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

ABSTRACT: 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.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Trypanosoma Brucei

SUBMITTER: Michael Urbaniak

LAB HEAD: Michael Urbaniak

PROVIDER: PXD000663 | Pride | 2014-05-13

REPOSITORIES: Pride

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			Action	DRS
	200313-2LG1-F3-F7.raw	Raw
	200313-2LG10-F18-F22.raw	Raw
	200313-2LG11-F3-F7.raw	Raw
	200313-2LG12-F8-F9.raw	Raw
	200313-2LG13-F10.raw	Raw

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Publications

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

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Nucleotide sugar biosynthesis occurs in the glycosomes of procyclic and bloodstream form Trypanosoma brucei

Project description:In Trypanosoma brucei, there are fourteen enzymatic biotransformations that collectively convert glucose into five essential nucleotide sugars: UDP-Glc, UDP-Gal, UDP-GlcNAc, GDP-Man and GDP-Fuc. These biotransformations are catalyzed by thirteen discrete enzymes, five of which possess putative peroxisome targeting sequences. Published experimental analyses using immunofluorescence microscopy and/or digitonin latency and/or subcellular fractionation and/or organelle proteomics have localized eight and six of these enzymes to the glycosomes of bloodstream form and procyclic form T. brucei, respectively. Here we increase these glycosome localizations to eleven in both lifecycle stages while noting that one, phospho-N-acetylglucosamine mutase, also localizes to the cytoplasm. In the course of these studies, the heterogeneity of glycosome contents was also noted. These data suggest that, unlike other eukaryotes, all of nucleotide sugar biosynthesis in T. brucei is compartmentalized to the glycosomes in both lifecycle stages.

2021-01-28 | PXD023124 | Pride

Genomic and Proteomic Studies on the Mode of Action of Oxaboroles against the African Trypanosome

Project description:SCYX-7158, an oxaborole, is currently in Phase I clinical trials for the treatment of human African trypanosomiasis. Here we investigate possible modes of action against Trypanosoma brucei using a chemo-proteomic approach. SILAC-based proteomic studies using an oxaborole analogue immobilised onto a resin was used either in competition with a soluble oxaborole or an immobilised inactive control to identify thirteen proteins common to both strategies. The diverse list of binding partners from the proteomic studies suggest a degree of polypharmacology that should reduce the risk of resistance to this compound class emerging in the field.

2015-11-25 | PXD002848 | Pride

Cell differentiation in Trypanosoma brucei is a bistable developmental switch requiring new protein synthesis to achieve signal memory

Project description:The life cycle of Trypanosoma brucei involves several cell differentiation transitions that allow transmission, survival and proliferation of these parasites. One of these transitions, the differentiation of growth-arrested stumpy forms in the mammalian blood into proliferating insect-stage procyclic forms, can be induced synchronously in vitro by addition of cis-aconitate (CA). Using single-cell analysis by flow-cytometry to follow differentiation, we show that this transition is an irreversible bistable switch where cells commit to differentiation after 1-3 hours of exposure to CA. This irreversibility implies the existence of positive feedback mechanisms that allow commitment to differentiation: i.e. the establishment of “memory” of exposure to the differentiation signal. Such mechanisms probably depend on post-translational modifications (e.g. phosphorylation) and/or synthesis of regulatory proteins. Using the reversible protein synthesis inhibitor cycloheximide, we find that protein synthesis is required for establishment of signal memory and normal commitment to differentiation. To characterize the ‘commitment proteome’, we performed SILAC phosphoproteomics to provide a detailed map of the protein expression and phosphorylation events during the early stages of differentiation in a synchronised parasite population. Using a rigorous candidate gene approach we have also demonstrated that the stumpy form enriched serine-throenine protein kinases TbNRKA/B stringently control the earliest events in differentiation identifying these kinases as major regulators of trypanosome development.

2018-05-08 | PXD002165 | Pride

Proteomic identification of the UDP-GlcNAc : PI α1-6 GlcNAc-transferase subunits of the glycosylphosphatidylinositol biosynthetic pathway of Trypanosoma brucei.

Project description:The first step of glycosylphosphatidylinositol (GPI) anchor biosynthesis in all eukaryotes is the addition of N-acetylglucosamine (GlcNAc) to phosphatidylinositol (PI) which is catalysed by a UDP-GlcNAc : PI α1-6 GlcNAc-transferase. This enzyme has been shown to be a complex of at least seven subunits in mammalian cells and a similar complex of homologous subunits has been postulated in yeast. Homologs of most of these mammalian and yeast subunits were identified in the Trypanosoma brucei predicted protein database. The putative catalytic subunit of the T. brucei complex,TbGPI3, was epitope tagged with three consecutive c-Myc sequences at its C-terminus. Immunoprecipitation of TbGPI3-3Myc followed by native polyacrylamide gel electrophoresis and anti-Myc Western blot showed that it is present in a ~240 kDa complex. Label-free quantitative proteomics were performed to compare anti-Myc pull-downs from lysates of TbGPI-3Myc expressing and wild type cell lines. TbGPI3-3Myc was the most highly enriched protein in the TbGPI3-3Myc lysate pull-down and partner proteins TbGPI15, TbGPI9, TbGPI2, TbGPI1 and TbERI1 were also identified with significant enrichment. Our proteomics data also suggest that an Arv-1 like protein (TbArv1) is a subunit of the T. brucei complex. Yeast and mammalian Arv1 have been previously implicated in GPI biosynthesis, but here we present the first experimental evidence for physical association of Arv1 with GPI biosynthetic machinery. A putative E2-ligase has also been tentatively identified as part of the T. brucei UDP-GlcNAc : PI α1-6 GlcNAc-transferase complex.

