Browsing by Subject "Trypanosoma brucei brucei"
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Item Essentiality and Regulation of Deoxyhypusination in Trypanosoma brucei(2014-07-01) Nguyen, Suong Thu; Orth, Kim; Phillips, Margaret A.; Goodman, Joel M.; De Brabander, Jef K.Human African trypanosomiasis is caused by protozoan parasite Trypanosoma brucei. T. brucei and other trypanosomatids require spermidine for the formation of trypanothione, a unique thiol-redox factor. In other eukaryotes, spermidine is essential for the (deoxy)hypusination of eukaryotic initiation factor 5A (eIF5A). Hypusination, a post-translational modification, occurs via two enzymatic reactions. First, deoxyhypusine synthase (DHS) transfers the aminobutyl moiety of spermidine onto the eIF5A-lysine generating deoxyhypusine which is then hydroxylated by deoxyhypusine hydroxylase to yield the final modification, hypusine. Modified eIF5A has been shown to alleviate ribosome stalling on polyproline tracts. Human and yeast encode two isoforms of eIF5A but only one gene was identified in T. brucei (Tb927.11.740). Herein, I show that TbeIF5A and its modified lysine are essential for parasite growth by gene knockdown and complementation experiments. I have also identified potential proteins whose translation is regulated by eIF5A using proteomic profiling for proline-rich T. brucei proteins. Interestingly, unlike most eukaryotes, trypanosomatids encode two divergent paralogs of DHS (DHSp: Tb927.1.870 and DHSc: Tb927.10.2580), only one of which (DHSc) contains the key catalytic lysine. I showed that both DHS genes are essential for growth of bloodstream-form T. brucei using conditional gene knockouts, further establishing the requirement for deoxyhypusine in these parasites. My biochemical characterization of TbDHS showed that the two T. brucei paralogs form a heterotetrameric complex and that DHSp enhances the activity of recombinant DHSc by 3000-fold. While the essentiality of eIF5A and DHS is consistent with other eukaryotes, the finding that the functional DHS complex is composed of an impaired catalytic subunit (DHSc) and a catalytically dead paralog (termed a prozyme) is novel. This mechanism reiterates the activation and regulation of S-adenosylmethionine decarboxylase by a catalytically dead paralog (AdoMetDC prozyme) in the trypanosomatids, and remarkably, it has independently evolved for two enzymes within the trypanosomatid spermidine biosynthetic pathway. T. brucei seemingly lack several classical eukaryotic transcriptional regulation mechanisms which creates selective pressure to evolve novel strategies to regulate enzyme function. We postulate that many additional examples of 'prozymes' remain to be discovered in the trypanosomatid parasites.Item The Function of Phosphatidylinositol 4-Kinase III-Beta in Trypanosoma Brucei(2006-12-20) Rodgers, Melissa Jeane; Phillips, Margaret A.Phosphatidylinositol 4-kinase phosphorylates phosphatidylinositol at the D4 position resulting in phosphatidylinositol 4-monophosphate. Subsequent phosphorylation events result in a group of molecules known as phosphoinositides. These molecules are important in signal transduction, endocytosis, exocytosis, and protein trafficking. Two phosphatidylinositol 4-kinases are found in Trypanosoma Brucei. We have cloned the gene encoding the Type III phosphatidylinositol 4-kinase Beta (TbPI4KIII-ß) in Trypanosoma Brucei. The protein was exogenously expressed in COS-7 cells and assayed for phosphatidylinositol kinase activity. The expressed protein migrated on SDS-PAGE near the predicted molecular weight of 66 kDa and phosphorylates phosphatidylinositol. Depletion of TbPI4KIII-ß in procyclic T. brucei by RNAi results in inhibition of cell growth and a distorted cellular morphology. Immunofluorescence studies revealed a distorted Golgi apparatus and mislocalization of lysosomal and flagellar pocket proteins. Ultrastructural analysis reveals internal accumulation of a heterogeneous population of vesicles, abnormal positioning of organelles and a loss of cell polarity. Scanning EM reveals a twisted morphology most likely due to an alteration in the microtubule cytoskeleton. Dividing TbPI4KIII-ß RNAi trypanosomes often exhibited a detached daughter flagellum and lacked a cleavage furrow, suggesting a defect in cell division and/or cytokinesis. Cell cycle analysis confirmed that cells depleted of TbPI4KIII-ß have a post-mitotic cytokinesis block. In summary, TbPI4KIII-ß is an essential protein in procyclic T. brucei, required for maintenance of Golgi structure, protein trafficking, normal cellular shape and cytokinesis.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 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 Glutathione Synthetase in Trypanothione Biosynthesis in Trypanosoma brucei(2013-07-25) Pratt, Chelsea BriAnne; Orth, Kim; Phillips, Margaret A.; Goodman, Joel M.; Ruben, LarryTrypanosoma brucei is the causative agent of Human African trypanosomiasis, commonly called sleeping sickness, which is a debilitating disease for which treatment is not currently ideal. Trypanosome parasites differ from their human host by utilizing a novel cofactor termed trypanothione instead of glutathione for protection against reactive oxygen species. Trypanothione is formed by the conjugation of two molecules of glutathione to spermidine forming a link between the polyamine and thiol biosynthetic pathways. My work has investigated the enzyme annotated as glutathione synthetase (GS) in T. brucei to determine if it indeed catalyzes the synthesis of glutathione and if so, to define its kinetic parameters, decipher whether it has any role in the regulation of these pathways, and assess if it is essential for growth. To determine whether the putative TbGS gene was correctly identified through sequence homology, I cloned and expressed the T. brucei gene (TbGS) in Escherichia coli, and purified the recombinant protein. Using an ATP-coupled spectrophotometric assay, I was able to measure TbGS kinetic activity and determine that it was comparable to activities of other published GS homologs, indicating that this gene was correctly annotated. To investigate the physiological role of TbGS in T. brucei, I used genetic approaches to manipulate TbGS levels, first by RNAi, and then by employing conditional knockout models. RNAi was used to decrease protein levels; however, even though TbGS protein levels were depleted by more than eighty percent, there was no altered growth phenotype, and parasites did not have increased sensitivity to known inhibitors of the pathway. I then constructed a TbGS conditional double knockout (cDKO) parasite cell line that contained a tetracycline (tet) regulated episomal copy of TbGS. By removing tet from the media and stopping TbGS protein production, parasites entered growth arrest by day five, which correlated with depleted thiol pools. Parasites remained in growth arrest until day eight after which they resumed growth. This resumption of growth also correlated with the return of low levels of TbGS and thiol pools indicating that loss of trypanothione caused growth arrest. To evaluate if the loss of TbGS had any regulatory effect, levels of biosynthetic pathway proteins were assessed by western blot analysis. A three-fold increase was seen in γ-GCS levels as well as a decrease in AdoMetDC prozyme and ODC levels. Thus our studies have shown that not only is TbGS essential for parasite growth but have also uncovered cross regulation between the polyamine and thiol pathways.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.