Amalgamating tools of molecular biology, genetics, biochemistry, structural biology, and immunochemistry, this proposal offers a thorough interdisciplinary analysis of three key enzymes of the purine salvage pathway of Leishmania. These enzymes are hypoxanthine-guanine phosphoribosyltransferase (HGPRT), adenine phosphoribosyltransferase (APRT), and xabthine phosphoribosyltransferase (XPRT). As protozoan parasites are auxotrophic for purines, HGPRT, APRT, and XPRT provide an important, if not vital, nutritional function for the parasite, and HGPRT initiates the intracellular metabolism of allopurinol, a lead compound that has shown therapeutic efficacy in both leishmaniasis and Chagas disease. The proposed investigations constitute a logical step in the validation of these enzymes as potential therapeutic targets and in the implementation of a rational, structure-based strategy of drug discovery, and ultimately drug design, for the treatment and prophylaxis of leishmaniasis and other diseases of parasitic origin. The first objective of this application is to determine the contributions of HGPRT, APRT, XPRT, and adenosine kinase (AK) to purine salvage in L. donovani promastigotes by phenotypic characterization of deltaxprt mutants that will be constructed by targeted gene replacement in wild type, deltahgprt, deltaaprt, deltahgprt/deltaaprt, deltahgprt/ak, deltaaprt/ak, and deltahgprt/deltaaprt/ak genetic backgrounds. Whether HGRPT, APRT, or XPRT function is essential for infectivity or virulence will be tested by generating null mutants in the infective M379 L. mexicana strain. The second specific aim entails a detailed biochemical and structural characterization of the HGRPT, APRT, and XPRT proteins. The first component of Specific Aim II consists of an evaluation of the HGPRT and XPRT molecular models and the APRT structure by site-directed mutagenesis of key amino acids that are conjectured to be involved in catalysis or substrate binding and biochemical characterization of the genetically altered proteins. The second aspect of this aim is to supplement the structure-function studies on HGPRT and XPRT via the introduction of crystallographic methods with the ultimate intention of resolving a 3-D structure of a leishmanial HGPRT or XPRT protein. The final specific aim is to ascertain the intracellular location of the APRT and XPRT proteins by subcellular fractionation of parasite lysates and by immunofluorescence and immunoelectron microscopy on intact cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AI023682-16
Application #
6373091
Study Section
Tropical Medicine and Parasitology Study Section (TMP)
Program Officer
Rogers, Martin J
Project Start
1983-06-01
Project End
2003-03-31
Budget Start
2001-04-01
Budget End
2002-03-31
Support Year
16
Fiscal Year
2001
Total Cost
$292,848
Indirect Cost
Name
Oregon Health and Science University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
009584210
City
Portland
State
OR
Country
United States
Zip Code
97239
Boitz, Jan M; Jardim, Armando; Ullman, Buddy (2016) GMP reductase and genetic uncoupling of adenylate and guanylate metabolism in Leishmania donovani parasites. Mol Biochem Parasitol 208:74-83
Ortiz, Diana; Forquer, Isaac; Boitz, Jan et al. (2016) Targeting the Cytochrome bc1 Complex of Leishmania Parasites for Discovery of Novel Drugs. Antimicrob Agents Chemother 60:4972-82
Smith, Sabrina; Boitz, Jan; Chidambaram, Ehzilan Subramanian et al. (2016) The cystathionine-?-synthase domains on the guanosine 5''-monophosphate reductase and inosine 5'-monophosphate dehydrogenase enzymes from Leishmania regulate enzymatic activity in response to guanylate and adenylate nucleotide levels. Mol Microbiol 100:824-40
Soysa, Radika; Tran, Khoa D; Ullman, Buddy et al. (2015) Integrating ribosomal promoter vectors that offer a choice of constitutive expression profiles in Leishmania donovani. Mol Biochem Parasitol 204:89-92
Rodriguez-Contreras, Dayana; Aslan, Hamide; Feng, Xiuhong et al. (2015) Regulation and biological function of a flagellar glucose transporter in Leishmania mexicana: a potential glucose sensor. FASEB J 29:11-24
Martin, Jessica L; Yates, Phillip A; Soysa, Radika et al. (2014) Metabolic reprogramming during purine stress in the protozoan pathogen Leishmania donovani. PLoS Pathog 10:e1003938
Soysa, Radika; Carter, Nicola S; Yates, Phillip A (2014) A dual luciferase system for analysis of post-transcriptional regulation of gene expression in Leishmania. Mol Biochem Parasitol 195:1-5
Boitz, Jan M; Ullman, Buddy (2013) Adenine and adenosine salvage in Leishmania donovani. Mol Biochem Parasitol 190:51-5
Soysa, Radika; Wilson, Zachary N; Elferich, Johannes et al. (2013) Substrate inhibition of uracil phosphoribosyltransferase by uracil can account for the uracil growth sensitivity of Leishmania donovani pyrimidine auxotrophs. J Biol Chem 288:29954-64
Arendt, Cassandra S (2013) Crithidia fasciculata adenosine transporter 1 (CfAT1), a novel high-affinity equilibrative nucleoside transporter specific for adenosine. Mol Biochem Parasitol 191:75-9

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