Mass Spectrometry of NOT gene-related purifications
Ontology highlight
ABSTRACT:
INSTRUMENT(S):
ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)
SUBMITTER: 
George Allen
LAB HEAD: Martine Collart
PROVIDER: PXD021580 | Pride | 2022-07-26
REPOSITORIES: Pride
Dataset's files
Items per page: 5 1 - 5 of 219 |
Similar Datasets
Project description:Kinetoplastids rely heavily on post-transcriptional mechanisms for control of gene expression, and on RNA-binding proteins that regulate mRNA splicing, translation and decay. Trypanosoma brucei ERBP1 (Tb927.10.14150) and ERBP2 (Tb927.9.9550) were previously identified as mRNA binding proteins that lack canonical RNA-binding domains. We here show that ERBP1 is associated with the endoplasmic reticulum, like ERBP2, and that the two proteins interact in vivo. Loss of ERBP1 from bloodstream-form T. brucei initially resulted in a growth defect but proliferation was restored after more prolonged cultivation. Results from a pull-down of tagged ERBP1 suggest that it preferentially binds to ribosomal protein mRNAs. The ERBP1 sequence resembles that of Saccharomyces cerevisiae Bfr1, which also localises to the endoplasmic reticulum and binds to ribosomal protein mRNAs. However, unlike Bfr1, ERBP1 does not bind to mRNAs encoding secreted proteins, and it is also not recruited to stress granules after starvation.
2020-01-16 | PXD016106 | Pride
Project description:An essential step in active host cell invasion by the obligate intracellular apicomplexan parasites is the formation of a moving junction (MJ), which joins both plasma membranes and connects the underlying host cortical cytoskeleton with the parasite actomyosin system. Invading Toxoplasma gondii secrete RON complex proteins from rhoptry organelles into the host cell which provide the MJ components on the cytoplasmic side of the host cell membrane. We investigated the role of an essential organelle-resident RON13 kinase and implicated in phosphorylation of rhoptry content. Structural biology and biochemistry demonstrated that RON13 is an atypical kinase inserted N-terminally in the organelle membrane and processed by the aspartyl protease 3. In the absence of RON13 function rhoptry discharge is unaffected but parasites fail to invade cells in vitro and become avirulent in a mouse model. Comparative phosphoproteomics revealed RON13-dependent phosphorylation of secreted RONs involved in anchoring the MJ in the host cell cytoskeleton.
2021-05-11 | PXD021516 | Pride
Project description:Kinetoplastids rely heavily on post-transcriptional mechanisms for control of gene expression, and on RNA-binding proteins that regulate mRNA splicing, translation and decay. Trypanosoma brucei ERBP1 (Tb927.10.14150) and ERBP2 (Tb927.9.9550) were previously identified as mRNA binding proteins that lack canonical RNA-binding domains. We here show that ERBP1 is associated with the endoplasmic reticulum, like ERBP2, and that the two proteins interact in vivo. Loss of ERBP1 from bloodstream-form T. brucei initially resulted in a growth defect but proliferation was restored after more prolonged cultivation. Results from a pull-down of tagged ERBP1 suggest that it preferentially binds to ribosomal protein mRNAs. The ERBP1 sequence resembles that of Saccharomyces cerevisiae Bfr1, which also localises to the endoplasmic reticulum and binds to ribosomal protein mRNAs. However, unlike Bfr1, ERBP1 does not bind to mRNAs encoding secreted proteins, and it is also not recruited to stress granules after starvation.
2020-05-27 | PXD016136 | Pride
Project description:In malaria parasites, evolution of parasitism has been linked to functional optimisation. Despite this optimisation, most members of a calcium-dependent protein kinase (CDPK) family show genetic redundancy during erythrocytic proliferation. To identify relationships between phospho-signalling pathways, we here screen 294 genetic interactions among protein kinases in Plasmodium berghei. This reveals a synthetic negative interaction between a hypomorphic allele of the protein kinase G (PKG) and CDPK4 to control erythrocyte invasion which is conserved in P. falciparum. CDPK4 becomes critical when PKG-dependent calcium signals are attenuated to phosphorylate proteins important for the stability of the inner membrane complex, which serves as an anchor for the acto-myosin motor required for motility and invasion. Finally, we show that multiple kinases functionally complement CDPK4 during erythrocytic proliferation and transmission to the mosquito vector. This study reveals how CDPKs are wired within a stage-transcending signalling network to control motility and host cell invasion in malaria parasites.
2018-09-17 | PXD011096 | Pride
Project description:Co-activator complexes regulate chromatin accessibility and transcription. SAGA (Spt-Ada-Gcn5 Acetyltransferase) is an evolutionary conserved multisubunit co-activator complex with modular organization. The core module of SAGA constitutes the structural heart, composed of a histone octamer like structure and two additional proteins. The central histone octamer like core structure consists of six histone fold domain (HFD)-containing proteins, forming three HF pairs (TADA1/TAF12, TAF6L/TAF9/9b, and TAF10/SUPT7L) in the mammalian SAGA complexes, to which adds SUPT3H, which contains an intramolecular HF pair. The yeast homologue of SUPT3H, called Spt3, was shown to interact genetically and biochemically with the TATA binding protein (TBP). Here we investigated the role of SUPT3H in human U2OS and mouse embryonic stem (ES) cells. Using immuno-purification coupled mass spectrometry experiments we show that both human and mouse SAGA can assemble without the double HFD-containing SUPT3H. Nascent RNA-seq experiments indicted that in either U2OS or mouse ESCs lacking SUPT3H only a small subset of genes is deregulated. Consequently, in mouse ESCs SUPT3H is not essential for mouse ESC survival, but is required for ESC growth and self-renewal. In addition, TBP recruitment experiments show no major change in TBP accumulation at gene promoters in the absence of SUPT3H in mouse and human cells. Taken together our data suggest in mammalian cells SUPT3H is not required for the assembly of SAGA, general TBP recruitment to genes and cell survival, but it is important for regulating a limited set of genes.
