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. 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. 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. We recently identified TbLpn, a protein homologous to yeast and human lipin, a phosphatidate phosphatase involved in membrane biogenesis, energy metabolism, and adipose tissue development. In yeast and mammals, lipin catalyzes the dephosphorylation of phosphatidic acid (PA) to diacylglycerol (DAG) which, in turn, is used for the synthesis of phospholipids. Careful examination of the predicted amino acid sequence of TbLpn has revealed the presence of two conserved domains characteristic of the lipin family of protein, as well as two aspartic acid residues essential for lipin enzymatic activity. This clearly suggests that TbLpn represents a functional homologue of lipin proteins. The aim of this research was to characterize the in vitro enzymatic activity of a mutant TbLpn in which two conserved aspartic acid residues were changed to alanines. A histidine-tagged mutant TbLpn was expressed in Escherichia coli, and purified over a nickel-resin column. The recombinant mutant protein will be assayed for its ability to carry out dephosphorylation of phosphatidic acid using a radioactive assay. We anticipate that the mutant TbLpn will show very little, if any, enzymatic activity compared to the wild type protein.
|Presenter:||Amber Karpie (Undergraduate Student)|
|Time:||3:15 pm Session IV|