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High-confidence glycosome proteome for procyclic form Trypanosoma brucei by epitope-tag organelle enrichment and SILAC proteomics.

ABSTRACT: The glycosome of the pathogenic African trypanosome Trypanosoma brucei is a specialized peroxisome that contains most of the enzymes of glycolysis and several other metabolic and catabolic pathways. The contents and transporters of this membrane-bounded organelle are of considerable interest as potential drug targets. Here we use epitope tagging, magnetic bead enrichment, and SILAC quantitative proteomics to determine a high-confidence glycosome proteome for the procyclic life cycle stage of the parasite using isotope ratios to discriminate glycosomal from mitochondrial and other contaminating proteins. The data confirm the presence of several previously demonstrated and suggested pathways in the organelle and identify previously unanticipated activities, such as protein phosphatases. The implications of the findings are discussed.

SUBMITTER: Guther ML

PROVIDER: S-EPMC4052807 | biostudies-other | 2014 Jun

REPOSITORIES: biostudies-other

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High-confidence glycosome proteome for procyclic form Trypanosoma brucei by epitope-tag organelle enrichment and SILAC proteomics.

Güther Maria Lucia S ML Urbaniak Michael D MD Tavendale Amy A Prescott Alan A Ferguson Michael A J MA

Journal of proteome research 20140513 6

The glycosome of the pathogenic African trypanosome Trypanosoma brucei is a specialized peroxisome that contains most of the enzymes of glycolysis and several other metabolic and catabolic pathways. The contents and transporters of this membrane-bounded organelle are of considerable interest as potential drug targets. Here we use epitope tagging, magnetic bead enrichment, and SILAC quantitative proteomics to determine a high-confidence glycosome proteome for the procyclic life cycle stage of the ...[more]

PMID: 24792668

Similar Datasets

A high-confidence glycosome proteome for procyclic form Trypanosoma brucei by epitope-tag organelle enrichment and SILAC

Project description:Epitope tagging and magnetic bead enrichment were combined with SILAC quantitative proteomics to determine a high-confidence glycosome proteome for the procyclic life cycle stage of the parasite Trypanosoma brucei.

2014-05-13 | PXD000663 | Pride

Proteome turnover in the bloodstream and procyclic forms of <i>Trypanosoma brucei</i> measured by quantitative proteomics.

Project description:Background: Cellular proteins vary significantly in both abundance and turnover rates. These parameters depend upon their rates of synthesis and degradation and it is useful to have access to data on protein turnover rates when, for example, designing genetic knock-down experiments or assessing the potential usefulness of covalent enzyme inhibitors. Little is known about the nature and regulation of protein turnover in Trypanosoma brucei, the etiological agent of human and animal African trypanosomiasis. Methods: To establish baseline data on T. brucei proteome turnover, a Stable Isotope Labelling with Amino acids in Cell culture (SILAC)-based mass spectrometry analysis was performed to reveal the synthesis and degradation profiles for thousands of proteins in the bloodstream and procyclic forms of this parasite. Results: This analysis revealed a slower average turnover rate of the procyclic form proteome relative to the bloodstream proteome. As expected, many of the proteins with the fastest turnover rates have functions in the cell cycle and in the regulation of cytokinesis in both bloodstream and procyclic forms. Moreover, the cellular localization of T. brucei proteins correlates with their turnover, with mitochondrial and glycosomal proteins exhibiting slower than average turnover rates. Conclusions: The intention of this study is to provide the trypanosome research community with a resource for protein turnover data for any protein or group of proteins. To this end, bioinformatic analyses of these data are made available via an open-access web resource with data visualization functions.

| S-EPMC6816455 | biostudies-literature

Lipidomic analysis of bloodstream and procyclic form Trypanosoma brucei.

Project description:The biological membranes of Trypanosoma brucei contain a complex array of phospholipids that are synthesized de novo from precursors obtained either directly from the host, or as catabolised endocytosed lipids. This paper describes the use of nanoflow electrospray tandem mass spectrometry and high resolution mass spectrometry in both positive and negative ion modes, allowing the identification of approximately 500 individual molecular phospholipids species from total lipid extracts of cultured bloodstream and procyclic form T. brucei. Various molecular species of all of the major subclasses of glycerophospholipids were identified including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol as well as phosphatidic acid, phosphatidylglycerol and cardolipin, and the sphingolipids sphingomyelin, inositol phosphoceramide and ethanolamine phosphoceramide. The lipidomic data obtained in this study will aid future biochemical phenotyping of either genetically or chemically manipulated commonly used bloodstream and procyclic strains of Trypanosoma brucei. Hopefully this will allow a greater understanding of the bizarre world of lipids in this important human pathogen.

| S-EPMC3744936 | biostudies-literature

Triacylglycerol Storage in Lipid Droplets in Procyclic Trypanosoma brucei.

