Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

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

ABSTRACT: 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.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Trypanosoma Brucei

SUBMITTER: Michele Tinti

LAB HEAD: Sir Michael Ferguson

PROVIDER: PXD007115 | Pride | 2019-09-17

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Publications

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

Tinti Michele M Güther Maria Lucia S MLS Crozier Thomas W M TWM Lamond Angus I AI Ferguson Michael A J MAJ

Wellcome open research 20191009

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 A ...[more]

PMID: 31681858

Similar Datasets

Global profiling and inhibition of protein lipidation in vector and host stages of the sleeping sickness parasite Trypanosoma brucei via chemical proteomics

Project description:The enzyme N-myristoyltransferase (NMT) catalyses the essential fatty acylation of substrate proteins with myristic acid in eukaryotes and is a validated drug target in the parasite Trypanosoma brucei, the causative agent of African trypanosomiasis (sleeping sickness). N-Myristoylation typically mediates membrane localisation of proteins and is essential to the function of many. However, only a handful of proteins are experimentally validated as N-myristoylated in T. brucei. Here, we perform metabolic labelling with an alkyne-tagged myristic acid analogue (“YnMyr”), enabling the capture of lipidated proteins in insect (PCF) and host (BSF) life stages of T. brucei. We further compare this with a longer chain palmitate analogue (“YnPal”) to explore the chain length-specific incorporation of fatty acids into proteins. Finally, we combine the alkynyl-myristate analogue with NMT inhibitors (Cpds 1 and 2) and quantitative chemical proteomics to globally define N-myristoylated proteins in the clinically relevant bloodstream form parasites.

2016-05-17 | PXD004053 | Pride

Mapping diversity in African trypanosomes using high resolution spatial proteomics

Project description:African trypanosomes are dixenous eukaryotic parasites that impose a significant human and veterinary disease burden on sub-Saharan Africa. Diversity between species and life-cycle stages is concomitant with distinct host and tissue tropisms within this group. Here, the spatial proteomes of two African trypanosome species, Trypanosoma brucei and Trypanosoma congolense, have been mapped, each in mammalian and insect life-stages represented by bloodstream form (BSF) and procyclic form (PCF) respectively. Using the hyperLOPIT (hyperplexed localisation of organelle proteins by isotope tagging) methodology, this work has provided four highly comprehensive spatial proteomes.

2023-10-22 | PXD035426 | Pride

T. Brucei, Nup-1 Depletion

Project description:Organization of the genome into compacted chromatin is a eukaryotic innovation facilitating increased sophistication in transcriptional regulation. In metazoa coiled-coil lamin proteins are major components of the chromatin organizer at the nuclear periphery and maintain nuclear integrity. While identifiable lamin homologues are restricted to metazoans, morphologically analogous structures maintaining nuclear organization in other eukaryotic lineages are known, but the molecular constituents remain undefined. Trypanosoma brucei NUP-1 is a large coiled-coil protein associated with fibrils at the inner face of the nuclear envelope. Using transcriptome analysis in combination with RNA interference and various imaging techniques, we demonstrate that NUP-1 forms a stable immobile cage around the nucleus, is required for viability and nuclear structural integrity, directs the positional organization of nuclear pore complexes, and serves to organize chromatin and specifically repress genes located at the nuclear periphery involved in immune evasion. Based on architectural similarity and functionality, we propose that NUP-1 is a novel, highly divergent lamin The effect of Nup-1 depletion on the transcriptome was examined in three independent experiments (A, B, & C). T. brucei cultures were either treated with RNAi (plus) or left untreated (minus) and RNA was extracted from each sample at the indicated time point (0h, 6h, 12h, 24h, or 48h). Two color microarrays were performed comparing treated and untreated samples at each time point. Dye swaps were performed and are indicated. Replicates of t=12h and t=24h for sample B were also included.

