Browsing by Subject "S-Adenosylmethionine"
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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 The Role of S-Adenosylmethionine Synthetase in Killing Effects of DFMO in African Trypanosomes(2013-01-17) Capota, Emanela; Phillips, Margaret A.T. brucei parasites cause a fatal disease that affects hundreds of thousands of people in sub-Saharan Africa. Since current treatment options show either poor efficacy or have safety concerns, it is necessary to identify new targets in the parasite for the development of better medication against this disease. The purpose of my work was to investigate the role of a functional AdoMetSyn in the toxic effect of eflornithine, a clinically used therapy for the treatment of the disease. Prior studies suggested that the toxicity of eflornithine may be due to the combined effect of polyamine depletion and detrimental effects of increased levels of AdoMet in the cell. In order to further study this effect I generated an RNAi cell line that allowed me to test the effects of decreasing S-Adenosylmethionine Synthetase (AdoMetSyn) levels on eflornithine toxicity. I found that knockdown of AdoMetSyn mRNA led to reduced growth rates and to a reduction in AdoMet and dcAdoMet levels in the cell. The potency of DFMO was increased when tested on the RNAi cells containing reduced AdoMet levels. Thus my studies do not provide support for the hypothesis that elevated levels of AdoMet lead to potentiation of the eflornithine anti-growth effects. These results can prove useful in improving DFMO treatment of trypanosomiasis in infected patients.Item SAM Homeostasis Is Regulated by CFIm-Mediated Splicing of MAT2A(August 2021) Scarborough, Anna Maurine; Tu, Benjamin; Mendell, Joshua T.; Green, Carla B.; Conrad, NicholasS-adenosylmethionine (SAM) is the methyl donor for nearly all cellular methylation events. Cells regulate intracellular SAM levels through intron detention of MAT2A, the only SAM synthetase expressed in most cells. The N6-adenosine methyltransferase METTL16 promotes splicing of the MAT2A detained intron by an unknown mechanism. Using an unbiased CRISPR knock-out screen, we identified CFIm25 (NUDT21) as a regulator of MAT2A intron detention and intracellular SAM levels. CFIm25 is a component of the cleavage factor Im (CFIm) complex that regulates poly(A) site selection, but we show it promotes MAT2A splicing independent of poly(A) site selection. CFIm25-mediated MAT2A splicing induction requires the RS domains of its binding partners, CFIm68 and CFIm59 as well as binding sites in the detained intron and 3´ UTR. These studies uncover mechanisms that regulate MAT2A intron detention and reveal a previously undescribed role for CFIm in splicing and SAM metabolism.Item The U6 snRNA m6A Methyltransferase METTL16 Regulates MAT2A Intron Retention Through Co-Transcriptional Splicing(2017-07-19) Pendleton, Kathryn Elizabeth; Corey, David R.; Conrad, Nicholas; Mendell, Joshua T.; Tu, BenjaminMaintenance of proper levels of the methyl donor S-adenosylmethionine (SAM) is critical for a wide variety of biological processes. We demonstrate that the N6-adenosine methyltransferase METTL16 regulates expression of human MAT2A, which encodes the SAM synthetase expressed in most cells. Upon SAM depletion by methionine starvation, cells induce MAT2A expression by enhanced splicing of a retained intron. Unlike previously studied intron retention events, we show that splicing induction of the MAT2A retained intron is regulated at the level of co-transcriptional splicing. This induction requires METTL16 and its methylation substrate, a vertebrate conserved hairpin (hp1) in the MAT2A 3´ UTR. Increasing METTL16 occupancy on the MAT2A 3´ UTR is sufficient to induce efficient splicing. We propose that under SAM-limiting conditions, METTL16 occupancy on hp1 increases due to inefficient enzymatic turnover, which promotes MAT2A splicing. We further show that METTL16 is the long-unknown methyltransferase for the U6 spliceosomal snRNA. These observations suggest that the conserved U6 snRNA methyltransferase evolved an additional function in vertebrates to regulate SAM homeostasis.