Browsing by Subject "Adenosylmethionine Decarboxylase"
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Item Regulation of Trypanosoma brucei Polyamine Biosynthesis(2018-07-23) Patel, Manish Mahesh; Conrad, Nicholas; Phillips, Margaret A.; Goodman, Joel M.; Reese, Michael L.; Tu, BenjaminHuman African Trypanosomiasis (HAT), also known as Sleeping Sickness, is a disease caused by the protozoan parasite Trypanosoma brucei. About 70 million people are at risk of infection in sub-Saharan Africa. While current treatments have efficacy, they are difficult to administer and serious toxicity is associated with one of the key compounds, emphasizing the need for novel therapeutic approaches. The polyamine biosynthetic pathway is vital for parasite viability and the first committed step catalyzed by ornithine decarboxylase is the target of Eflornithine, which is used to treat late stage disease. Our lab has investigated this pathway to identify other potential targets for drug development. During this work I also demonstrated that the parasite employs unique regulatory strategies to control metabolism through this pathway. Among them, I found T. brucei S-adenosylmethionine decarboxylase (TbAdoMetDC), which is a homodimer in other eukaryotes, is a heterodimer in the trypanosomatids. The active enzyme is composed of two paralogs, one with limited activity (AdoMetDC) and the other inactive, which I termed prozyme. Prozyme activates trypanosomatid AdoMetDC through an allosteric mechanism that involves relief of autoinhibition through a conformational change. Prozyme is found in limited quantities relative to TbAdoMetDC and upregulated upon knockdown or chemical inhibition of TbAdoMetDC, providing a mechanism to regulate polyamine flux in the cell. The cellular mechanism of regulation is not fully understood, as gene expression in T. brucei differs significantly from that of mammals. Herein, I show prozyme is regulated at the level of protein translation. I found that TbAdoMetDC suppresses prozyme expression at the protein level in an enzyme activity-independent manner. Upon its loss, prozyme is expressed constitutively in an unregulated manner. I also show that the enzymatic product of TbAdoMetDC, dcAdoMet, acts as a metabolic signal for prozyme upregulation. Under chemical inhibition of AdoMetDC by Genz-644131 I show dcAdoMet is the only significant metabolite to correlate with prozyme protein upregulation. I further support this hypothesis by characterizing the effects of knockdown of S-adenosylmethionine synthetase by RNAi and through methionine starvation to correlate dcAdoMet depletion with prozyme upregulation independent of AdoMetDC manipulation. Through this work I also demonstrated that TbAdoMetSyn is an essential enzyme and validate the activity of its allele. Taken together, I expand upon our mechanistic understanding of this complex regulatory paradigm between an enzyme and pseudoenzyme in T. brucei.Item Regulation of Trypanosoma Cruzi S-Adenosylmethionine Decarboxylase Activity(2005-12-15) Beswick, Tracy; Phillips, Margaret A.Chagas' disease, caused by Trypanosoma cruzi, is a major health problem for which there is no good chemotherapy. Development of new drugs is crucial. One strategy is to develop inhibitors of enzymes in essential metabolic pathways. Trypanosomes need polyamines to survive; thus, the enzymes in the polyamine biosynthetic pathway are targets for drug development. Characterization of S-adenosylmethionine decarboxylase (the rate-limiting enzyme in the pathway) activity in trypanosomes and comparison to human AdoMetDC will aid the discovery of novel, species specific inhibitors. I have characterized putrescine stimulation of T. cruzi AdoMetDC activity as well as the allostery between the putrescine binding site and the active site of the enzyme. Putrescine is capable of stimulating T. cruzi AdoMetDC decarboxylation but not proenzyme processing. This contrasts with the human enzyme, for which processing and decarboxylation are both modestly stimulated by putrescine. R13, a Leu in the human enzyme, was shown to be essential for processing in the T. cruzi enzyme. Additionally, D174 is an important binding determinant for putrescine in both species of AdoMetDC. Other mutations near the putative putrescine site (S111R and F285H) indicate that the putrescine binding site for T. cruzi AdoMet DC is more surface-exposed than the human enzyme. Putrescine stimulates decarboxylase activity 41 fold for T. cruzi AdoMetDC (compared to 1.7 fold for the human enzyme). This stimulation is achieved through allosteric binding to a site approximately 15 ?ay from the active site. To examine this allostery, we used putrescine and active site mutations as well as an active site inhibitor, CGP 40215. This inhibitor unexpectedly exhibited noncompetitive inhibition with a Ki of 32muM and 3muM in the absence and presence of putrescine, respectively (Ki for the human enzyme is 10nM and 6nM). Residues important for communication between the two sites include L221, H5, D174, S111 and F285. L221, a Thr residue in the human enzyme, is partially responsible for some of the species specificity as the Ki for L221T was 800nM. The differences in putrescine stimulation and CGP 40215 inhibition indicate development of species specific inhibitors is possible and could be an effective strategy for novel antitrypanosomal chemotherapy.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.