Comprehensive landscape of active deubiquitinating enzymes profiled by advanced chemoproteomics
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA556874 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:Enzymes that bind and process ubiquitin, a small 76 amino acid protein, have been recognized as pharmacological targets in oncology, immunological disorders and neurodegeneration. Mass spectrometry technology has now reached the capacity to cover the proteome with enough depth to interrogate entire biochemical pathways including those that contain DUBs and E3 ligase substrates. We have recently characterized the breast cancer cell (MCF7) deep proteome by detecting and quantifying ~10,000 proteins, and within this data set, we can detect endogenous expression of 65 deubiquitylating enzymes (DUBs), whereas matching transcriptomics detected 78 DUB mRNAs. Since enzyme activity provides another meaningful layer of information in addition of the expression levels, we have combined advanced mass spectrometry technology, pre-fractionation and more potent/selective ubiquitin active-site probes with propargyl based electrophiles to profile 74 DUBs including distinguishable isoforms for five DUBs in MCF7 crude extract material. Competition experiments with cysteine alkylating agents, pan-DUB inhibitors ubiquitin combined with probe labelling revealed the proportion of active cellular DUBs directly engaged with probes by label-free quantitative (LFQ) mass spectrometry, demonstrating that USP13, 39 and USP40 are non-reactive to probe, reflecting no, low or restricted enzymatic activity under these cellular conditions. Our extended chemoproteomics workflow increases depth of covering the active DUBome, including isoform-specific resolution, and provides the framework for more comprehensive cell-based small molecule DUB selectivity profiling.
2019-08-31 | GSE134954 | GEO
Project description:Enzymes that bind and process ubiquitin, a small 76 amino acid protein, have been recognized as pharmacological targets in oncology, immunological disorders and neurodegeneration. Mass spectrometry technology has now reached the capacity to cover the proteome with enough depth to interrogate entire biochemical pathways including those that contain DUBs and E3 ligase substrates. We have recently characterized the breast cancer cell (MCF7) deep proteome by detecting and quantifying ~10,000 proteins, and within this data set, we can detect endogenous expression of 65 deubiquitylating enzymes (DUBs), whereas matching transcriptomics detected 78 DUB mRNAs. Since enzyme activity provides another meaningful layer of information in addition of the expression levels, we have combined advanced mass spectrometry technology, pre-fractionation and more potent/selective ubiquitin active-site probes with propargylic based electrophiles to profile 74 DUBs including distinguishable isoforms for five DUBs in MCF7 crude extract material. Competition experiments with cysteine alkylating agents, pan-DUB inhibitors combined with probe labelling revealed the proportion of active cellular DUBs directly engaged with probes by label-free quantitative (LFQ) mass spectrometry. This demonstrated that USP13, 39 and 40 are non-reactive to probe, indicating restricted enzymatic activity under these cellular conditions. Our extended chemoproteomics workflow increases depth of covering the active DUBome, including isoform-specific resolution, and provides the framework for more comprehensive cell-based small molecule DUB selectivity profiling.
2019-11-12 | PXD014391 | Pride
Project description: Not available
| S-EPMC6727631 | biostudies-literature
Project description:Comprehensive Profiling of Cancer Cells and Their Microenvironment in Advanced NSCLC
| PRJNA622993 | ENA
Project description:A precise understanding of DNA methylation dynamics on a genome wide scale is of great importance for the comprehensive investigation of a variety of biological processes such as reprogramming of somatic cells to iPSCs, cell differentiation and also cancer development. To date, a complex integration of multiple and distinct genome wide data sets is required to derive the global activity of DNA modifying enzymes. We present GwEEP - Genome-wide Epigenetic Efficiency Profiling as a versatile approach to infer dynamic efficiency changes of DNA modifying enzymes at base pair resolution on a genome wide scale. GwEEP relies on genome wide oxidative Hairpin Bisulfite sequencing (HPoxBS) data sets, which are translated by a sophisticated hidden Markov model into quantitative enzyme efficiencies with reported confidence around the estimates. GwEEP in its present form predicts de novo and maintenance methylation efficiencies of Dnmts, as well as the hydroxylation efficiency of Tets but its purposefully flexible design allows to capture further oxidation processes such as formylation and carboxylation given available data in the future. Applied to a well characterized ES cell model, GwEEP precisely predicts the complex epigenetic changes following a Serum-to-2i shift i.e., (i) instant reduction in maintenance efficiency (ii) gradually decreasing de novo methylation efficiency and (iii) increasing Tet efficiencies. In addition, a complementary analysis of Tet triple knock-out ES cells confirms the previous hypothesized mutual interference of Dnmts and Tets. GwEEP is applicable to a wide range of biological samples including cell lines, but also tissues and primary cell types.
