Journal articles: 'Ubiquitin protease'

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Yip, Matthew C. J., Nicholas O. Bodnar, and Tom A. Rapoport. "Ddi1 is a ubiquitin-dependent protease." Proceedings of the National Academy of Sciences 117, no. 14 (March 19, 2020): 7776–81. http://dx.doi.org/10.1073/pnas.1902298117.

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TheSaccharomyces cerevisiaeprotein Ddi1 and its homologs in higher eukaryotes have been proposed to serve as shuttling factors that deliver ubiquitinated substrates to the proteasome. Although Ddi1 contains both ubiquitin-interacting UBA and proteasome-interacting UBL domains, the UBL domain is atypical, as it binds ubiquitin. Furthermore, unlike other shuttling factors, Ddi1 and its homologs contain a conserved helical domain (helical domain of Ddi1, HDD) and a retroviral-like protease (RVP) domain. The RVP domain is probably responsible for cleavage of the precursor of the transcription factor Nrf1 in higher eukaryotes, which results in the up-regulation of proteasomal subunit genes. However, enzymatic activity of the RVP domain has not yet been demonstrated, and the function of Ddi1 remains poorly understood. Here, we show that Ddi1 is a ubiquitin-dependent protease, which cleaves substrate proteins only when they are tagged with long ubiquitin chains (longer than about eight ubiquitins). The RVP domain is inactive in isolation, in contrast to its retroviral counterpart. Proteolytic activity of Ddi1 requires the HDD domain and is stimulated by the UBL domain, which mediates high-affinity interaction with the polyubiquitin chain. Compromising the activity of Ddi1 in yeast cells results in the accumulation of polyubiquitinated proteins. Aside from the proteasome, Ddi1 is the only known endoprotease that acts on polyubiquitinated substrates. Ddi1 and its homologs likely cleave polyubiquitinated substrates under conditions where proteasome function is compromised.

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Drag, Marcin, Jowita Mikolajczyk, Miklos Bekes, Francisca E. Reyes-Turcu, Jonathan A. Ellman, Keith D. Wilkinson, and Guy S. Salvesen. "Positional-scanning fluorigenic substrate libraries reveal unexpected specificity determinants of DUBs (deubiquitinating enzymes)." Biochemical Journal 415, no. 3 (October 15, 2008): 367–75. http://dx.doi.org/10.1042/bj20080779.

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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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Gardner, Richard G., Zara W. Nelson, and Daniel E. Gottschling. "Ubp10/Dot4p Regulates the Persistence of Ubiquitinated Histone H2B: Distinct Roles in Telomeric Silencing and General Chromatin." Molecular and Cellular Biology 25, no. 14 (July 2005): 6123–39. http://dx.doi.org/10.1128/mcb.25.14.6123-6139.2005.

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ABSTRACT We previously discovered that the ubiquitin protease Ubp10/Dot4p is important for telomeric silencing through its interaction with Sir4p. However, the mechanism of Ubp10p action was unknown. We now provide evidence that Ubp10p removes ubiquitin from histone H2B; cells with UBP10 deleted have increased steady-state levels of H2B ubiquitination. As a consequence, ubp10Δ cells also have increased steady-state levels of histone H3 Lys4 and Lys79 methylation. Consistent with its role in silencing, Ubp10p is preferentially localized to silent chromatin where its ubiquitin protease activity maintains low levels of H3 Lys4 and Lys79 methylation to allow optimal Sir protein binding to telomeres and global telomeric silencing. The ubiquitin protease Ubp8p has also been shown to remove ubiquitin from H2B, and ubp8Δ cells have increased steady-state levels of H2B ubiquitination similar to those in ubp10Δ cells. Unlike ubp10Δ cells, however, ubp8Δ cells do not have increased steady-state levels of H3 Lys4 and Lys79 methylation, nor is telomeric silencing affected. Despite their separate functions in silencing and SAGA-mediated transcription, respectively, deletion of both UBP10 and UBP8 results in a synergistic increase in the steady-state levels of H2B ubiquitination and in the number of genes with altered expression, indicating that Ubp10p and Ubp8p likely overlap in some of their target chromatin regions. We propose that Ubp10p and Ubp8p are the only ubiquitin proteases that normally remove monoubiquitin from histone H2B and, while there are regions of the genome to which each is specifically targeted, both combine to regulate the global balance of H2B ubiquitination.

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Schairer, Rebekka, Gareth Hall, Ming Zhang, Richard Cowan, Roberta Baravalle, Frederick W. Muskett, Peter J. Coombs, et al. "Allosteric activation of MALT1 by its ubiquitin-binding Ig3 domain." Proceedings of the National Academy of Sciences 117, no. 6 (January 24, 2020): 3093–102. http://dx.doi.org/10.1073/pnas.1912681117.

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The catalytic activity of the protease MALT1 is required for adaptive immune responses and regulatory T (Treg)-cell development, while dysregulated MALT1 activity can lead to lymphoma. MALT1 activation requires its monoubiquitination on lysine 644 (K644) within the Ig3 domain, localized adjacent to the protease domain. The molecular requirements for MALT1 monoubiquitination and the mechanism by which monoubiquitination activates MALT1 had remained elusive. Here, we show that the Ig3 domain interacts directly with ubiquitin and that an intact Ig3-ubiquitin interaction surface is required for the conjugation of ubiquitin to K644. Moreover, by generating constitutively active MALT1 mutants that overcome the need for monoubiquitination, we reveal an allosteric communication between the ubiquitination site K644, the Ig3-protease interaction surface, and the active site of the protease domain. Finally, we show that MALT1 mutants that alter the Ig3-ubiquitin interface impact the biological response of T cells. Thus, ubiquitin binding by the Ig3 domain promotes MALT1 activation by an allosteric mechanism that is essential for its biological function.

