Positional-scanning fluorigenic substrate libraries reveal unexpected specificity determinants of DUBs (deubiquitinating enzymes)

DUBs (deubiquitinating enzymes) are a family of proteases responsible for the specific removal of ubiquitin attached to target proteins and thus control the free cellular pools of this molecule. DUB activity is usually assayed using full-length ubiquitin, and these enzymes generally show low activity towards small substrates that constitute the P4–P1 LRGG (Lys-Arg-Gly-Gly) C-terminal motif of ubiquitin. To gain insight into the C-terminal recognition region of ubiquitin by DUBs, we synthesized positional scanning libraries of fluorigenic tetrapeptides and tested them on three examples of human DUBs [OTU-1 (ovarian tumour 1), Iso-T (isopeptidase T) and UCH-L3 (ubiquitin C-terminal hydrolase L3)] and one viral ubiquitin-specific protease, namely PLpro (papain-like protease) from SARS (severe acute respiratory syndrome) virus. In most cases the results show flexibility in the P4 position, very high specificity for arginine in the P3 position and glycine in the P2 position, in accord with the sequence of the natural substrate, ubiquitin. Surprisingly, screening of the P2 position revealed that UCH-L3, in contrast with all the other tested DUBs, demonstrates substantial tolerance of alanine and valine at P2, and a parallel analysis using the appropriate mutation of the full-length ubiquitin confirms this. We have also used an optimal tetrapeptide substrate, acetyl-Lys-Arg-Gly-Gly-7-amino-4-methylcoumarin, to investigate the activation mechanism of DUBs by ubiquitin and elevated salt concentration. Together, our results reveal the importance of the dual features of (1) substrate specificity and (2) the mechanism of ubiquitin binding in determining deubiquitination by this group of proteases.

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USP48 Sustains Chemoresistance and Metastasis in Ovarian Cancer

Current Cancer Drug Targets ◽

10.2174/1568009620666200503045400 ◽

2020 ◽

Vol 20 (9) ◽

pp. 689-699

Author(s):

Xuemeng Lei ◽

Xukun Li ◽

Hongyan Chen ◽

Zhihua Liu

Keyword(s):

Ovarian Cancer ◽

Cancer Patients ◽

Migration And Invasion ◽

Clinical Samples ◽

Deubiquitinating Enzymes ◽

Potential Therapeutic Target ◽

Kaplan Meier ◽

Specific Protease ◽

Ubiquitin Specific Protease

Background: Ubiquitin specific protease 48 (USP48) is a member of the deubiquitinating enzymes (DUBs) family. However, the function of USP48 in ovarian cancer remains unclear. Objective: The present study reveals that USP48 knockdown could significantly inhibit cell migration and invasion in ES2, 3AO and A2780 cells, without affecting cell proliferation. Methods: After carboplatin (CBP) treatment, the USP48 ablation increases the apoptosis rate, and the cleaved PARP and cleaved caspase 3 expression levels in ES2, 3AO and A2780 cells. The subcutaneous tumor and intraperitoneally injected experiments demonstrated that the USP48 knockdown significantly increases responsiveness to CBP, and alleviates the metastasis in vivo. Meanwhile, USP48 deficiency results in the improved survival of mice. Results: Finally, the analysis of clinical samples and the TCGA and Kaplan-Meier Plot database revealed that the high expression of USP48 in ovarian cancer patients is associated with poor survival and resistance to CBP therapy. Conclusion: In summary, USP48 may be a potential therapeutic target for ovarian cancer patients.

