Grant: $81,750 - National Institutes of Health - May. 15, 2009
100% voted satisfied - 0% voted not satisfied - 1 vote(s) cast
Award Description: The goal of this proposal is to identify the enzymes implicated in phosphatidylcholine biosynthesis from the precursor phosphatidylethanolamine in the human parasite Leishmania, to assess their importance in Leishmania viability, and to establish how the anti-leishmanial drug miltefosine interferes with this metabolic route. We expect to demonstrate that two candidate genes, LmPEM1 and LmPEM2, are involved in the conversion of phosphatidylethanolamine into phosphatidylcholine, are inhibited by miltefosine, and are essential for parasite viability. It is anticipated that results of this project will contribute to a better understanding of how the parasite produces its most abundant lipids and a deeper knowledge of the mechanism of action of the phosphocholine analog, miltefosine, currently tested in clinical trials against several forms of leishmaniasis.
Project Description: The goal of this proposal is to identify the enzymes implicated in phosphatidylcholine (PC) biosynthesis from the precursor phosphatidylethanolamine (PE), assess their importance in Leishmania viability, and establish how the anti-leishmanial drug miltefosine interferes with this metabolic route. Specific Aim 1: determine the subcellular localization of LmPEM1 and LmPEM2. Antisera were obtained after immunization with the recombinant proteins GST-LmPEM1 536-582 and GST-LmPEM2 187-225 that were expressed in Eschericchia coli. Using parasite whole cell extracts, we demonstrated that the respective antisera reacted with LmPEM1 and LmPEM2, respectively. Both LmPEM1 and LmPEM2 bear a putative C-terminal endoplasmic reticulum retention motif, suggesting that these proteins reside in this organelle. Immunofluorescence assays with LmPEM1 and LmPEM2 purified antisera revealed an intracellular, punctuated stain that showed significant overlap with that of the recombinant protein GFP-KDEL, an endoplasmic reticulum resident. This assay demonstrates that PC biosynthesis from the precursor PE occurs in the endoplasmic reticulum. Specific Aim 2: determine LmPEM1 and LmPEM2 enzymatic activity and inhibition profile in the presence of miltefosine. We expressed in a constitutive fashion L. major LmPEM1 and LmPEM2 in the choline auxotroph yeast strain ?scpem1?scpem2, which lacks PE methylase transferase activity due to deletion of ScPEM1 and ScPEM2. We demonstrated that LmPEM1 and LmPEM2 (alone and in combination) restored choline autotrophy of the ?scpem1?scpem2 strain, suggesting that both LmPEM1 and LmPEM2 are able to methylate PE. Future studies are aimed at characterizing the enzymatic properties and reaction products of these enzymes. Specific Aim 3: creation of null mutants of LmPEM1 and LmPEM2. Plasmids for the inactivation of LmPEM1 and LmPEM2 are in progress.
Infrastructure Description: Not applicable
Jobs Summary: N/A (Total jobs reported: 0)
Project Status: Less Than 50% Completed
This award's data was last updated on May. 15, 2009. Help expand these official descriptions using the wiki below.