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Vertegaal, Alfred C. O. "SUMO chains: polymeric signals." Biochemical Society Transactions 38, no. 1 (January 19, 2010): 46–49. http://dx.doi.org/10.1042/bst0380046.

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Ubiquitin and ubiquitin-like proteins are conjugated to a wide variety of target proteins that play roles in all biological processes. Target proteins are conjugated to ubiquitin monomers or to ubiquitin polymers that form via all seven internal lysine residues of ubiquitin. The fate of these target proteins is controlled in a chain architecture-dependent manner. SUMO (small ubiquitin-related modifier) shares the ability of ubiquitin to form chains via internal SUMOylation sites. Interestingly, a SUMO-binding site in Ubc9 is important for SUMO chain synthesis. Similar to ubiquitin–polymer cleavage by USPs (ubiquitin-specific proteases), SUMO chain formation is reversible. SUMO polymers are cleaved by the SUMO proteases SENP6 [SUMO/sentrin/SMT3 (suppressor of mif two 3)-specific peptidase 6], SENP7 and Ulp2 (ubiquitin-like protease 2). SUMO chain-binding proteins including ZIP1, SLX5/8 (synthetic lethal of unknown function 5/8), RNF4 (RING finger protein 4) and CENP-E (centromere-associated protein E) have been identified that interact non-covalently with SUMO chains, thereby regulating target proteins that are conjugated to SUMO multimers. SUMO chains play roles in replication, in the turnover of SUMO targets by the proteasome and during mitosis and meiosis. Thus signalling via polymers is an exciting feature of the SUMO family.

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Fieulaine, Sonia, Martin D. Witte, Christopher S. Theile, Maya Ayach, Hidde L. Ploegh, Isabelle Jupin, and Stéphane Bressanelli. "Turnip yellow mosaic virus protease binds ubiquitin suboptimally to fine-tune its deubiquitinase activity." Journal of Biological Chemistry 295, no. 40 (July 30, 2020): 13769–83. http://dx.doi.org/10.1074/jbc.ra120.014628.

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Single-stranded, positive-sense RNA viruses assemble their replication complexes in infected cells from a multidomain replication polyprotein. This polyprotein usually contains at least one protease, the primary function of which is to process the polyprotein into mature proteins. Such proteases also may have other functions in the replication cycle. For instance, cysteine proteases (PRO) frequently double up as ubiquitin hydrolases (DUB), thus interfering with cellular processes critical for virus replication. We previously reported the crystal structures of such a PRO/DUB from Turnip yellow mosaic virus (TYMV) and of its complex with one of its PRO substrates. Here we report the crystal structure of TYMV PRO/DUB in complex with ubiquitin. We find that PRO/DUB recognizes ubiquitin in an unorthodox way: It interacts with the body of ubiquitin through a split recognition motif engaging both the major and the secondary recognition patches of ubiquitin (Ile44 patch and Ile36 patch, respectively, including Leu8, which is part of the two patches). However, the contacts are suboptimal on both sides. Introducing a single-point mutation in TYMV PRO/DUB aimed at improving ubiquitin-binding led to a much more active DUB. Comparison with other PRO/DUBs from other viral families, particularly coronaviruses, suggests that low DUB activities of viral PRO/DUBs may generally be fine-tuned features of interaction with host factors.

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Mielech, Anna M., Xufang Deng, Yafang Chen, Eveline Kindler, Dorthea L. Wheeler, Andrew D. Mesecar, Volker Thiel, Stanley Perlman, and Susan C. Baker. "Murine Coronavirus Ubiquitin-Like Domain Is Important for Papain-Like Protease Stability and Viral Pathogenesis." Journal of Virology 89, no. 9 (February 18, 2015): 4907–17. http://dx.doi.org/10.1128/jvi.00338-15.