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USP19 is a ubiquitin-specific protease regulated in rat skeletal muscle during catabolic states

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00281.2004 ◽

2005 ◽

Vol 288 (4) ◽

pp. E693-E700 ◽

Cited By ~ 61

Author(s):

Lydie Combaret ◽

Olasunkanmi A. J. Adegoke ◽

Nathalie Bedard ◽

Vickie Baracos ◽

Didier Attaix ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Mass ◽

De Novo ◽

Deubiquitinating Enzyme ◽

Mrna Levels ◽

Deubiquitinating Enzymes ◽

Rat Skeletal Muscle ◽

Ubiquitin System ◽

Specific Protease ◽

Ubiquitin Specific Protease

Ubiquitin-dependent proteolysis is activated in skeletal muscle atrophying in response to various catabolic stimuli. Previous studies have demonstrated activation of ubiquitin conjugation. Because ubiquitination can also be regulated by deubiquitinating enzymes, we used degenerate oligonucleotides derived from conserved sequences in the ubiquitin-specific protease (UBP) family of deubiquitinating enzymes in RT-PCR with skeletal muscle RNA to amplify putative deubiquitinating enzymes. We identified USP19, a 150-kDa deubiquitinating enzyme that is widely expressed in various tissues including skeletal muscle. Expression of USP19 mRNA increased by ∼30–200% in rat skeletal muscle atrophying in response to fasting, streptozotocin-induced diabetes, dexamethasone treatment, and cancer. Increased mRNA levels during fasting returned to normal with refeeding, but 1 day later than the normalization of rates of proteolysis and coincided instead with recovery of muscle mass. Indeed, in all catabolic treatments, USP19 mRNA was inversely correlated with muscle mass and provided an index of muscle mass that may be useful in many pathological conditions, using small human muscle biopsies. The increased expression of this deubiquitinating enzyme under conditions of increased proteolysis suggests that it may play a role in regeneration of free ubiquitin either coincident with or after proteasome-mediated degradation of substrates. USP19 may also be involved in posttranslational processing of polyubiquitin produced de novo in response to induction of the polyubiquitin genes seen under these conditions. Deubiquitinating enzymes thus appear involved in muscle wasting and implicate a widening web of regulation of genes in the ubiquitin system in this process.

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Identification of a single nucleotide promoter polymorphism regulating the transcription of ubiquitin specific protease 18 gene related to the resistance to porcine reproductive and respiratory syndrome virus infection

Veterinary Immunology and Immunopathology ◽

10.1016/j.vetimm.2014.09.007 ◽

2014 ◽

Vol 162 (3-4) ◽

pp. 65-71 ◽

Cited By ~ 9

Author(s):

Yanping Li ◽

Yi Sun ◽

Feng Xing ◽

Li Kang ◽

Pengfei Wang ◽

...

Keyword(s):

Virus Infection ◽

Promoter Polymorphism ◽

Respiratory Syndrome Virus ◽

Single Nucleotide ◽

Specific Protease ◽

Ubiquitin Specific Protease

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The role of the deubiquitinating enzyme DUB3/USP17 in cancer: a narrative review

Cancer Cell International ◽

10.1186/s12935-021-02160-y ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Guang-Fei Yang ◽

Xin Zhang ◽

Yi-Ge Su ◽

Ren Zhao ◽

Yan-Yang Wang

Keyword(s):

Central Nervous System ◽

Cancer Research ◽

Soft Tissue Sarcomas ◽

Regulatory Mechanisms ◽

Deubiquitinating Enzyme ◽

Biological Functions ◽

Deubiquitinating Enzymes ◽

Specific Protease ◽

Ubiquitin Specific Protease

AbstractThe balance between ubiquitination and deubiquitination is critical for the degradation, transport, localization, and activity of proteins. Deubiquitinating enzymes (DUBs) greatly contribute to the balance of ubiquitination and deubiquitination, and they have been widely studied due to their fundamental role in cancer. DUB3/ubiquitin-specific protease 17 (USP17) is a type of DUB that has attracted much attention in cancer research. In this review, we summarize the biological functions and regulatory mechanisms of USP17 in central nervous system, head and neck, thoracic, breast, gastrointestinal, genitourinary, and gynecologic cancers as well as bone and soft tissue sarcomas, and we provide new insights into how USP17 can be used in the management of cancer.

