• Characterization of in vitro and in vivo enzymatic activity of TbLpn and its role in phospholipids biosynthesis in Trypanosoma brucei

      Munini, Dominic Nyamai (2012-06-20)
      Trypanosoma brucei belongs to a group of parasitic protozoan called flagellates. It causes African sleeping sickness and is transmitted by tsetse fly of genus Glossina. African sleeping is a devastating vector-borne disease which threatens over 60 million people in 36 Sub-Saharan Africa. It causes over 70,000 deaths annually and is fatal unless treated. Reemergence of the disease in recent years has been a concern for scientists. There is concern on design of new drugs for the treatment of the disease because of the toxic effects of drugs designed around 50 years ago. Trypanosomes have great importance over the other parasitic organisms. They are able to synthesize phospholipids de novo. This makes the trypanosome phospholipid biosynthesis mechanism a very attractive target for design of new drugs. TbLpn which has been identified in T. brucei is a protein homolog to yeast and mammalian Lipins. Yeast and mammalian lipins are phosphatidate phosphatases involved in membrane biogenesis, energy metabolism and adipose tissue development. The lipin protein family in mammals and yeast catalyzes the dephosphorylation of phosphatidic acid to diacylglycerol, which is in tum used to synthesize phospholipids. Two conserved domains as well as the two aspartic acid residues necessary for enzymatic activity in lipin protein family have also been identified in TbLpn. This is a clear indication that TbLpn might represent functional homolog of lipin proteins. The goal of this research project is to demonstrate in vivo and in vitro enzymatic activity of TbLpn. Recombinant His-TbLpn expressed in Escherichia coli was purified over Nickel column and used to test the enzymatic activity of TbLpn. Native TbLpn was also immunoprecipitated from T. brucei cytosolic extract and used to carry out the dephosphorylation of phosphatidic acid.