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ABSTRACTUbiquitin-like domains (Ubls) now are recognized as common elements adjacent to viral and cellular proteases; however, their function is unclear. Structural studies of the papain-like protease (PLP) domains of coronaviruses (CoVs) revealed an adjacent Ubl domain in severe acute respiratory syndrome CoV, Middle East respiratory syndrome CoV, and the murine CoV, mouse hepatitis virus (MHV). Here, we tested the effect of altering the Ubl adjacent to PLP2 of MHV on enzyme activity, viral replication, and pathogenesis. Using deletion and substitution approaches, we identified sites within the Ubl domain, residues 785 to 787 of nonstructural protein 3, which negatively affect protease activity, and valine residues 785 and 787, which negatively affect deubiquitinating activity. Using reverse genetics, we engineered Ubl mutant viruses and found that AM2 (V787S) and AM3 (V785S) viruses replicate efficiently at 37°C but generate smaller plaques than wild-type (WT) virus, and AM2 is defective for replication at higher temperatures. To evaluate the effect of the mutation on protease activity, we purified WT and Ubl mutant PLP2 and found that the proteases exhibit similar specific activities at 25°C. However, the thermal stability of the Ubl mutant PLP2 was significantly reduced at 30°C, thereby reducing the total enzymatic activity. To determine if the destabilizing mutation affects viral pathogenesis, we infected C57BL/6 mice with WT or AM2 virus and found that the mutant virus is highly attenuated, yet it replicates sufficiently to elicit protective immunity. These studies revealed that modulating the Ubl domain adjacent to the PLP reduces protease stability and viral pathogenesis, revealing a novel approach to coronavirus attenuation.IMPORTANCEIntroducing mutations into a protein or virus can have either direct or indirect effects on function. We asked if changes in the Ubl domain, a conserved domain adjacent to the coronavirus papain-like protease, altered the viral protease activity or affected viral replication or pathogenesis. Our studies using purified wild-type and Ubl mutant proteases revealed that mutations in the viral Ubl domain destabilize and inactivate the adjacent viral protease. Furthermore, we show that a CoV encoding the mutant Ubl domain is unable to replicate at high temperature or cause lethal disease in mice. Our results identify the coronavirus Ubl domain as a novel modulator of viral protease stability and reveal manipulating the Ubl domain as a new approach for attenuating coronavirus replication and pathogenesis.

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Békés, Miklós, Wioletta Rut, Paulina Kasperkiewicz, Monique P. C. Mulder, Huib Ovaa, Marcin Drag, Christopher D. Lima, and Tony T. Huang. "SARS hCoV papain-like protease is a unique Lys48 linkage-specific di-distributive deubiquitinating enzyme." Biochemical Journal 468, no. 2 (May 22, 2015): 215–26. http://dx.doi.org/10.1042/bj20141170.

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We compare processing proteases from two human coronaviruses - the severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome (MERS) hCoVs - with respect to their activities and substrate specificities for ubiquitin (Ub)-like signaling molecules, Ub and ISG15 (interferon stimulated gene 15); and doing so, we uncover a unique mode of polyUb recognition by the SARS protease.

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9

Gilchrist, Catherine A., Douglas A. Gray, and Rohan T. Baker. "A Ubiquitin-specific Protease That Efficiently Cleaves the Ubiquitin-Proline Bond." Journal of Biological Chemistry 272, no. 51 (December 19, 1997): 32280–85. http://dx.doi.org/10.1074/jbc.272.51.32280.

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10

Luna‐Vargas, Mark P. A., Alex C. Faesen, Willem J. van Dijk, Michael Rape, Alexander Fish, and Titia K. Sixma. "Ubiquitin‐specific protease 4 is inhibited by its ubiquitin‐like domain." EMBO reports 12, no. 4 (March 18, 2011): 365–72. http://dx.doi.org/10.1038/embor.2011.33.

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Schneider, Tatjana, Daniel Schneider, Daniel Rösner, Saurav Malhotra, Franziska Mortensen, Thomas U. Mayer, Martin Scheffner, and Andreas Marx. "Dissecting Ubiquitin Signaling with Linkage-Defined and Protease Resistant Ubiquitin Chains." Angewandte Chemie International Edition 53, no. 47 (September 4, 2014): 12925–29. http://dx.doi.org/10.1002/anie.201407192.

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12

Moree, Shannon E., Laure Maneix, Polina Iakova, Fabio Stossi, Ergun Sahin, and Andre Catic. "Imaging-Based Screening of Deubiquitinating Proteases Identifies Otubain-1 as a Stabilizer of c-MYC." Cancers 14, no. 3 (February 4, 2022): 806. http://dx.doi.org/10.3390/cancers14030806.

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The ubiquitin–proteasome pathway precisely controls the turnover of transcription factors in the nucleus, playing an important role in maintaining appropriate quantities of these regulatory proteins. The transcription factor c-MYC is essential for normal development and is a critical cancer driver. Despite being highly expressed in several tissues and malignancies, the c-MYC protein is also continuously targeted by the ubiquitin–proteasome pathway, which can either facilitate or inhibit c-MYC degradation. Deubiquitinating proteases can remove ubiquitin chains from target proteins and rescue them from proteasomal digestion. This study sought to determine novel elements of the ubiquitin–proteasome pathway that regulate c-MYC levels. We performed an overexpression screen with 41 human proteases to identify which deubiquitinases stabilize c-MYC. We discovered that the highly expressed Otubain-1 (OTUB1) protease increases c-MYC protein levels. Confirming its role in enhancing c-MYC activity, we found that elevated OTUB1 correlates with inferior clinical outcomes in the c-MYC-dependent cancer multiple myeloma, and overexpression of OTUB1 accelerates the growth of myeloma cells. In summary, our study identifies OTUB1 as a novel amplifier of the proto-oncogene c-MYC.

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Lorenzo, Mayra E., Jae U. Jung, and Hidde L. Ploegh. "Kaposi's Sarcoma-Associated Herpesvirus K3 Utilizes the Ubiquitin-Proteasome System in Routing Class I Major Histocompatibility Complexes to Late Endocytic Compartments." Journal of Virology 76, no. 11 (June 1, 2002): 5522–31. http://dx.doi.org/10.1128/jvi.76.11.5522-5531.2002.