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A novel frameshift mutation in ubiquitin-specific protease 26 gene in a patient with severe oligozoospermia

Bioscience Reports ◽

10.1042/bsr20191902 ◽

2020 ◽

Vol 40 (4) ◽

Author(s):

Leilei Li ◽

Qi Xi ◽

Hongguo Zhang ◽

Jia Fei ◽

Yuting Jiang ◽

...

Keyword(s):

Male Infertility ◽

Frameshift Mutation ◽

Bioinformatics Analysis ◽

Chinese Patient ◽

Deubiquitinating Enzyme ◽

Deubiquitinating Enzymes ◽

Severe Oligozoospermia ◽

Specific Protease ◽

Ubiquitin Specific Protease ◽

Testis Tissue

Abstract Ubiquitin-specific protease 26 (USP26) encodes a predicted protein containing his- and cys- domains that are conserved among deubiquitinating enzymes. USP26 is specifically expressed in testis tissue and is a potential infertility gene. In the present study, we performed genetic testing related to spermatogenesis impairment in a patient with idiopathic severe oligozoospermia to identify the cause. The patient underwent clinical examination and reproductive hormone testing. Genes associated with male infertility, including USP26, were assessed by targeted exome sequencing. A novel frameshift mutation, c.2195delT (p.Phe732Serfs*14), was identified in USP26. This frameshift mutation was located in residue 732 of USP26 gene, leading to loss of the conserved deubiquitinating enzyme His-domain and producing a truncated protein of 744 amino acids. Bioinformatics analysis revealed this mutation to be pathogenic. A novel framshift mutation c.2195delT (p.Phe732Serfs*14) in USP26 gene was reported to be associated with male infertility in a Chinese patient with severe oligozoospermia.

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USP47 Promotes Tumorigenesis by Negative Regulation of p53 through Deubiquitinating Ribosomal Protein S2

Cancers ◽

10.3390/cancers12051137 ◽

2020 ◽

Vol 12 (5) ◽

pp. 1137 ◽

Cited By ~ 1

Author(s):

Jinhong Cho ◽

Jinyoung Park ◽

Sang Chul Shin ◽

Mihue Jang ◽

Jae-Hong Kim ◽

...

Keyword(s):

Ribosomal Protein ◽

Xenograft Model ◽

Deubiquitinating Enzymes ◽

Potential Therapeutic Target ◽

Mouse Xenograft Model ◽

Specific Protease ◽

Important Regulator ◽

Ubiquitin Specific Protease ◽

Cellular Stresses ◽

Ribosomal Stress

p53 is activated in response to cellular stresses such as DNA damage, oxidative stress, and especially ribosomal stress. Although the regulations of p53 by E3 ligase and deubiquitinating enzymes (DUBs) have been described, the cellular roles of DUB associated with ribosomal stress have not been well studied. In this study, we report that Ubiquitin Specific Protease 47 (USP47) functions as an important regulator of p53. We show that ubiquitinated ribosomal protein S2 (RPS2) by Mouse double minute 2 homolog (MDM2) is deubiquitinated by USP47. USP47 inhibits the interaction between RPS2 and MDM2 thereby alleviating RPS2-mediated suppression of MDM2 under normal conditions. However, dissociation of USP47 leads to RPS2 binding to MDM2, which is required for the suppression of MDM2, consequently inducing up-regulation of the p53 level under ribosomal stress. Finally, we show that depletion of USP47 induces p53 and therefore inhibits cell proliferation, colony formation, and tumor progression in cancer cell lines and a mouse xenograft model. These findings suggest that USP47 could be a potential therapeutic target for cancer.

