Browsing by Subject "Trypanosomiasis, African"
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Item Identification and Characterization of Drug Targets in the Pyrimidine and Purine Pathways of Trypanosoma brucei(2016-11-29) Leija, Christopher Luis; Bruick, Richard K.; Phillips, Margaret A.; Tu, Benjamin; Kohler, Jennifer J.; Reese, Michael L.The single-celled extracellular parasite Trypanosoma brucei causes Human African Trypanosomiasis (HAT), which is fatal if untreated. Current therapies result in severe side effects and require complex treatment regimens. In an effort to spur the development of effective, safe, and simple to administer drugs, my work sought to identify and characterize novel drug targets in the parasite pyrimidine and purine pathways. The pyrimidine de novo biosynthetic pathway has been well characterized, however little work had been done to evaluate the importance of pyrimidine salvage enzymes. Specifically, my research validates the essentiality two seemingly redundant enzymes: thymidine kinase (TK) and cytidine deaminase (CDA). Using a combination of genetic and analytical techniques, a novel pathway linking cytosine and thymine nucleotides was discovered. This pathway is composed of the salvage enzymes TK and CDA in addition to a newly discovered 5'-nucleotidase. I demonstrate that the function of this pathway is to convert de novo synthesized cytosine deoxynucleotides into the deoxycytidine, which is ultimately converted to thymine deoxynucleotides. The vital role for TK in bridging pyrimidine nucleotide pools may represent a shared vulnerability unique to kinetoplastids, providing an opportunity to target multiple human pathogens. In contrast to the pyrimidine pathway, the parasite lacks the ability to generate purine nucleotides de novo. As a consequence, they are dependent on the salvage of purine nucleosides/bases from the host through a redundant and interconnected network of purine salvage and interconversion enzymes. In theory, any single precursor is capable of sustaining the formation of all purine nucleotides. We demonstrate that strategic inhibition of key metabolic routes circumvents the redundant nature of this pathway. The enzyme guanosine-5'-monophosphate synthase (GMPS) catalyzes the formation of GMP from xanthosine-5'-monophosphate. The generation of a GMPS null cell line restricts the parasite to the salvage of guanine to maintain GMP nucleotide pools, which is only viable in supraphysiological concentrations of guanine. Using a similar approach, we also genetically validated the essentiality of adenylosuccinate lyase (ADSL), which catalyzes the formation of AMP and fumarate from adenylosuccinate. In this case, depletion of this enzyme is lethal in all conditions. These two novel drug targets offer a solution to bypassing the redundancy in the purine pathway for the development of anti-trypanosomal therapies.Item The Role of S-Adenosylmethionine Decarboxylase on Regulation of Polyamine and Trypanothione Metabolism in Trypanosoma Brucei(2008-05-13) Willert, Erin Kathleen; Phillips, Margaret A.Trypanosoma brucei is the causative agent of Human African Trypanosomiasis (HAT), a fatal and neglected disease affecting Sub-Saharan Africa. Current therapeutics are limited for several reasons, underscoring the need for new and improved drugs. The polyamine/trypanothione pathway is essential for T. brucei, and the biosynthetic enzymes in this pathway are potential drug targets. We have characterized T. brucei S-adenosylmethionine decarboxylase (AdoMetDC), a key enzyme required for the synthesis of spermidine and trypanothione, and examined the role of AdoMetDC on the regulation of polyamine and trypanothione metabolism. The recombinant T. brucei AdoMetDC enzyme displays low catalytic efficiency as compared to the human enzyme (1000 fold lower). Also, the specific activity in trypanosome cell lysates is about 400 fold higher than that of the recombinant enzyme. We have discovered that the product of a second gene, which we have named prozyme, is required for full activity. Prozyme arose through gene duplication and mutational drift, and has no intrinsic decarboxylase activity. AdoMetDC and prozyme form a tight heterodimer, and have a catalytic efficiency that is 1,200 fold higher than AdoMetDC alone. The heterodimeric organization may be a means for polyamine regulation in T. brucei, and the differences between host and parasite enzymes suggest that AdoMetDC is an intriguing drug target. In order to better understand the role of AdoMetDC, we created a stable T. brucei cell line that can be induced to knockdown AdoMetDC expression by RNAi. AdoMetDC knockdown cells are auxotrophic for spermidine. In these cells, putrescine, the precursor of spermidine, is increased five fold, and spermidine levels drop to about 50% of uninduced cells. Levels of glutathionyl-spermidine and trypanothione are almost completely abolished, indicating that the trypanosomes are maintaining spermidine levels at the expense of trypanothione. Protein levels of prozyme, ornithine decarboxylase and trypanothione synthetase are increased during AdoMetDC knockdown. Therefore AdoMetDC has a central role in the biosynthesis and metabolism of polyamines and trypanothione.Item Structural Basis for the Allosteric Activation of Trypanosoma Brucei S-adenosylmethionine Decarboxylase by a Catalytically Dead Homolog(2012-12-06) Velez, Nahir Aimee 1983-; Orth, Kim; Albanesi, Joseph P.; De Brabander, Jef K.; Roth, Michael G.; Phillips, Margaret A.Human African Trypanosomiasis (HAT) is caused by single-celled parasites, Trypanosoma brucei, which are transmitted to humans by infected tsetse flies. Trypanosomiasis has a profound impact on the health of a large number of people in sub-Saharan Africa and it is fatal when untreated. Unfortunately, current drug therapy is limited mostly because of toxic effects on the patients. The polyamine biosynthetic pathway is a validated target for the development of drugs. Enzymes involved in polyamine biosynthesis exhibit features that differ significantly between the parasites and the human host. Therefore, exploitation of such differences can lead to the design of new inhibitors that can selectively kill the parasites. My work is focused on S-adenosylmethionine decarboxylase (AdoMetDC), which in the trypanosomatids is regulated by a unique mechanism, heterodimer formation with a catalytically dead homolog. This protein, designated prozyme, forms a high-affinity heterodimer with AdoMetDC and increases its activity by >1000-fold. The heterodimer is confirmed to be the functional enzyme in vivo. Therefore, understanding the mechanisms that regulate T. brucei AdoMetDC activation by prozyme can provide essential information for more effective inhibitory strategies. The role of specific residues involved in the process was studied by deletion and site-directed mutagenesis. Results indicate that 12 key amino acids at the N-terminal portion of the enzyme, which are fully conserved in the trypanosomatids but absent from other eukaryotic homologs, play a crucial role since there is more than 50 percent less activation by prozyme when they are either removed or mutated to alanine. AdoMetDC L8 and L10 seem to be the strongest determinants for stimulation by prozyme in this region. Analytical ultracentrigugation analyses in the sedimentation velocity mode indicated that dimerization is not impaired when these essential residues are removed, since binding affinities between wildtype and mutant heterodimers remain similar (Kd= <0.5 and 1μM, respectively). Thus, these results imply that key residues in the area must be acting through an allosteric regulatory mechanism. I have also characterized the activity of the L. major AdoMetDC/prozyme complex, the catalytic efficency from which increases by 170-fold upon binding of the homolog. Swapped complexes containing AdoMetDC and prozyme from different trypanosomatids (T. brucei, T. cruzi and L.major) are functional, supporting the idea that amino acid residues essential for the activation mechanism are conserved in all species.