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ABSTRACT Human herpesvirus 8 (HHV8) downregulates major histocompatibility complex (MHC) class I complexes from the plasma membrane via two of its genes, K3 and K5. The N termini of K3 and K5 contain a plant homeodomain (PHD) predicted to be structurally similar to RING domains found in E3 ubiquitin ligases. In view of the importance of the ubiquitin-proteasome system in sorting within the endocytic pathway, we analyzed its role in downregulation of MHC class I complexes in cells expressing K3. Proteasome inhibitors as well as cysteine and aspartyl protease inhibitors stabilize MHC class I complexes in cells expressing K3. However, proteasome inhibitors differentially affect sorting of MHC class I complexes within the endocytic pathway and prevent their delivery to a dense endosomal compartment. In this compartment, the cytoplasmic tail of MHC class I complexes is cleaved by cysteine proteases. The complex is then cleaved within the plane of the membrane by an aspartyl protease, resulting in a soluble MHC class I fragment composed of the lumenal domain of the heavy chain, β2-microglobulin (β2m), and peptide. We conclude that K3 not only directs internalization, but also targets MHC class I complexes to a dense endocytic compartment on the way to lysosomes in a ubiquitin-proteasome-dependent manner.

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Colland, Frédéric. "The therapeutic potential of deubiquitinating enzyme inhibitors." Biochemical Society Transactions 38, no. 1 (January 19, 2010): 137–43. http://dx.doi.org/10.1042/bst0380137.

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Proteases play a key role in various pathological processes and several protease inhibitors are already available for treatment. DUBs (deubiquitinating enzymes) constitute one of the largest classes of human proteases and are key effectors of the ubiquitin–proteasome system. This pathway regulating cellular protein turnover has been implicated in the pathogenesis of many human diseases, including neurodegenerative disorders, viral diseases and cancer. The therapeutic efficacy of the proteasome inhibitor Velcade® (bortezomib) for treating multiple myeloma and mantle cell lymphoma establishes this system as a valid target for cancer treatment. A promising alternative to targeting the proteasome itself would be to target the upstream, ubiquitin conjugation/deconjugation system, to generate more specific, less toxic anticancer agents. Advances in small molecule-based inhibitors specifically targeting DUBs are presented in this review.

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Bacchi, Marine, Benjamin Fould, Magali Jullian, Aude Kreiter, Amélie Maurras, Olivier Nosjean, Thibault Coursindel, Karine Puget, Gilles Ferry, and Jean A. Boutin. "Screening ubiquitin specific protease activities using chemically synthesized ubiquitin and ubiquitinated peptides." Analytical Biochemistry 519 (February 2017): 57–70. http://dx.doi.org/10.1016/j.ab.2016.12.014.

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Sarkari, Feroz, Keith Wheaton, Anthony La Delfa, Majda Mohamed, Faryal Shaikh, Rahima Khatun, Cheryl H. Arrowsmith, Lori Frappier, Vivian Saridakis, and Yi Sheng. "Ubiquitin-specific Protease 7 Is a Regulator of Ubiquitin-conjugating Enzyme UbE2E1." Journal of Biological Chemistry 288, no. 23 (April 19, 2013): 16975–85. http://dx.doi.org/10.1074/jbc.m113.469262.

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Ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme found in all eukaryotes that catalyzes the removal of ubiquitin from specific target proteins. Here, we report that UbE2E1, an E2 ubiquitin conjugation enzyme with a unique N-terminal extension, is a novel USP7-interacting protein. USP7 forms a complex with UbE2E1 in vitro and in vivo through the ASTS USP7 binding motif within its N-terminal extension in an identical manner with other known USP7 binding proteins. We show that USP7 attenuates UbE2E1-mediated ubiquitination, an effect that requires the N-terminal ASTS sequence of UbE2E1 as well as the catalytic activity of USP7. Additionally, USP7 is critical in maintaining the steady state levels of UbE2E1 in cells. This study reveals a new cellular mechanism that couples the opposing activities of the ubiquitination machinery and a deubiquitinating enzyme to maintain and modulate the dynamic balance of the ubiquitin-proteasome system.

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Lindner, Holger A., Nasser Fotouhi-Ardakani, Viktoria Lytvyn, Paule Lachance, Traian Sulea, and Robert Ménard. "The Papain-Like Protease from the Severe Acute Respiratory Syndrome Coronavirus Is a Deubiquitinating Enzyme." Journal of Virology 79, no. 24 (December 15, 2005): 15199–208. http://dx.doi.org/10.1128/jvi.79.24.15199-15208.2005.