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The role of UBL domains in ubiquitin-specific proteases

Biochemical Society Transactions ◽

10.1042/bst20120004 ◽

2012 ◽

Vol 40 (3) ◽

pp. 539-545 ◽

Cited By ~ 39

Author(s):

Alex C. Faesen ◽

Mark P.A. Luna-Vargas ◽

Titia K. Sixma

Keyword(s):

Catalytic Activity ◽

Catalytic Domain ◽

Mechanisms Of Action ◽

Deubiquitinating Enzymes ◽

Activated State ◽

Ubiquitin Binding ◽

Specific Protease ◽

Ubiquitin Conjugation ◽

Ubiquitin Specific Protease

Ubiquitin conjugation and deconjugation provides a powerful signalling system to change the fate of its target enzymes. Ubiquitination levels are organized through a balance between ubiquitinating E1, E2 and E3 enzymes and deubiquitination by DUBs (deubiquitinating enzymes). These enzymes are tightly regulated to control their activity. In the present article, we discuss the different ways in which DUBs of the USP (ubiquitin-specific protease) family are regulated by internal domains with a UBL (ubiquitin-like) fold. The UBL domain in USP14 is important for its localization at the proteasome, which enhances catalysis. In contrast, a UBL domain in USP4 binds to the catalytic domain and competes with ubiquitin binding. In this process, the UBL domain mimics ubiquitin and partially inhibits catalysis. In USP7, there are five consecutive UBL domains, of which the last two affect catalytic activity. Surprisingly, they do not act like ubiquitin and activate catalysis rather than inhibiting it. These C-terminal UBL domains promote a conformational change that allows ubiquitin binding and organizes the catalytic centre. Thus it seems that UBL domains have different functions in different USPs. Other proteins can modulate the roles of UBL domains in USP4 and USP7. On one hand, the inhibition of USP4 can be relieved when the UBL is sequestered by another USP. On the other, the activation of USP7 is increased, when the UBL-activated state is stabilized by allosteric binding of GMP synthetase. Altogether, UBL domains appear to be able to regulate catalytic activity in USPs, but they can use widely different mechanisms of action, in which they may, as in USP4, or may not, as in USP7, use the direct resemblance to ubiquitin.

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Divergence in Ubiquitin Interaction and Catalysis among the Ubiquitin-Specific Protease Family Deubiquitinating Enzymes

Biochemistry ◽

10.1021/acs.biochem.6b00033 ◽

2016 ◽

Vol 55 (33) ◽

pp. 4708-4719 ◽

Cited By ~ 7

Author(s):

Adam H. Tencer ◽

Qin Liang ◽

Zhihao Zhuang

Keyword(s):

Deubiquitinating Enzymes ◽

Specific Protease ◽

Ubiquitin Specific Protease

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Emerging Roles of Ubiquitin-Specific Protease 25 in Diseases

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.698751 ◽

2021 ◽

Vol 9 ◽

Author(s):

Wenjing Zhu ◽

Dandan Zheng ◽

Dandan Wang ◽

Lehe Yang ◽

Chengguang Zhao ◽

...

Keyword(s):

Immune Responses ◽

Viral Infections ◽

Disease Onset ◽

Mechanisms Of Action ◽

Deubiquitinating Enzyme ◽

Deubiquitinating Enzymes ◽

Specific Protease ◽

Ubiquitin Specific Protease ◽

Similarities And Differences

The balance of ubiquitination and deubiquitination plays diverse roles in regulating protein stability and cellular homeostasis. Deubiquitinating enzymes catalyze the hydrolysis and removal of ubiquitin chains from target proteins and play critical roles in various disease processes, including cancer, immune responses to viral infections and neurodegeneration. This article aims to summarize roles of the deubiquitinating enzyme ubiquitin-specific protease 25 (USP25) in disease onset and progression. Previous studies have focused on the role of USP25 in antiviral immunity and neurodegenerative diseases. Recently, however, as the structural similarities and differences between USP25 and its homolog USP28 have become clear, mechanisms of action of USP25 in cancer and other diseases have been gradually revealed.

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