Browsing by Subject "Gene Expression Regulation, Enzymologic"

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Ral G-Proteins and the Exocyst Complex are Mediators of the Cellular Response to Nutrients
(2012-07-20) Bodemann, Brian Oliver; White, Michael A.
The small G-proteins, RalA and RalB, are important mediators of cellular responses to viral infection and nutrient availability. Prior work has demonstrated that the exocyst complex is an important effector of Ral G-protein signaling. The eight member exocyst contains two Ral effector proteins, Exo84 and Sec5, which contribute to distinct cellular responses. During viral infection, RalB promotes the activation of the innate immunity signaling kinase, TBK1, through direct assembly on a Sec5-containing subcomplex of the exocyst. Macroautophagy is an important cellular process which facilitates cellular adaptation to nutrient deprivation as well as the clearance of intracellular pathogens. The study of macroautophagy in mammalian cells has described induction, vesicle nucleation, and membrane elongation complexes as key signaling intermediates driving autophagosome biogenesis. How these components are recruited to nascent autophagosomes is poorly understood, and although much is known about signaling mechanisms that restrain autophagy, the nature of positive inductive signals that can promote autophagy remain cryptic. I report that RalB is localized to nascent autophagosomes. RalB and its effector Exo84 are required for nutrient starvation-induced autophagocytosis, and RalB activation is sufficient to promote autophagosome formation. Through direct binding to Exo84, RalB induces the assembly of ULK1 and Beclin1-VPS34 complexes on the exocyst, which are required for isolation membrane formation and maturation. Thus, RalB-Exo84 signaling is a primary adaptive response to nutrient limitation that directly engages autophagocytosis through mobilization of the core vesicle nucleation machinery. Conversely, I find that Sec5 associates with mTORC1, a key inhibitor of autophagy. Intriguingly, I find that the Ral–Sec5 activated kinase, TBK1, is necessary for amino acid stimulation of mTORC1 activity. Thus, distinct Ral-dependent subcomplexes of the exocyst mediate the cellular response to nutrient availability.
Regulation of Trypanosoma brucei Polyamine Biosynthesis
(2018-07-23) Patel, Manish Mahesh; Conrad, Nicholas; Phillips, Margaret A.; Goodman, Joel M.; Reese, Michael L.; Tu, Benjamin
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.