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ABSTRACT The severe acute respiratory syndrome coronavirus papain-like protease (SARS-CoV PLpro) is involved in the processing of the viral polyprotein and, thereby, contributes to the biogenesis of the virus replication complex. Structural bioinformatics has revealed a relationship for the SARS-CoV PLpro to herpesvirus-associated ubiquitin-specific protease (HAUSP), a ubiquitin-specific protease, indicating potential deubiquitinating activity in addition to its function in polyprotein processing (T. Sulea, H. A. Lindner, E. O. Purisima, and R. Menard, J. Virol. 79:4550-4551, 2005). In order to confirm this prediction, we overexpressed and purified SARS-CoV PLpro (amino acids [aa]1507 to 1858) from Escherichia coli. The purified enzyme hydrolyzed ubiquitin-7-amino-4-methylcoumarin (Ub-AMC), a general deubiquitinating enzyme substrate, with a catalytic efficiency of 13,100 M−1s−1, 220-fold more efficiently than the small synthetic peptide substrate Z-LRGG-AMC, which incorporates the C-terminal four residues of ubiquitin. In addition, SARS-CoV PLpro was inhibited by the specific deubiquitinating enzyme inhibitor ubiquitin aldehyde, with an inhibition constant of 210 nM. The purified SARS-CoV PLpro disassembles branched polyubiquitin chains with lengths of two to seven (Ub2-7) or four (Ub4) units, which involves isopeptide bond cleavage. SARS-CoV PLpro processing activity was also detected against a protein fused to the C terminus of the ubiquitin-like modifier ISG15, both in vitro using the purified enzyme and in HeLa cells by coexpression with SARS-CoV PLpro (aa 1198 to 2009). These results clearly establish that SARS-CoV PLpro is a deubiquitinating enzyme, thereby confirming our earlier prediction. This unexpected activity for a coronavirus papain-like protease suggests a novel viral strategy to modulate the host cell ubiquitination machinery to its advantage.

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Driessen, Christoph, Jeanette Gogel, Marianne Kraus, Huib Ovaa, Alexander Beck, and Thomas Rückrich. "Acquired Bortezomib (Velcade®)-Resistance Is Accompanied by Downmodulation of Proteasome Activity and Upregulation of Tripeptidyl Peptidase II (TPPII) and a Ubiquitin-Specific Protease." Blood 106, no. 11 (November 16, 2005): 3375. http://dx.doi.org/10.1182/blood.v106.11.3375.3375.

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Abstract Because cell lines can be adapted to proteasome inhibition in vitro (Pfeifer G. et al., Science 283: 978–981, 1999.), secondary resistance of malignant cells towards Bortezomib (Velcade®) in vivo is a likely scenario. We have succeeded to adapt the human AML cell line HL-60 to Bortezomib in vitro, so that the adapted subline (HL-60a) shows normal viability and growth rate at 40 nM Velcade®, while cell death is induced above 10nM in the parental line. We hypothesized that alternative proteolytic pathways might allow the continued proliferation and survival of Bortezomib-adapted cells, and assessed the activity-profiles of proteasomal subunits, ubiquitin-specific proteases and lysosomal cysteine proteases in a functional proteomics approach, using recently developed synthetic affinity probes. These tools for the first time allow semiquantitative visualization of the individual members of these protease families, based on their activity, in contrast to western blot which lacks activity information or to the turnover of fluorogenic substrates, which is not truly protease-specific. After 72h of culture in Bortezomib-free medium (wash out phase), HL-60a cells contained significantly reduced levels of active proteasomal β1,β2, and β5 subunits, compared to the parental line, as confirmed by a reduced turnover of the β5-selective fluorogenic substrate Suc-LLVY-AMC. A panel of 7 different ubiquitin-specific proteases (USP) was visualized in both types of cells, using the affinity probe HAUbVS. Of these, a 97 kD USP that we have identified as USP14 by mass spectrometry-based sequencing, was significantly upregulated in HL-60a cells. By contrast, the activity of lysosomal cysteine proteases remained unchanged in HL-60a cells. Because the cytosolic protease tripeptidyl peptidase II (TPPII) can partially substitute for proteasome activity in lactacystine-treated cell lines, we assessed TPPII-activity using a fluorogenic substrate. We observed a significant upregulation of TPPII-activity in HL-60a cells, compared to non-adapted controls. Inhibition of TPPII, however, was not sufficient to restore Bortezomib-sensitivity in HL-60a cells. Interestingly, the combination of Bortezomib with the HIV protease inhibitor Ritonavir induced synergistic cytotoxicity both in HL-60 and HL-60a cells. Thus, Bortezomib-resistance is accompabied by upregulation of protease activities in alternative pathways. Combining different protease inhibitors like Bortezomib and Ritonavir might be a promising option to overcome primary or secondary Bortezomib resistance.

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Clemente, Valentino, Padraig D’Arcy, and Martina Bazzaro. "Deubiquitinating Enzymes in Coronaviruses and Possible Therapeutic Opportunities for COVID-19." International Journal of Molecular Sciences 21, no. 10 (May 15, 2020): 3492. http://dx.doi.org/10.3390/ijms21103492.

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Following the outbreak of novel severe acute respiratory syndrome (SARS)-coronavirus (CoV)2, the majority of nations are struggling with countermeasures to fight infection, prevent spread and improve patient survival. Considering that the pandemic is a recent event, no large clinical trials have been possible and since coronavirus specific drug are not yet available, there is no strong consensus on how to treat the coronavirus disease 2019 (COVID-19) associated viral pneumonia. Coronaviruses code for an important multifunctional enzyme named papain-like protease (PLP), that has many roles in pathogenesis. First, PLP is one of the two viral cysteine proteases, along with 3-chymotripsin-like protease, that is responsible for the production of the replicase proteins required for viral replication. Second, its intrinsic deubiquitinating and deISGylating activities serve to antagonize the host’s immune response that would otherwise hinder infection. Both deubiquitinating and deISGylating functions involve the removal of the small regulatory polypeptides, ubiquitin and ISG15, respectively, from target proteins. Ubiquitin modifications can regulate the innate immune response by affecting regulatory proteins, either by altering their stability via the ubiquitin proteasome pathway or by directly regulating their activity. ISG15 is a ubiquitin-like modifier with pleiotropic effects, typically expressed during the host cell immune response. PLP inhibitors have been evaluated during past coronavirus epidemics, and have showed promising results as an antiviral therapy in vitro. In this review, we recapitulate the roles of PLPs in coronavirus infections, report a list of PLP inhibitors and suggest possible therapeutic strategies for COVID-19 treatment, using both clinical and preclinical drugs.

