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Deubiquitinating enzyme mutagenesis screens identify a USP43 driven HIF-1 transcriptional response [CRISPR]

ABSTRACT: The ubiquitination and proteasome-mediated degradation of Hypoxia Inducible Factors (HIFs) is central to metazoan oxygen-sensing, but the involvement of deubiquitinating enzymes (DUBs) in HIF signalling is less clear. Here, using a bespoke DUBs sgRNA library we conduct CRISPR/Cas9 mutagenesis screens to determine how DUBs are involved in HIF signalling. Alongside defining DUBs involved in HIF activation or suppression, we identify USP43 as a DUB required for efficient activation of a HIF response. USP43 is hypoxia regulated and selectively associates with the HIF-1 isoform, and while USP43 does not alter HIF-1 stability, it facilitates HIF-1 nuclear accumulation and binding to its target genes. Mechanistically, USP43 associates with 14-3-3 proteins in a hypoxia-dependent manner to increase the nuclear pool of HIF-1, independently of DUB activity. Together, our results unveil the DUB landscape in HIF signalling, and highlight the multifunctionality of DUBs, illustrating that they can provide important signalling roles outside of their catalytic activity.

ORGANISM(S): Homo sapiens

PROVIDER: GSE252651 | GEO | 2024/05/17

REPOSITORIES: GEO

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Deubiquitinating enzyme mutagenesis screens identify a USP43 driven HIF-1 transcriptional response [RNA-seq]

Project description:The ubiquitination and proteasome-mediated degradation of Hypoxia Inducible Factors (HIFs) is central to metazoan oxygen-sensing, but the involvement of deubiquitinating enzymes (DUBs) in HIF signalling is less clear. Here, using a bespoke DUBs sgRNA library we conduct CRISPR/Cas9 mutagenesis screens to determine how DUBs are involved in HIF signalling. Alongside defining DUBs involved in HIF activation or suppression, we identify USP43 as a DUB required for efficient activation of a HIF response. USP43 is hypoxia regulated and selectively associates with the HIF-1 isoform, and while USP43 does not alter HIF-1 stability, it facilitates HIF-1 nuclear accumulation and binding to its target genes. Mechanistically, USP43 associates with 14-3-3 proteins in a hypoxia-dependent manner to increase the nuclear pool of HIF-1, independently of DUB activity. Together, our results unveil the DUB landscape in HIF signalling, and highlight the multifunctionality of DUBs, illustrating that they can provide important signalling roles outside of their catalytic activity.

2024-05-17 | GSE252654 | GEO

Deubiquitinating enzymes and the proteasome regulate unique sets of ubiquitin substrates.

Project description:The ubiquitin-proteasome axis has been extensively explored at a system-wide level, but the impact of deubiquitinating enzymes (DUBs) on the ubiquitinome remains largely unknown. Using UbiSite technology and inhibitors, we have compared the contributions of the proteasome and DUBs on the ubiquitinome. We uncovered large differential dynamic Ub signalling networks with substrates and sites uniquely regulated by DUBs or by the proteasome, highlighting the role of DUBs in degradation-independent ubiquitin signalling. DUBs regulate substrates via nearly 40,000 unique sites. Moreover, we found that ubiquitin conjugated to SUMO2/3 forms a unidirectional proteasomal degradation signal with strikingly rapid kinetics compared to ubiquitin polymers only. We found that PARP1, is hyper ubiquitinated in response to DUB inhibition, increasing its enzymatic activity. Our findings highlight the key regulatory roles of DUBs on ubiquitin dynamics.

2022-05-23 | PXD030644 | Pride

Deubiquitinating enzymes and the proteasome regulate unique sets of ubiquitin substrates.

2022-05-23 | PXD027328 | Pride

Deubiquitinating enzymes and the proteasome regulate unique sets of ubiquitin substrates.

Project description:The ubiquitin-proteasome axis has been extensively explored at a system-wide level, but the impact of deubiquitinating enzymes (DUBs) on the ubiquitinome remains largely unknown. Using UbiSite technology and inhibitors, we have compared the contributions of the proteasome and DUBs on the ubiquitinome. We uncovered large differential dynamic Ub signalling networks with substrates and sites uniquely regulated by DUBs or by the proteasome, highlighting the role of DUBs in degradation-independent ubiquitin signalling. DUBs regulate substrates via nearly 40,000 unique sites. Moreover, we found that ubiquitin conjugated to SUMO2/3 forms a unidirectional proteasomal degradation signal with strikingly rapid kinetics compared to ubiquitin polymers only. We found that PARP1 is hyper ubiquitinated in response to DUB inhibition, increasing its enzymatic activity. Our findings highlight the key regulatory roles of DUBs on ubiquitin dynamics.

