Phospholipids are a major structural component of biological membranes, forming lipid bilayers. In addition, these molecules play roles in signal transduction and vesicular trafficking, serve as membrane anchors for proteins and carbohydrates, and often regulate enzymes and transport proteins. Phosphatidylcholine (PC), the predominant lipid in eukaryotic membranes, can be synthesized by methylation of phosphatidylethanolamine and via the CDP-choline pathway. The aim of this project is to determine the role of the CDP-choline pathway in phosphatidylcholine synthesis in Leishmania, a genus of trypanosome protozoa. The contribution of several synthetic routes to PC will be evaluated in cultured cells and confocal microscopy will be employed to determine the cellular location of enzymes involved in PC synthesis. The scope of the research project also includes characterization of recombinant forms of two enzymes of the CDP-choline pathway from Leishmania species, choline kinase, which phosphorylates choline to form phosphocholine, and CTP:phosphocholine cytidylyltransferase, which transfers a cytidine nucleotide to phosphocholine.
Broader Impacts This project will contribute to the greater body of knowledge with respect to phospholipid synthesis, providing information important for understanding synthesis of phosphatidylcholine in trypanosomes. Completion of the research relies on significant participation from undergraduate students majoring in chemistry, biochemistry, and/or molecular biology. The students will benefit from participation in independent research and exposure to biochemical and molecular biological laboratory techniques. A major goal of the project is to provide the opportunity for students to present research results at regional and national meetings upon completion of their projects. A quality research experiences gives students the confidence they can succeed and serves as a driving force for the remainder of their careers. Support for research projects that target undergraduates is vital for keeping our nation's students excited about being a scientist at the pivotal undergraduate stage of their academic careers.
Leishmania is a protozoan parasite that is the causative agent of leishmaniasis, a disease which threatens 350 million people, mostly in third world countries. There are different types of leishmaniasis including cutaneous, visceral, and mucocutaneous. Cutaneous leishmaniasis results in skin lesions, whereas visceral leishmaniasis causes swelling of the spleen and liver and can be fatal. Mucocutaneous leishmaniasis affects the nasal area and the lining of the mucosa. Overall, the drugs available are either costly, have severe side effects or Leishmania species are becoming resistant towards them. Additional research is needed in order to find additional drugs and drug targets for the treatment of this disease. This research project hopes to contribute by characterizing properties of enzymes of phospholipid synthesis in Leishmania which are potential drug targets. Phosphatidylcholine is the most abundant phospholipid in protozoans and this phospholipid is likely critical to organism survival. In addition to a central role as a structural component of membrane bilayers, phospholipids participate in cellular signaling, vesicular trafficking, as membrane anchors, and regulate enzymes and transport proteins. Phosphatidylcholine is synthesized via two pathways in eukaryotes, either three successive methylations catalyzed by the enzyme phosphatidylethanolamine N-methyltransferase or by the CDP-choline pathway. Genes that appear to encode enzymes of the CDP-choline pathway have been found in genome sequences from Leishmania. The intellectual merit of this research is to determine the pathways used for phosphatidylcholine synthesis in Leishmania as well as characterize enzymes of the CDP-choline pathway. In this research the enzymes choline kinase, CTP:phosphocholine cytidylyltransferase, and CTP:phosphoethanolamine cytidylyltransferase were studied and catalytic properties characterized. Future elucidation of the differences between catalysis and regulation of Leishmania and mammalian enzymes has the potential to provide a valuable contribution to the field of phospholipid biosynthesis.The broader impacts of this research include the potential benefit to society from training future scientists. Funding from the National Science Foundation is vital for keeping our nation’s students excited about being a scientist at the pivotal undergraduate stage of their academic careers. A significant portion of the funded research was conducted by undergraduate students. An important aspect of independent laboratory research is the extension of research experience beyond topics covered in biochemistry laboratory courses. Students obtained research experience that included cell culture, cellular fractionation, nucleic acid isolation, agarose gel electrophoresis, polymerase chain reaction, protein expression, SDS-PAGE, Western analysis, enzyme assay, and use of radioisotopes. In addition, students gained instrumentation experience including centrifugation, electrophoresis, microscopy, scintillation counting, and spectrophotometry. Upon graduation, students supported through NSF funding obtained permanent, full-time research positions in both government and academic laboratories. Providing research opportunities for students at the interface of chemistry and biology produces scientists of the next generation with laboratory skills and thinking ability necessary to facilitate future scientific discovery.