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Shin, Ji Yeong, Srinivasan Muniyappan, Non-Nuoc Tran, Hyeonjeong Park, Sung Bae Lee, and Byung-Hoon Lee. "Deubiquitination Reactions on the Proteasome for Proteasome Versatility." International Journal of Molecular Sciences 21, no. 15 (July 27, 2020): 5312. http://dx.doi.org/10.3390/ijms21155312.

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The 26S proteasome, a master player in proteolysis, is the most complex and meticulously contextured protease in eukaryotic cells. While capable of hosting thousands of discrete substrates due to the selective recognition of ubiquitin tags, this protease complex is also dynamically checked through diverse regulatory mechanisms. The proteasome’s versatility ensures precise control over active proteolysis, yet prevents runaway or futile degradation of many essential cellular proteins. Among the multi-layered processes regulating the proteasome’s proteolysis, deubiquitination reactions are prominent because they not only recycle ubiquitins, but also impose a critical checkpoint for substrate degradation on the proteasome. Of note, three distinct classes of deubiquitinating enzymes—USP14, RPN11, and UCH37—are associated with the 19S subunits of the human proteasome. Recent biochemical and structural studies suggest that these enzymes exert dynamic influence over proteasome output with limited redundancy, and at times act in opposition. Such distinct activities occur spatially on the proteasome, temporally through substrate processing, and differentially for ubiquitin topology. Therefore, deubiquitinating enzymes on the proteasome may fine-tune the degradation depending on various cellular contexts and for dynamic proteolysis outcomes. Given that the proteasome is among the most important drug targets, the biology of proteasome-associated deubiquitination should be further elucidated for its potential targeting in human diseases.

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Sy, S. M. H., J. Jiang, W. S. O, Y. Deng, and M. S. Y. Huen. "The ubiquitin specific protease USP34 promotes ubiquitin signaling at DNA double-strand breaks." Nucleic Acids Research 41, no. 18 (July 17, 2013): 8572–80. http://dx.doi.org/10.1093/nar/gkt622.

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Nakamura, Nobuhiro, Kumi Harada, Masako Kato, and Shigehisa Hirose. "Ubiquitin-specific protease 19 regulates the stability of the E3 ubiquitin ligase MARCH6." Experimental Cell Research 328, no. 1 (October 2014): 207–16. http://dx.doi.org/10.1016/j.yexcr.2014.07.025.

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Wang, Hui, Donghong Ju, Dhong-Hyo Kho, Huanjie Yang, Li Li, Avraham Raz, Fei Sun, and Youming Xie. "The ubiquitin specific protease USP34 protects the ubiquitin ligase gp78 from proteasomal degradation." Biochemical and Biophysical Research Communications 509, no. 2 (February 2019): 348–53. http://dx.doi.org/10.1016/j.bbrc.2018.12.141.

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Brew, Christine T., and Tim C. Huffaker. "The Yeast Ubiquitin Protease, Ubp3p, Promotes Protein Stability." Genetics 162, no. 3 (November 1, 2002): 1079–89. http://dx.doi.org/10.1093/genetics/162.3.1079.

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Abstract Stu1p is a microtubule-associated protein required for spindle assembly. In this article we show that the temperature-sensitive stu1-5 allele is synthetically lethal in combination with ubp3, gim1-gim5, and kem1 mutations. The primary focus of this article is on the stu1-5 ubp3 interaction. Ubp3 is a deubiquitination enzyme and a member of a large family of cysteine proteases that cleave ubiquitin moieties from protein substrates. UBP3 is the only one of 16 UBP genes in yeast whose loss is synthetically lethal with stu1-5. Stu1p levels in stu1-5 cells are several-fold lower than the levels in wild-type cells and the stu1-5 temperature sensitivity can be rescued by additional copies of stu1-5. These results indicate that the primary effect of the stu1-5 mutation is to make the protein less stable. The levels of Stu1p are even lower in ubp3Δ stu1-5 cells, suggesting that Ubp3p plays a role in promoting protein stability. We also found that ubp3Δ produces growth defects in combination with mutations in other genes that decrease protein stability. Overall, these data support the idea that Ubp3p has a general role in the reversal of protein ubiquitination.

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Chandrasekaran, Arun Pandian, Sang Hyeon Woo, Neha Sarodaya, Byung Ho Rhie, Apoorvi Tyagi, Soumyadip Das, Bharathi Suresh, et al. "Ubiquitin-Specific Protease 29 Regulates Cdc25A-Mediated Tumorigenesis." International Journal of Molecular Sciences 22, no. 11 (May 28, 2021): 5766. http://dx.doi.org/10.3390/ijms22115766.