2011-12-15 | E-GEOD-26256 | 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

The Trypanosoma brucei MISP family of invariant proteins are co-expressed with BARP as triple helical bundle structures on the surface of salivary gland forms, but is dispensable for parasite development within the tsetse vector

Project description:Trypanosoma brucei spp. develop into mammalian-infectious metacyclic trypomastigotes inside tsetse salivary glands. Besides acquiring a variant surface glycoprotein (VSG) coat, little is known about the metacyclic expression of invariant surface antigens. Proteomics analyses of saliva from T. brucei-infected flies identified, in addition to VSG and Brucei Alanine-Rich Protein (BARP) peptides, a family of GPIanchored surface proteins herein named Metacyclic Invariant Surface Proteins (MISP). The MISP family is encoded by five paralog genes with >80% protein identity, which are exclusively expressed by salivary gland stages of the parasite and peak in metacyclic stage, as shown by confocal microscopy and immuno-high resolution scanning electron microscopy. Crystallographic analysis of a MISP isoform (MISP360) and a high confidence model of BARP revealed a triple helical bundle architecture commonly found in other trypanosome surface proteins. Molecular modelling combined with live fluorescent microscopy suggests that MISP N-termini are extended above the VSG coat. However, vaccination with recombinant MISP360 isoform did not protect mice against a T. brucei infectious tsetse bite. Lastly, both RNAi knock down and CRISPR-Cas9-driven knock out of all MISP paralogues suggest they are not essential for parasite development in the tsetse vector. We speculate that MISP may be relevant during trypanosome inoculation or establishment in the vertebrate’s skin.

2023-05-10 | PXD039684 | Pride

Genome-scale RNA interference profiling of Trypanosoma brucei sensitivity determinants for experimental phthalimide-based trypanocidal drug PHT-39

Project description:In a phenotypic screening approach of novel molecules composed of a synergistic combination of phthalimide, benzimidazole, and triazole scaffolds we discovered compounds with potent anti-leishmanial activity. The resulting early-lead compound PHT-39, which contains a trifluoromethyl substitution, demonstrated the highest efficacy in a Leishmania infantum intramacrophage assay, with an EC50 of 1.2+/- 3.2 μM.Cytotoxicity testing of PHT-39 in Hep-G2 cells indicated high selectivity of over 90-fold. To investigate the mechanism of action we carried out experiments in Trypanosoma brucei, which is also sensitive to PHT-39. Here we used a genome-wide RNAi library approach (PMID: 22278056; PMID: 21363968) to detect sensitivity determinants. This high-throughput phenotyping approach identified sensitivity determinants for PHT-39, which included a P-type ATPase that is crucial for the uptake of miltefosine and amphotericin, strongly indicating a shared route for cellular entry.

2024-03-08 | E-MTAB-13844 | biostudies-arrayexpress

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

Effect of truncation mutants of the trypanosomatid nuclear lamina protein NUP-1 on the whole cell proteome in T. brucei

Project description:The nuclear lamina has multiple functions, including maintaining nuclear structural integrity and differential gene expression. Correct spatial and temporal lamina assembly is essential to meet these and other roles. Recently, it emerged that multiple lamina systems exist that are likely products of independent origins, while all these systems share remarkably analogous functions. Several lamina proteins are known in trypanosomes, two of which, NUP-1 and NUP-2, are essential, coiled-coil proteins with a molecular mass 450 and 250 kDa, respectively. Sequence analysis indicates distinct quaternary structures when compared to the ~60 kDa lamin proteins of multiple lineages, including metazoa. To uncover organisational principles of the trypanosome lamina we generated NUP-1 deletion mutants (N=N-terminal domain; C= C-terminal domain; NC: fusion of the N- and C-terminal domain with entire repeat region deletedd))designed to identify domains of NUP-1 responsible for oligomerisation. We find that both N- and C-termini act as interaction domains and disruption of these interactions impacts additional components of the lamina, the nuclear envelope and nucleoporin TbNup98. By contrast there is remarkably little impact on transcription, crucially including silencing of telomeric variant surface glycoprotein genes. These data indicate that both terminal domains of NUP-1 have roles in assembling the trypanosome lamina and suggest an architecture distinct to the lamin system is based on a ‘hub and spoke’ configuration.

2021-06-03 | PXD019978 | Pride

Transcription profiling of T. brucei bloodstream and procyclic forms to look at differential expression of membrane-trafficking genes

Project description:To look at differential expression of membrane-trafficking genes, we extracted RNA from exponentially growing cultures of T. brucei bloodstream (BSF) and procyclic (PCF) forms, labelled and competitively hybridised the cDNA to the microarray. 8 arrays, representing 6 BSF and 5 PCF biological replicates, as well as dye swaps were used.

2008-06-26 | E-MEXP-1529 | biostudies-arrayexpress

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