2022-05-23 | PXD027330 | Pride

Comprehensive landscape of active deubiquitinating enzymes profiled by advanced chemoproteomics

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

Characterisation of HIF-dependent alternative isoforms in pancreatic cancer

Project description:Intra-tumor hypoxia is a common feature in many solid cancers. Although transcriptional targets of hypoxia-inducible factors (HIFs) have been well characterized, alternative splicing or processing of pre-mRNA transcripts which occurs during hypoxia and subsequent HIF stabilization is much less understood. Here, we identify HIF-dependent alternative splicing events after whole transcriptome sequencing in pancreatic cancer cells exposed to hypoxia with and without downregulation of the aryl hydrocarbon receptor nuclear translocator (ARNT), a protein required for HIFs to form a transcriptionally active dimer.

2019-11-01 | GSE139673 | GEO

Expression data from SNU-449 cells transfected with USP48

Project description:Deubiquitinating enzymes (DUBs) are divided into seven subgroups based on sequence and structure and perform the roles of mediating the deubiquitination of substrate proteins, regulating protein function, and participating in various cellular life activities. Among them, the ubiquitin-specific protease family (USP) is the DUB subtype with the most members and structural diversity discovered so far. USP48 is a member of the USP family of ubiquitin-specific proteases and has been found to promote glioblastoma by deubiquitinating the Gli1 transcription factor and to promote the progression of non-small-cell lung cancer through inhibition of the Wnt/β-catenin signaling pathway. We used microarrays to detail changes in gene expression regulated by USP48 overexpression in HCC cells.

2024-04-20 | GSE264186 | GEO

alternative promoter array HeLa HIF1 ChIP-chip

Project description:DNA binding profiling of endogenous HIF1A on proximal promoters in human HeLa cells exposed to 1% oxygen (hypoxia), using normoxic cells (21% oxygen) as reference. Biological background: The Hypoxia Inducible Factor Family of transcription factors is proposed as the main orchestrator of the cellular response to hypoxia. HIFs are heterodimers of a HIF alpha and a HIF beta subunit. HIF alpha protein stability is regulated by oxygen-dependent proteasomal degradation, and hence HIFs are strongly stabilized in hypoxia. Purpose of the study: a number of HIF1 ChIP-chip studies have been reported, employing various cell types, array platforms and HIF antibodies, and the overlap of HIF binding locations in these studies is relatively small. The aim of this study was to characterize HIF1 binding in an additional cell line (HeLa), and employing a different HIFalpha antibody. Experimental design: We conducted a total of six hybridizations employing four biological replicates. For two biological replicates, we performed dye-swap technical replicate experiments. Results summary: We identified 55 HIF binding locations in HeLa cells (FDR<2%). While this number is relatively low compared to previous studies, presumably due to limiting antibody sensitivity, the overlap with data from other cell lines is comparable to our HeLa data.

2012-05-20 | E-MEXP-3499 | biostudies-arrayexpress

Proteomics-based identification of DUBsubstrates using selective inhibitors

Project description:Deubiquitinating enzymes (DUBs) catalyze the removal of ubiquitin, thereby reversing the activity of E3 ubiquitin ligases and are central to the control of protein abundance and function. Despite the growing interest in DUBs as therapeutic targets, cellular functions for DUBs remain largely unknown and technical challenges often preclude the identification of DUB substrates in a comprehensive manner. Here we demonstrate that recently developed potent DUB inhibitors coupled to mass spectrometry-based proteomics can identify DUB substrates at a proteome-wide scale. We applied this methodology to USP7, a DUB widely investigated as a therapeutic target and identified novel substrates. We demonstrate that USP7 substrates are enriched for DNA repair enzymes and E3 ubiquitin ligases. This work provides not only a comprehensive annotation of USP7 substrates, but a general methodology widely applicable to other DUBs, which is critical for translational development of DUB targeted agents.

2021-01-18 | PXD020882 | Pride

Proteomics-based identification of DUBsubstrates using selective inhibitors

2021-01-18 | PXD020865 | Pride

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