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Cell division cycle 25A (Cdc25A) is a dual-specificity phosphatase that is overexpressed in several cancer cells and promotes tumorigenesis. In normal cells, Cdc25A expression is regulated tightly, but the changes in expression patterns in cancer cells that lead to tumorigenesis are unknown. In this study, we showed that ubiquitin-specific protease 29 (USP29) stabilized Cdc25A protein expression in cancer cell lines by protecting it from ubiquitin-mediated proteasomal degradation. The presence of USP29 effectively blocked polyubiquitination of Cdc25A and extended its half-life. CRISPR-Cas9-mediated knockdown of USP29 in HeLa cells resulted in cell cycle arrest at the G0/G1 phase. We also showed that USP29 knockdown hampered Cdc25A-mediated cell proliferation, migration, and invasion of cancer cells in vitro. Moreover, NSG nude mice transplanted with USP29-depleted cells significantly reduced the size of the tumors, whereas the reconstitution of Cdc25A in USP29-depleted cells significantly increased the tumor size. Altogether, our results implied that USP29 promoted cell cycle progression and oncogenic transformation by regulating protein turnover of Cdc25A.

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Mohideen, Firaz, and Christopher D. Lima. "SUMO Takes Control of a Ubiquitin-Specific Protease." Molecular Cell 30, no. 5 (June 2008): 539–40. http://dx.doi.org/10.1016/j.molcel.2008.05.010.

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Tsai, Yien Che, and Allan M. Weissman. "A Ubiquitin-Binding Rhomboid Protease Aimed at ERADication." Developmental Cell 23, no. 3 (September 2012): 454–56. http://dx.doi.org/10.1016/j.devcel.2012.08.015.

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Schneider, Tatjana, Daniel Schneider, Andreas Marx, and Martin Scheffner. "Analyse des „Ubiquitin-Codes“ mithilfe Protease-resistenter Ubiquitinketten." BIOspektrum 21, no. 4 (June 2015): 382–84. http://dx.doi.org/10.1007/s12268-015-0589-4.

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Kwon, Seul-Ki, Eun-Hea Kim, and Kwang-Hyun Baek. "RNPS1 is modulated by ubiquitin-specific protease 4." FEBS Letters 591, no. 2 (January 2017): 369–81. http://dx.doi.org/10.1002/1873-3468.12531.

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Mukhopadhyay, Debaditya, Ferhan Ayaydin, Nagamalleswari Kolli, Shyh-Han Tan, Tadashi Anan, Ai Kametaka, Yoshiaki Azuma, Keith D. Wilkinson, and Mary Dasso. "SUSP1 antagonizes formation of highly SUMO2/3-conjugated species." Journal of Cell Biology 174, no. 7 (September 21, 2006): 939–49. http://dx.doi.org/10.1083/jcb.200510103.

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Small ubiquitin-related modifier (SUMO) processing and deconjugation are mediated by sentrin-specific proteases/ubiquitin-like proteases (SENP/Ulps). We show that SUMO-specific protease 1 (SUSP1), a mammalian SENP/Ulp, localizes within the nucleoplasm. SUSP1 depletion within cell lines expressing enhanced green fluorescent protein (EGFP) fusions to individual SUMO paralogues caused redistribution of EGFP-SUMO2 and -SUMO3, particularly into promyelocytic leukemia (PML) bodies. Further analysis suggested that this change resulted primarily from a deficit of SUMO2/3-deconjugation activity. Under these circumstances, PML bodies became enlarged and increased in number. We did not observe a comparable redistribution of EGFP-SUMO1. We have investigated the specificity of SUSP1 using vinyl sulfone inhibitors and model substrates. We found that SUSP1 has a strong paralogue bias toward SUMO2/3 and that it acts preferentially on substrates containing three or more SUMO2/3 moieties. Together, our findings argue that SUSP1 may play a specialized role in dismantling highly conjugated SUMO2 and -3 species that is critical for PML body maintenance.

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Wang, Zhe, Xuwen Wang, Yu Kang, Haiyang Zhong, Chao Shen, Xiaojun Yao, Dongsheng Cao, and Tingjun Hou. "Binding affinity and dissociation pathway predictions for a series of USP7 inhibitors with pyrimidinone scaffold by multiple computational methods." Physical Chemistry Chemical Physics 22, no. 10 (2020): 5487–99. http://dx.doi.org/10.1039/d0cp00370k.

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Barretto, Naina, Dalia Jukneliene, Kiira Ratia, Zhongbin Chen, Andrew D. Mesecar, and Susan C. Baker. "The Papain-Like Protease of Severe Acute Respiratory Syndrome Coronavirus Has Deubiquitinating Activity." Journal of Virology 79, no. 24 (December 15, 2005): 15189–98. http://dx.doi.org/10.1128/jvi.79.24.15189-15198.2005.