2023-03-01 | GSE169070 | GEO
Project description:Characterizing Advanced Breast Cancer Heterogeneity and Treatment Resistance through Serial Biopsies and Comprehensive Analytics
| PRJNA688545 | ENA
Project description:Characterizing Advanced Breast Cancer Heterogeneity and Treatment Resistance through Serial Biopsies and Comprehensive Analytics
| PRJNA688546 | ENA
Project description:Ubiquitination is a reversible protein modification involved in various cellular processes in eukaryotic cells. Deubiquitinating enzymes, the proteins responsible for the removal of ubiquitin, act as essential regulators to maintain ubiquitin homeostasis and to exquisitely regulate protein degradation via the ubiquitination pathway. Cryptococcus neoformans is an important basidiomycete pathogen that causes life-threatening meningoencephalitis primarily within the immunocompromised population. In order to understand the possible influence deubiquitinating enzymes have on growth and virulence of the model pathogenic yeast Cryptococcus neoformans, we generated deletion mutants of 7 putative deubiquitinase genes. Compared to other deubiquitinating enzyme mutants, a ubp5M-bM-^HM-^F mutant exhibited severely attenuated virulence and many distinct phenotypes, including decreased capsule formation, hypomelanization, defective sporulation, and elevated sensitivity to several external stressors (such as high temperature, oxidative and nitrosative stresses, high salts, and antifungal agents). Ubp5 appears to be the major deubiquitinating enzyme in C. neoformans for maintaining a pool of free ubiquitin for stress responses, supports the evolutionary divergence of Cryptococcus sp. from the model yeast S. cerevisiae, and provides an important paradigm for understanding the potential role of deubiquitination in virulence by other pathogenic fungi. In addition, other putative deubiquitinase mutants (doa4M-bM-^HM-^F and ubp13M-bM-^HM-^F) exhibit similar phenotypes to the ubp5M-bM-^HM-^F mutant, illustrating possible functional overlap among deubiquitinating enzymes in C. neoformans. Certain functioning deubiquitinating enzymes are essential for the virulence composite of C. neoformans and provide an additional yeast survival and propagation advantage in the host. In this study, transcription profiles of ubp5 mutant and WT Cryptococcus neoformans strains were compared in a dye-swap experiment following 1hr exposure to either 30C or 37C.
2012-04-10 | E-GEOD-35067 | biostudies-arrayexpress
Project description:Ubiquitination is a reversible protein modification involved in various cellular processes in eukaryotic cells. Deubiquitinating enzymes, the proteins responsible for the removal of ubiquitin, act as essential regulators to maintain ubiquitin homeostasis and to exquisitely regulate protein degradation via the ubiquitination pathway. Cryptococcus neoformans is an important basidiomycete pathogen that causes life-threatening meningoencephalitis primarily within the immunocompromised population. In order to understand the possible influence deubiquitinating enzymes have on growth and virulence of the model pathogenic yeast Cryptococcus neoformans, we generated deletion mutants of 7 putative deubiquitinase genes. Compared to other deubiquitinating enzyme mutants, a ubp5∆ mutant exhibited severely attenuated virulence and many distinct phenotypes, including decreased capsule formation, hypomelanization, defective sporulation, and elevated sensitivity to several external stressors (such as high temperature, oxidative and nitrosative stresses, high salts, and antifungal agents). Ubp5 appears to be the major deubiquitinating enzyme in C. neoformans for maintaining a pool of free ubiquitin for stress responses, supports the evolutionary divergence of Cryptococcus sp. from the model yeast S. cerevisiae, and provides an important paradigm for understanding the potential role of deubiquitination in virulence by other pathogenic fungi. In addition, other putative deubiquitinase mutants (doa4∆ and ubp13∆) exhibit similar phenotypes to the ubp5∆ mutant, illustrating possible functional overlap among deubiquitinating enzymes in C. neoformans. Certain functioning deubiquitinating enzymes are essential for the virulence composite of C. neoformans and provide an additional yeast survival and propagation advantage in the host. New Zealand White rabbits were inoculated with WT strain H99 and harvested by intracisternal spinal tap on days 1, 3, 4, and 7. For the human sample, RNA was extracted from CSF obtained from a de-identified patient with cryptococcal meningitis.
2012-04-10 | E-GEOD-36183 | biostudies-arrayexpress
Project description:Ubiquitination is a reversible protein modification involved in various cellular processes in eukaryotic cells. Deubiquitinating enzymes, the proteins responsible for the removal of ubiquitin, act as essential regulators to maintain ubiquitin homeostasis and to exquisitely regulate protein degradation via the ubiquitination pathway. Cryptococcus neoformans is an important basidiomycete pathogen that causes life-threatening meningoencephalitis primarily within the immunocompromised population. In order to understand the possible influence deubiquitinating enzymes have on growth and virulence of the model pathogenic yeast Cryptococcus neoformans, we generated deletion mutants of 7 putative deubiquitinase genes. Compared to other deubiquitinating enzyme mutants, a ubp5M-bM-^HM-^F mutant exhibited severely attenuated virulence and many distinct phenotypes, including decreased capsule formation, hypomelanization, defective sporulation, and elevated sensitivity to several external stressors (such as high temperature, oxidative and nitrosative stresses, high salts, and antifungal agents). Ubp5 appears to be the major deubiquitinating enzyme in C. neoformans for maintaining a pool of free ubiquitin for stress responses, supports the evolutionary divergence of Cryptococcus sp. from the model yeast S. cerevisiae, and provides an important paradigm for understanding the potential role of deubiquitination in virulence by other pathogenic fungi. In addition, other putative deubiquitinase mutants (doa4M-bM-^HM-^F and ubp13M-bM-^HM-^F) exhibit similar phenotypes to the ubp5M-bM-^HM-^F mutant, illustrating possible functional overlap among deubiquitinating enzymes in C. neoformans. Certain functioning deubiquitinating enzymes are essential for the virulence composite of C. neoformans and provide an additional yeast survival and propagation advantage in the host. WT Cryptococcus neoformans strain H99 was incubated in pooled ex vivo human CSF obtained from de-identified patients and harvested after 24hr for RNA extraction. New Zealand White rabbits were inoculated with WT strain H99 and harvested by intracisternal spinal tap on days 1 and 7 for RNA extraction.
2012-04-10 | E-GEOD-36182 | biostudies-arrayexpress