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Proteomics of broad deubiquitylase inhibition unmasks redundant enzyme function to reveal substrates and assess enzyme specificity.

ABSTRACT: Deubiquitylating enzymes (DUBs) counteract ubiquitylation to control stability or activity of substrates. Identification of DUB substrates is challenging because multiple DUBs can act on the same substrate, thwarting genetic approaches. Here, we circumvent redundancy by chemically inhibiting multiple DUBs simultaneously in Xenopus egg extract. We used quantitative mass spectrometry to identify proteins whose ubiquitylation or stability is altered by broad DUB inhibition, and confirmed their DUB-dependent regulation with human orthologs, demonstrating evolutionary conservation. We next extended this method to profile DUB specificity. By adding recombinant DUBs to extract where DUB activity was broadly inhibited, but ubiquitylation and degradation were active at physiological rates, we profiled the ability of DUBs to rescue degradation of these substrates. We found that USP7 has a unique ability to broadly antagonize degradation. Together, we present an approach to identify DUB substrates and characterize DUB specificity that overcomes challenges posed by DUB redundancy.

SUBMITTER: Rossio V

PROVIDER: S-EPMC8052291 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Proteomics of broad deubiquitylase inhibition unmasks redundant enzyme function to reveal substrates and assess enzyme specificity

Project description:Deubiquitylating enzymes (DUBs) counteract ubiquitylation to control stability or activity of substrates. Identification of DUB substrates is challenging because multiple DUBs can act on the same substrate, thwarting genetic approaches. Here, we circumvent redundancy by chemically inhibiting multiple DUBs simultaneously in Xenopus egg extract. We discovered a set of proteins that depends on DUBs for their stability and we confirmed their DUB-dependent regulation with human orthologs, demonstrating evolutionary conservation. We next extended this approach, developing a new method to profile DUB specificity. By adding recombinant DUBs to extract where DUB activity was broadly inhibited, but ubiquitylation and degradation were active at physiological rates, we profiled the ability of DUBs to rescue degradation of these new substrates. We found that USP7 has a unique ability to broadly antagonize their degradation. Together, we identify novel DUB substrates and present an approach to characterize DUB specificity that overcomes challenges posed by DUB redundancy.

2022-09-02 | PXD022762 | Pride

Proteomics of broad deubiquitylase inhibition unmasks redundant enzyme function to reveal substrates and assess enzyme specificity

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USP7 substrates identified by proteomics analysis reveal the specificity of USP7

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USP7 substrates identified by proteomics analysis reveal the specificity of USP7.

Project description:Deubiquitylating enzymes (DUBs) remove ubiquitin chains from proteins and regulate protein stability and function. USP7 is one of the most extensively studied DUBs, since USP7 has several well-known substrates important for cancer progression, such as MDM2, N-MYC, and PTEN. Thus, USP7 is a promising drug target. However, systematic identification of USP7 substrates has not yet been performed. In this study, we carried out proteome profiling with label-free quantification in control and single/double-KO cells of USP7and its closest homolog, USP47 Our proteome profiling for the first time revealed the proteome changes caused by USP7 and/or USP47 depletion. Combining protein profiling, transcriptome analysis, and tandem affinity purification of USP7-associated proteins, we compiled a list of 20 high-confidence USP7 substrates that includes known and novel USP7 substrates. We experimentally validated MGA and PHIP as new substrates of USP7. We further showed that MGA deletion reduced cell proliferation, similar to what was observed in cells with USP7 deletion. In conclusion, our proteome-wide analysis uncovered potential USP7 substrates, providing a resource for further functional studies.

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Single-mutation fitness landscapes for an enzyme on multiple substrates reveal specificity is globally encoded.

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USP7 substrates identified by proteomics analysis reveal the specificity of USP7

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Enzyme Kinetics for Complex System Enables Accurate Determination of Specificity Constants of Numerous Substrates in a Mixture by Proteomics Platform.

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Project description:Protein arginine methyltransferases (PRMTs) introduce arginine methylation, a post-translational modification with the increasingly eminent role in normal physiology and disease. PRMT4 or coactivator-associated arginine methyltransferase 1 (CARM1) is a propitious target for cancer therapy; however, few CARM1 substrates are known, and its mechanism of substrate recognition is poorly understood. Here we employed a quantitative mass spectrometry approach to globally profile CARM1 substrates in breast cancer cell lines. We identified >130 CARM1 protein substrates and validated in vitro >90% of sites they encompass. Bioinformatics analyses reveal enrichment of proline-containing motifs, in which both methylation sites and their proximal sequences are frequently targeted by somatic mutations in cancer. Finally, we demonstrate that the N-terminus of CARM1 is involved in substrate recognition and nearly indispensable for substrate methylation. We propose that development of CARM1-specific inhibitors should focus on its N-terminus and predict that other PRMTs may employ similar mechanism for substrate recognition.

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Phosphoproteome dynamics reveal novel ERK1/2 MAP kinase substrates with broad spectrum of functions.

Project description:The ERK1/2 MAP kinase pathway is an evolutionarily conserved signaling module that controls many fundamental physiological processes. Deregulated activity of ERK1/2 MAP kinases is associated with developmental syndromes and several human diseases. Despite the importance of this pathway, a comprehensive picture of the natural substrate repertoire and biochemical mechanisms regulated by ERK1/2 is still lacking. In this study, we used large-scale quantitative phosphoproteomics and bioinformatics analyses to identify novel candidate ERK1/2 substrates based on their phosphorylation signature and kinetic profiles in epithelial cells. We identified a total of 7936 phosphorylation sites within 1861 proteins, of which 155 classify as candidate ERK1/2 substrates, including 128 new targets. Candidate ERK1/2 substrates are involved in diverse cellular processes including transcriptional regulation, chromatin remodeling, RNA splicing, cytoskeleton dynamics, cellular junctions and cell signaling. Detailed characterization of one newly identified substrate, the transcriptional regulator JunB, revealed that ERK1/2 phosphorylate JunB on a serine adjacent to the DNA-binding domain, resulting in increased DNA-binding affinity and transcriptional activity. Our study expands the spectrum of cellular functions controlled by ERK1/2 kinases.

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