated with CIAP (dephosphorylates proteins) was compared to non-CIAP treated TbLpn using an SDS/Western blot. The cellular localization of TbLpn was also determined by first preparing both cytosolic and nuclear extracts. TbPRMT1 and 7 were downregulated by RNA interference in both the cytosolic and nuclear extracts and compared to normal TbPRMT1 and 7 using SDS/Western blot. The results show that whether or not TbPRMT1 and 7 are downregulated or not, TbLpn is primarily found in the cytosol. Thus, it can be concluded that TbLpn is primarily found in the cytosol, however its location within the cytosol, due to phosphorylation, is unknown. The results also show that TbLpn is in fact phosphorylated in vivo, due to the difference in the mobility shift between the treated and non-treated CIAP samples. The CIAP treated sample should have been higher because in general, if a protein is dephosphorylated, its shift should be lower than usual. However, its mobility shift was higher than that of the non-CIAP treated. It is unknown why that is, but phosphorylation could be linked to another process that affects its mobility shift in some way. Learning that TbLpn is phosphorylated is of great importance, since it could play a key factor in which TbLpn catalyzes the beginning stages of the formation of PE and PC, which are found in high abundance on the plasma membrane.