Browsing by Subject "Protozoan Proteins"
Now showing 1 - 4 of 4
- Results Per Page
- Sort Options
Item Acanthamoeba spp. Secrete a Mannose-Induced Protein that Correlates with Ability to Cause Acanthamoeba Keratitis(2004-01-14) Hurt, Michael Allen; Niederkorn, Jerry Y.Acanthamoeba spp. are ubiquitously distributed in the environment. The trophozoite form can infect the cornea and cause sight-threatening corneal inflammation known as Acanthamoeba keratitis. The pathogenic cascade of Acanthamoeba keratitis begins when Acanthamoebae bind to mannose expressed on traumatized corneas. Published reports indicate that mannose is upregulated on the corneal surface during wound healing. Experiments in laboratory animals have shown that corneal abrasion prior to infection is essential for generating Acanthamoeba keratitis. Furthermore, supernatants from AcanthamoebaItem 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 Protein Composition of the Chlamydomonas Flagellar Membrane Is Dynamically Regulated by Cilium-Generated Signaling During Fertilization(2012-07-09) Belzile, Olivier; Snell, William J.The cellular and molecular mechanisms specifying the membrane protein composition of cilia and flagella at steady state and during cilium-generated signaling are poorly understood. Our laboratory uses the biflagellated green alga Chlamydomonas as a model system to study regulated movement of the flagellar adhesion receptor SAG1 from the cell body to the flagella. Interactions between the plus flagellar receptor (agglutinin) SAG1 and its cognate receptor SAD1 on flagella of minus gametes induces flagellar adhesion and activation of a cAMP-dependent signaling pathway ultimately leading to cell-cell fusion. Although previous work from our laboratory and others suggested that pathway activation triggers mobilization of a pool of SAG1 from the cell body to the flagella, those studies depended on indirect adhesion bioassays detecting the activity of SAG1, not the protein. Here, I report use of new tools to study directly the regulation of SAG1 localization. I show that the SAG1 gene bearing a C-terminal HA tag rescues flagellar adhesion and cell fusion in the flagellar adhesion mutant, sag1-5. Immunofluorescence studies of resting SAG1-HA/sag1-5 mt+ plus gametes show that the protein is present mostly on cell bodies. Biochemical studies show that only gametes express SAG1-HA. Detection of precursor forms indicates SAG1 undergoes cleavage soon after its synthesis to yield an HA-tagged 65 kDa, C-terminal portion (SAG1-HA-C65), and that SAG1-HA-C65 is on the cell surface. Consistent with the predicted 3 transmembrane domains at the C-terminus, release of SAG1-HA-C65 in a soluble form requires detergent and is not achieved upon mechanical disruption, high salt, or high pH treatments. Cell fractionation demonstrates that in resting gametes the majority of SAG1-HA-C65 is present on cell bodies and only a small amount is on flagella. Within minutes after signaling is triggered by flagellar adhesion, however, SAG1-HA-C65 is mobilized from the cell body to the flagella and the organelles become highly enriched in the protein, where it forms large detergent-resistant complexes. I show that Chlamydomonas can regulate the amount of SAG1 in the flagella through cilium-generated signaling, and thus provides the first system for studying regulation of the membrane protein composition of the cilium/flagellum in a biochemically tractable system.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.