2022-07-26 | PXD026991 | Pride
Project description:Using strain NCTC 11168 in carbon (serine)-limited continuous cultures we report the first detailed examination of oxygen-dependent changes in gene and protein expression in Campylobacter jejuni under conditions where the growth rate is fixed. We show that in steady-states established at μ = 0.2 h-1 over a wide-range of oxygen inputs, a perceived aerobiosis scale can be calibrated by the acetate excretion flux, which becomes zero when metabolism is fully aerobic (100% aerobiosis = 5% v/v oxygen in the gas inflow). A label-free proteomic analysis compared protein abundance (reported as the emPAI ratio) at 40% aerobiosis (= 1.88% v/v oxygen in the gas inflow) and 150% aerobiosis (= 7.5% v/v oxygen in the gas inflow) aerobiosis. This identified 857 proteins. Of 223 proteins more abundant at 40% aerobiosis, those involved in host colonisation and alternative pathways of electron transport were prominent. At 150% aerobiosis, 129 proteins were more abundant, including those involved in oxidative stress protection, citric-acid cycle, lactate and proline oxidation.
2017-10-04 | PXD006467 | Pride
Project description:The client-specificity of TMX3, TMX4 and TMX5 in cellula, was assessed by expressing mutant forms of the enzymes, where the last cysteine residue of the TMX’s CXXC catalytic sites has been mutated to alanine, which stabilizes the mixed disulfide that oxidoreductases establish with clients. Clients remain disulfide-bonded to the oxidoreductase and are identified upon co-immunoprecipitation and mass spectrometry analyses.
2023-06-12 | PXD041156 | Pride
Project description:The endoplasmic reticulum (ER) is the organelle of nucleated cells that produces lipids, sugars and proteins. More than 20 ER-resident members of the Protein Disulfide Isomerase (PDI) family regulate formation, isomerization and disassembly of covalent bonds in newly synthesized polypeptides. The PDI family includes few membrane-bound members. Among these, TMX1, TMX2, TMX3, TMX4 and TMX5 belong to the thioredoxin-related transmembrane (TMX) protein family. TMX5 is the least known member of the family. Here, we establish that TMX5 covalently engages via its active site cysteine residue at position 220 a subset of secretory proteins, mainly single- and multi-pass Golgi-resident polypeptides. TMX5 also interacts non-covalently, and covalently, via non-catalytic cysteine residues, with the PDI family members PDI, ERp57 and ERp44. The association of TMX5 and ERp44 requires formation of a mixed disulfide between the catalytic cysteine residue 29 of ERp44 and the non-catalytic cysteine residues 114 and/or 124 of TMX5 and controls the ER retention of TMX5. Thus, TMX5 belongs to the family of proteins including Ero1, Ero1, Prx4, ERAP1, SUMF1 that do not display ER retention sequences and rely on ERp44 engagement for proper inter-compartmental distribution. The client-specificity of TMX5 in cellulo was assessed by expressing mutant forms of the enzymes, where the last cysteine residue of the TMX’s CXXC catalytic sites has been mutated to alanine, which stabilizes the mixed disulfide that oxidoreductases establish with clients. Clients remain disulfide-bonded to the oxidoreductase and are identified upon co-immunoprecipitation and mass spectrometry analyses.
2024-09-27 | PXD054716 | Pride
Project description:Ovarian cancer is a major cause of cancer mortality among women largely due to late diagnosis of advanced stage metastatic disease. More extensive molecular analysis of metastatic ovarian cancer is needed to identify post-translational modifications of proteins particularly associated with metastatic disease so that we can better understand the metastatic process and identify potential therapeutic targets. Proteins are the target of many recently developed cancer treatments, but an often neglected aspect of biomarker discovery is protein glycosylation, especially those involving tumor-associated carbohydrate antigens (TACAs) such as sialyl-Lewis(x) (SLe(x) and sialyl-Lewis(a) (SLe(a), which have been identified in various cancers. Although it is already known that considerable changes in protein glycosylation are major contributors to the initiation, progression and metastasis of tumors, specifics about glycosylation changes particularly important to the metastatic process are still lacking. In this report we describe the results of a combined glycomic and proteomic study of metastatic ovarian cancer (OC) ascites fluids. Glycoproteins in ascites fluid were enriched by affinity binding to lectins (ConA or WGA) and other affinity matrices. Separate glycomic and proteomic analyses were performed as well as glycopeptide analyses. Relative abundances of different N-glycan groups and proteins were identified from original ascites fluids and corresponding lectin bound samples. Levels of biomarkers CA125, MUC1 and fibronectin were also monitored in these samples by Western blot analysis. N-glycan analysis of ascites fluids showed the presence of large, highly fucosylated and sialylated, complex and hybrid glycans, some of which were not observed in normal serum. Proteins in OC ascites that were more abundant or not present in the serum control, were haptoglobin, fibronectin, lumican, fibulin, hemopexin, ceruloplasmin, alpha-1-antitrypsin and alpha-1-antichymotrypsin. Glycopeptide analysis identified N- and O-glycans in clusterin, hemopexin, and fibulin that were present in OC ascites.
2016-08-15 | PXD003794 | Pride
Project description:Tandem affinity purification of MGA-interacting proteins and mass spectrometry
2021-07-06 | PXD025930 | Pride