Human African trypanosomiasis (HAT) is caused by the parasitic protozoan Trypanosoma brucei. HAT exerts a large burden in both health and economic costs to the affected regions. There is a great need to translate recent advances in the understanding ofthe basic biology ofthe parasite into new drugs. Polyamines are essential metabolites that are required for cell growth. The polyamine biosynthetic enzymes, including ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) are essential to the parasite, and ODC is the target of a currently utilized anti-trypanosomal drug called eflornithine. In order to better understand the pathway and its potential to be exploited for additional drug discovery my lab has been using genetic, biochemical and structural approaches to determine which enzymes in the pathway are essential, to characterize regulatory mechanisms used to control pathway flux, to understand structure function relationships influencing catalysis and ligand binding to ODC and AdoMetDC, and to discover new inhibitors of pathway enzymes. In this fund period we have shown that spermidine synthase and glutathione synthase are essential enzymes, we have built on our previous discovery that that AdoMetDC is regulated by a novel mechanism by identifying both cis and trans factors that may contribute to translational regulation ofthe AdoMetDC regulatory subunit prozyme and we have identified new inhibitors of both ODC and AdoMetDC. Finally in a new direction we have begun studies on the role of deoxyhypusine synthase (DHS) in T. brucei. We remarkedly discovered that similarly to AdoMetDC, DHS is activated by oligomerization with a catalytically dead paralog, showing that this novel mechanism has evolved twice within the trypanosomatid polyamine metabolic pathway. Future studies will build on these findings to: 1) characterize cis/trans regulatory factors influencing translation of prozyme, 2) solve the X-ray structure sof both the AdoMetDC and DHS 3) identify other proteins in T. brucei that are regulated by inactive paralogs, and 4) study the role of deoxyhypusine modification of elF5A in T. brucei.
Human African sleeping sickness is a fatal insect borne disease and current drug therapies are toxic and difficult to administer. The work described in this proposal characterizes the biology of an essential metabolic pathway (polyamine and trypanothione biosynthesis) that has unique features in the parasite, with the goal of providing insight into the potential to target this pathway for drug discovery.
|Pratt, Chelsea; Nguyen, Suong; Phillips, Margaret A (2014) Genetic validation of Trypanosoma brucei glutathione synthetase as an essential enzyme. Eukaryot Cell 13:614-24|
|Nguyen, Suong; Jones, Deuan C; Wyllie, Susan et al. (2013) Allosteric activation of trypanosomatid deoxyhypusine synthase by a catalytically dead paralog. J Biol Chem 288:15256-67|
|Velez, Nahir; Brautigam, Chad A; Phillips, Margaret A (2013) Trypanosoma brucei S-adenosylmethionine decarboxylase N terminus is essential for allosteric activation by the regulatory subunit prozyme. J Biol Chem 288:5232-40|
|Xiao, Yanjing; Nguyen, Suong; Kim, Sok Ho et al. (2013) Product feedback regulation implicated in translational control of the Trypanosoma brucei S-adenosylmethionine decarboxylase regulatory subunit prozyme. Mol Microbiol 88:846-61|
|Kalidas, Savitha; Li, Qiong; Phillips, Margaret A (2011) A Gateway® compatible vector for gene silencing in bloodstream form Trypanosoma brucei. Mol Biochem Parasitol 178:51-5|
|Smithson, David C; Lee, Jeongmi; Shelat, Anang A et al. (2010) Discovery of potent and selective inhibitors of Trypanosoma brucei ornithine decarboxylase. J Biol Chem 285:16771-81|
|Smithson, David C; Shelat, Anang A; Baldwin, Jeffrey et al. (2010) Optimization of a non-radioactive high-throughput assay for decarboxylase enzymes. Assay Drug Dev Technol 8:175-85|
|Deng, Xiaoyi; Lee, Jeongmi; Michael, Anthony J et al. (2010) Evolution of substrate specificity within a diverse family of beta/alpha-barrel-fold basic amino acid decarboxylases: X-ray structure determination of enzymes with specificity for L-arginine and carboxynorspermidine. J Biol Chem 285:25708-19|
|Shaw, Frances L; Elliott, Katherine A; Kinch, Lisa N et al. (2010) Evolution and multifarious horizontal transfer of an alternative biosynthetic pathway for the alternative polyamine sym-homospermidine. J Biol Chem 285:14711-23|
|Lee, Jeongmi; Sperandio, Vanessa; Frantz, Doug E et al. (2009) An alternative polyamine biosynthetic pathway is widespread in bacteria and essential for biofilm formation in Vibrio cholerae. J Biol Chem 284:9899-907|
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