Project description:Carbon storage is likely to enable adaptation of trypanosomes to nutritional challenges or bottlenecks during their stage development and migration in the tsetse. Lipid droplets are candidates for this function. This report shows that feeding of T. brucei with oleate results in a 4-5 fold increase in the number of lipid droplets, as quantified by confocal fluorescence microscopy and by flow cytometry of BODIPY 493/503-stained cells. The triacylglycerol (TAG) content also increased 4-5 fold, and labeled oleate is incorporated into TAG. Fatty acid carbon can thus be stored as TAG in lipid droplets under physiological growth conditions in procyclic T. brucei. β-oxidation has been suggested as a possible catabolic pathway for lipids in T. brucei. A single candidate gene, TFEα1 with coding capacity for a subunit of the trifunctional enzyme complex was identified. TFEα1 is expressed in procyclic T. brucei and present in glycosomal proteomes, Unexpectedly, a TFEα1 gene knock-out mutant still expressed wild-type levels of previously reported NADP-dependent 3-hydroxyacyl-CoA dehydrogenase activity, and therefore, another gene encodes this enzymatic activity. Homozygous Δtfeα1/Δtfeα1 null mutant cells show a normal growth rate and an unchanged glycosomal proteome in procyclic T. brucei. The decay kinetics of accumulated lipid droplets upon oleate withdrawal can be fully accounted for by the dilution effect of cell division in wild-type and Δtfeα1/Δtfeα1 cells. The absence of net catabolism of stored TAG in procyclic T. brucei, even under strictly glucose-free conditions, does not formally exclude a flux through TAG, in which biosynthesis equals catabolism. Also, the possibility remains that TAG catabolism is completely repressed by other carbon sources in culture media or developmentally activated in post-procyclic stages in the tsetse.

| S-EPMC4262433 | biostudies-literature

Chemical perturbation of Trypanosoma brucei procyclic form cells

Project description:T. brucei PF cells were treated with several chemical reagents and anti-trypanosomatid drugs. The effect of each chemical perturbation on the transcriptome of T. brucei was examined by transcript profiling of treated vs. control cells. The results indicated widespread changes, suggesting that the transcriptome of T. brucei is highly responsive to environmental factors that perturb its metabolic and biological pathways.

2012-05-30 | GSE37593 | GEO

Trypanosoma brucei mitochondrial respiratome: composition and organization in procyclic form.

Project description:The mitochondrial respiratory chain is comprised of four different protein complexes (I-IV), which are responsible for electron transport and generation of proton gradient in the mitochondrial intermembrane space. This proton gradient is then used by F?F?-ATP synthase (complex V) to produce ATP by oxidative phosphorylation. In this study, the respiratory complexes I, II, and III were affinity purified from Trypanosoma brucei procyclic form cells and their composition was determined by mass spectrometry. The results along with those that we previously reported for complexes IV and V showed that the respiratome of Trypanosoma is divergent because many of its proteins are unique to this group of organisms. The studies also identified two mitochondrial subunit proteins of respiratory complex IV that are encoded by edited RNAs. Proteomics data from analyses of complexes purified using numerous tagged component proteins in each of the five complexes were used to generate the first predicted protein-protein interaction network of the Trypanosoma brucei respiratory chain. These results provide the first comprehensive insight into the unique composition of the respiratory complexes in Trypanosoma brucei, an early diverged eukaryotic pathogen.

| S-EPMC3186196 | biostudies-literature

Regulation of Fructose 1,6-Bisphosphatase in Procyclic Form Trypanosoma brucei.

