Regulation of Trypanosoma brucei Polyamine Biosynthesis




Patel, Manish Mahesh

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Human 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.

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