2021-02-26 | PXD022979 | Pride

Proteome Turnover in Bloodstream and Procyclic form Trypanosoma brucei Measured by Quantitative Proteomics

Project description: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 look-up 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 human and animal African trypanosomiasis.

2019-09-17 | PXD007115 | Pride

An Msp1-containing complex removes orphaned proteins in the mitochondrial outer membrane of trypanosomes (SILAC-IP data)

Project description:The AAA-ATPase Msp1 extracts mislocalized outer membrane proteins and thus contributes to mitochondrial proteostasis. Using pull down experiments, we show that trypanosomal Msp1 localizes to both the glycosome and the mitochondrial outer membrane, where it forms a stable complex with four outer membrane proteins. The trypanosome-specific pATOM36 mediates complex assembly of alpha-helically anchored mitochondrial outer membrane proteins, such as protein translocase subunits. Inhibition of complex assembly triggers a pathway that results in the proteasomal digestion of unassembled substrates. Using inducible single, double and triple RNAi cell lines combined with proteomic analyses, we demonstrate that both TbMsp1 and the trypanosomal homolog of a cytosolic AAA-ATPase, VCP, are implicated in this quality control pathway. Moreover, in the absence of TbVCP, three out of the four TbMsp1-interacting mitochondrial proteins are required for efficient proteasomal digestion of pATOM36 substrates, suggesting they act in concert with TbMsp1. pATOM36 is a functional analogue of the yeast MIM complex, and possibly of human MTCH2, suggesting that similar mitochondrial quality control pathways linked to Msp1 might also exist in yeast and humans.

2023-07-26 | PXD039631 | Pride

Mode of action studies for a novel anti-leishmania compound

Project description:We describe the development of a novel anti-leishmanial drug-like chemical series based on a pyrazolopyrimidine scaffold. The leading compound is efficacious in a mouse model of visceral leishmaniasis, and has suitable physicochemical, pharmacokinetic and toxicological properties for further development and has been declared a preclinical candidate. Detailed mode of action studies indicate that compounds from this series act principally by inhibiting the parasite protein kinases.

2018-11-30 | PXD009711 | Pride

Immunoisolation of the trypanosome decapping enzyme ALPH1 delivers a potential subcomplex

Project description:Removal of mRNA 5’ caps primes transcripts for degradation and is central for regulating gene expression in eukaryotes. The canonical decapping enzyme DCP2 is stringently controlled by assembly into a dynamic multi-protein complex together with the 5´-3´exoribonuclease Xrn1. Kinetoplastida lack DCP2 orthologues but instead rely on the ApaH-like phosphatase ALPH1 for decapping. The enzyme is composed of a catalytic domain flanked by C-terminal and N-terminal extensions. In our related deposition PXD038550 we analysed the ALPH1 interactome by BioID proximity labelling for the full length protein and truncated versions in order to assign domain specific interactions. We showed that Trypanosoma brucei ALPH1 acts in a complex composed of the trypanosome XRN1 ortholog XRNA and four proteins that are unique to Kinetoplastida. The interactome was validated by reverse experiments targeting T. brucei and T. cruzi XRNA by affinity capture and, additionally, the ALPH1 interacting CMGC-family kinase by BioID. Here we carried out affinity capture with T. brucei and T. cruzi ALPH1, which delivers a potential sub-complex missing the C-terminal interactor XRNA.

2023-06-14 | PXD042322 | Pride

Transcription profiling of procyclic Trypanosoma brucei cultured at 27oC or under heat shock stress conditions at 41oC

Project description:Procyclic trypanosomes (strain 427 lister) were grown in culture under standard conditions at 27ºC in SDM79 medium with 10% foetal bovine serume (Brun and Schnenberger, 1979), in a gazed incubator (5% CO2). Logarithmically growing procyclic cells (at about 5*10^6 cells/ml, at 27°C) were added to one volume medium that had been heated to 53°C and incubated at 41ºC for 60 minutes in a waterbath in a closed tube (41ºC sample). The control cells were added to one volume medium at 27ºC and also incubated for 60 minutes in a closed tube at 27°C. Cells were harvested and washed once in PBS. The harvesting was done within 8 minutes.

2008-09-10 | E-MEXP-1476 | biostudies-arrayexpress

Proximity labelling and cryomill affinity purification compared for Trypanosoma brucei proteins with unconfined, semi-confined and confined localisation

Project description:Identification of protein-protein interactions is a major contributor for understanding protein function and elucidating molecular and cellular mechanisms. Two strategies are currently popular for identification of protein interaction partners directly from the cellular environment. Affinity purification (pulldown) isolates the protein of interest with the aid of a matrix that specifically captures the target and potential partners. In BioID, the protein of interest is fused to biotin ligase facilitating proximity biotinylation and biotinylated proteins are isolated under stringent conditions with streptavidin. Whilst clear that these two methods can provide insight, it is also apparent that they can reveal distinct interactomes, but the basis for these differences is less obvious. We compare these methods using four different trypanosome proteins as baits: the cytoplasmic poly(A) binding proteins PABP1 and PABP2, mRNA export receptor MEX67, that shuttles between the nucleus and the cytoplasm with predominant localisation at the nuclear pores, and the nucleoporin NUP158.

2022-11-30 | PXD031245 | Pride

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