Project description:Glycolysis is well described in Trypanosoma brucei, while the importance of gluconeogenesis and one of the key enzymes in that pathway, fructose 1,6-bisphosphatase, is less understood. Using a sensitive and specific assay for FBPase, we demonstrate that FBPase activity in insect stage, procyclic form (PF), parasite changes with parasite cell line, extracellular glucose levels, and cell density. FBPase activity in log phase PF 2913 cells was highest in high glucose conditions, where gluconeogenesis is expected to be inactive, and was undetectable in low glucose, where gluconeogenesis is predicted to be active. This unexpected relationship between FBPase activity and extracellular glucose levels suggests that FBPase may not be exclusively involved in gluconeogenesis and may play an additional role in parasite metabolism. In stationary phase cells, the relationship between FBPase activity and extracellular glucose levels was reversed. Furthermore, we found that monomorphic PF 2913 cells had significantly higher FBPase levels than pleomorphic PF AnTat1.1 cells where the activity was undetectable except when cells were grown in standard SDM79 media, which is glucose-rich and commonly used to grow PF trypanosomes in vitro. Finally, we observed several conditions where FBPase activity changed while protein levels did not, suggesting that the enzyme may be regulated via post-translational modifications.

| S-EPMC8157246 | biostudies-literature

Novel sterol metabolic network of Trypanosoma brucei procyclic and bloodstream forms.

Project description:Trypanosoma brucei is the protozoan parasite that causes African trypanosomiasis, a neglected disease of people and animals. Co-metabolite analysis, labelling studies using [methyl-2H3]-methionine and substrate/product specificities of the cloned 24-SMT (sterol C24-methyltransferase) and 14-SDM (sterol C14demethylase) from T. brucei afforded an uncommon sterol metabolic network that proceeds from lanosterol and 31-norlanosterol to ETO [ergosta-5,7,25(27)-trien-3β-ol], 24-DTO [dimethyl ergosta-5,7,25(27)-trienol] and ergosterol [ergosta-5,7,22(23)-trienol]. To assess the possible carbon sources of ergosterol biosynthesis, specifically 13C-labelled specimens of lanosterol, acetate, leucine and glucose were administered to T. brucei and the 13C distributions found were in accord with the operation of the acetate-mevalonate pathway, with leucine as an alternative precursor, to ergostenols in either the insect or bloodstream form. In searching for metabolic signatures of procyclic cells, we observed that the 13C-labelling treatments induce fluctuations between the acetyl-CoA (mitochondrial) and sterol (cytosolic) synthetic pathways detected by the progressive increase in 13C-ergosterol production (control<[2-(13)C]leucine<[2-(13)C]acetate<[1-(13)C]glucose) and corresponding depletion of cholesta-5,7,24-trienol. We conclude that anabolic fluxes originating in mitochondrial metabolism constitute a flexible part of sterol synthesis that is further fluctuated in the cytosol, yielding distinct sterol profiles in relation to cell demands on growth.

| S-EPMC3491665 | biostudies-literature

pH regulation in glycosomes of procyclic form <i>Trypanosoma brucei</i>.

Project description:Here we report the use of a fluorescein-tagged peroxisomal targeting sequence peptide (F-PTS1, acetyl-C{K(FITC)}GGAKL) for investigating pH regulation of glycosomes in live procyclic form Trypanosoma brucei When added to cells, this fluorescent peptide is internalized within vesicular structures, including glycosomes, and can be visualized after 30-60 min. Using F-PTS1 we are able to observe the pH conditions inside glycosomes in response to starvation conditions. Previous studies have shown that in the absence of glucose, the glycosome exhibits mild acidification from pH 7.4 ± 0.2 to 6.8 ± 0.2. Our results suggest that this response occurs under proline starvation as well. This pH regulation is found to be independent from cytosolic pH and requires a source of Na+ ions. Glycosomes were also observed to be more resistant to external pH changes than the cytosol; placement of cells in acidic buffers (pH 5) reduced the pH of the cytosol by 0.8 ± 0.1 pH units, whereas glycosomal pH decreases by 0.5 ± 0.1 pH units. This observation suggests that regulation of glycosomal pH is different and independent from cytosolic pH regulation. Furthermore, pH regulation is likely to work by an active process, because cells depleted of ATP with 2-deoxyglucose and sodium azide were unable to properly regulate pH. Finally, inhibitor studies with bafilomycin and EIPA suggest that both V-ATPases and Na+/H+ exchangers are required for glycosomal pH regulation.

| S-EPMC5427261 | biostudies-literature

RNAi of TbNMD3 in procyclic form Trypanosoma brucei lines

Project description:A procyclic form Trypansome brucei RNAi line (PTT parental line, transfected with pALC14 incorporating a TbNMD3 gene fragment) capable of inducing depletion of TbNMD3 was analysed for mRNA expression by RNAseq

2015-04-28 | GSE67246 | GEO

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