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Phosphoproteomic analysis of mammalian infective Trypanosoma brucei subjected to heat shock suggest atypical mechanisms for thermotolerance

ABSTRACT: A phosphoproteomic analysis of heat shock response in the mammalian infective bloodstream form Trypanosoma brucei was conducted using SILAC-based quantitation. Treatment at 41 0C for 1h produced significantly altered phosphorylation at 193 sites and significantly altered the abundance of 20 proteins.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Trypanosoma Brucei

DISEASE(S): Trypanosomiasis

SUBMITTER: Michael Urbaniak

LAB HEAD: Michael D Urbaniak

PROVIDER: PXD016482 | Pride | 2020-03-31

REPOSITORIES: Pride

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			Action	DRS
	CB-HS-F1pep.raw	Raw
	CB-HS-F1phos.raw	Raw
	CB-HS-F2pep.raw	Raw
	CB-HS-F2phos.raw	Raw
	CB-HS-F3pep.raw	Raw

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Publications

Phosphoproteomic analysis of mammalian infective Trypanosoma brucei subjected to heat shock suggests atypical mechanisms for thermotolerance.

Ooi Cher P CP Benz Corinna C Urbaniak Michael D MD

Journal of proteomics 20200319

The symptoms of African sleeping sickness, caused by the parasite Trypanosoma brucei, can include periods of fever as high as 41 <sup>°</sup>C which triggers a heat shock response in the parasite. To capture events involved in sensing and responding to heat shock in the mammalian infective form we have conducted a SILAC-based quantitative proteomic and phosphoproteomic analysis of T. brucei cells treated at 41 <sup>°</sup>C for 1h. Our analysis identified 193 heat shock responsive phosphorylatio ...[more]

PMID: 32198071

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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

Organising the cell cycle in the absence transcriptional control: Dynamic phosphorylation co-ordinates the Trypanosoma brucei cell cycle post-transcriptionally

Project description:The cell division cycle of the unicellular eukaryote Trypanosome brucei is tightly regulated despite the paucity of transcriptional control that results from the arrangement of genes in polycistronic units and lack of dynamically regulated transcription factors. To identify the contribution of dynamic phosphorylation to T. brucei cell cycle control we have combined cell cycle synchronisation by centrifugal elutriation with SILAC quantitative phosphoproteomic analysis. Cell cycle regulated changes in phosphorylation site abundance (917 sites, average 5-fold change) were more widespread and of a larger magnitude than changes in protein abundance (443 proteins, average 2-fold change) and were mostly independent of each other. Hierarchical clustering of co-regulated phosphorylation sites according to their cell cycle profile revealed a bulk increase in phosphorylation occurs across the cell cycle, with a significant enrichment of known cell cycle regulators and RNA binding proteins (RBPs) within the largest clusters. Cell cycle regulated changes in essential cell cycle kinases is temporally co-ordinated with differential phosphorylation of components of the kinetochore and eukaryotic initiation factors, along with many RBPs not previously linked to the cell cycle such as eight PSP1-C terminal domain containing proteins. The temporal profiles demonstrate the importance of dynamic phosphorylation in co-ordinating progression through the cell cycle, and provides evidence that RBPs play a central role in post-transcriptional gene regulation of the T. brucei cell cycle.

2019-10-22 | PXD013488 | Pride

Single locus phosphoproteomics reveals phosphorylation of RPA-1 is required for generation of single-strand DNA following a break at a subtelomeric locus

Project description:Damage to the genetic material of the cell poses a universal threat to all forms of life. Central to the DNA damage response (DDR) is a phosphorylation signalling cascade that leads to the co-ordination of the cellular response to a DNA break. Identifying the proteins that are phosphorylated is crucial to understanding the mechanisms underlying this DDR. We have used SILAC-based quantitative phosphoproteomics to profile changes in phosphorylation site abundance following a single double strand break (DSB) at a chromosome internal locus and the subtelomeric bloodstream form expression site in Trypanosoma brucei. We report >6500 phosphorylation sites, including a core set of 211 DSB responsive phosphorylation sites. Along with phosphorylation of canonical DNA damage factors, we find that there is a striking distinction between the proteins phosphorylated in response to a chromosome internal DSB and one at the active BES and describe a single phosphorylation event on Replication factor A (RPA) 1 that is required for efficient resection at a bloodstream form expression site

2024-09-04 | PXD034455 | Pride

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

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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

Trypanosoma brucei whole cell proteome changes upon exposure to the aminomethyl-benzoxaborole AN3057

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2024-03-25 | PXD049328 | 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.

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Project description:p166 is a central component of a protein complex that is essential for mitochondrial genome segregation in Trypanosoma brucei. The last 34 aa (the C-tail) of p166 are absolutely essential for its function of p166. The prupose of this experiment is to find even putative binding partners interacting with p166. For this purpose, two CoIP experiments with a truncated soluble version of p166 were performed. One uses p166 with an intact C-tail whereas the C-tail is removed in a second experiment. Differences between the two CoIPs indicate potential interactors of the C-tail.

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Characterisation of a highly diverged putative mitochondrial ATP synthase peripheral stalk subunit b in Trypanosoma brucei

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