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ABSTRACT Replication of the genomic RNA of severe acute respiratory syndrome coronavirus (SARS-CoV) is mediated by replicase polyproteins that are processed by two viral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro). Previously, we showed that SARS-CoV PLpro processes the replicase polyprotein at three conserved cleavage sites. Here, we report the identification and characterization of a 316-amino-acid catalytic core domain of PLpro that can efficiently cleave replicase substrates in trans-cleavage assays and peptide substrates in fluorescent resonance energy transfer-based protease assays. We performed bioinformatics analysis on 16 papain-like protease domains from nine different coronaviruses and identified a putative catalytic triad (Cys1651-His1812-Asp1826) and zinc-binding site. Mutagenesis studies revealed that Asp1826 and the four cysteine residues involved in zinc binding are essential for SARS-CoV PLpro activity. Molecular modeling of SARS-CoV PLpro suggested that this catalytic core may also have deubiquitinating activity. We tested this hypothesis by measuring the deubiquitinating activity of PLpro by two independent assays. SARS CoV-PLpro hydrolyzed both diubiquitin and ubiquitin-7-amino-4-methylcoumarin (AMC) substrates, and hydrolysis of ubiquitin-AMC is approximately 180-fold more efficient than hydrolysis of a peptide substrate that mimics the PLpro replicase recognition sequence. To investigate the critical determinants recognized by PLpro, we performed site-directed mutagenesis on the P6 to P2′ residues at each of the three PLpro cleavage sites. We found that PLpro recognizes the consensus cleavage sequence LXGG, which is also the consensus sequence recognized by cellular deubiquitinating enzymes. This similarity in the substrate recognition sites should be considered during the development of SARS-CoV PLpro inhibitors.

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Yang, Wei, Liangli Wang, and Wulf Paschen. "Development of a High-Throughput Screening Assay for Inhibitors of Small Ubiquitin-Like Modifier Proteases." Journal of Biomolecular Screening 18, no. 5 (March 7, 2013): 621–28. http://dx.doi.org/10.1177/1087057113479971.

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Small ubiquitin-like modifier (SUMO1–3) is a small group of proteins that are ligated to lysine residues in target proteins. SUMO conjugation is a highly dynamic process, as SUMOylated proteins are rapidly deconjugated by SUMO proteases. SUMO conjugation/deconjugation plays pivotal roles in major cellular pathways and is associated with a number of pathological conditions. It is therefore of significant clinical interest to develop new strategies to screen for compounds to specifically interfere with SUMO conjugation/deconjugation. Here, we describe a novel high-throughput screening (HTS)–compatible assay to identify inhibitors of SUMO proteases. The assay is based on AlphaScreen technology and uses His-tagged SUMO2 conjugated to Strep-tagged SUMO3 as a SUMO protease substrate. A bacterial SUMOylation system was used to generate this substrate. A three-step purification strategy was employed to yield substrate of high quality. Our data indicated that this unique substrate can be readily detected in the AlphaScreen assays in a dose-dependent manner. Cleavage reactions by SUMO protease with or without inhibitor were monitored based on AlphaScreen signals. Furthermore, the assay was adapted to a 384-well format, and the interplate and interday variability was evaluated in eight 384-well plates. The average Z′ factor was 0.83 ± 0.04, confirming the suitability for HTS applications.

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van der Knaap, Jan A., Elena Kozhevnikova, Karin Langenberg, Yuri M. Moshkin, and C. Peter Verrijzer. "Biosynthetic Enzyme GMP Synthetase Cooperates with Ubiquitin-Specific Protease 7 in Transcriptional Regulation of Ecdysteroid Target Genes." Molecular and Cellular Biology 30, no. 3 (December 7, 2009): 736–44. http://dx.doi.org/10.1128/mcb.01121-09.

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ABSTRACT Drosophila GMP synthetase binds ubiquitin-specific protease 7 (USP7) and is required for its ability to deubiquitylate histone H2B. Previously, we showed that the GMPS/USP7 complex cooperates with the Polycomb silencing system through removal of the active ubiquitin mark from histone H2B (H2Bub). Here, we explored the interplay between GMPS and USP7 further and assessed their role in hormone-regulated gene expression. Genetic analysis established a strong cooperation between GMPS and USP7, which is counteracted by the histone H2B ubiquitin ligase BRE1. Loss of either GMPS or USP7 led to increased levels of histone H2Bub in mutant animals. These in vivo analyses complement our earlier biochemical results, establishing that GMPS/USP7 mediates histone H2B deubiquitylation. We found that GMPS/USP7 binds ecdysone-regulated loci and that mutants display severe misregulation of ecdysone target genes. Ecdysone receptor (EcR) interacts biochemically and genetically with GMPS/USP7. Genetic and gene expression analyses suggested that GMPS/USP7 acts as a transcriptional corepressor. These results revealed the cooperation between a biosynthetic enzyme and a ubiquitin protease in developmental gene control by hormone receptors.

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Wang, Shaoyu, Kui Li, Hui Gao, Zepeng Liu, Shuang Shi, Qiang Tan, and Zhengguang Wang. "Ubiquitin-specific peptidase 8 regulates proliferation and early differentiation of sheep skeletal muscle satellite cells." Czech Journal of Animal Science 66, No. 3 (March 2, 2021): 87–96. http://dx.doi.org/10.17221/105/2020-cjas.

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Ubiquitin-specific protease 8 (USP8), a member of the ubiquitin-specific protease (USP) family, was originally identified as playing a role in the regulation of growth and cell cycle. However, its functional role in myogenesis is unknown. In this study, we investigated the role of USP8 in proliferation and differentiation of sheep skeletal muscle satellite cells. The results showed that the expression level of USP8 was significantly increased on days 2 and 3 following the induction of the differentiation process. Furthermore, knocking down USP8 resulted in a significant increase in myogenin-positive cells, and promoted early differentiation of satellite cells by regulating the expression level of paired box 7 (PAX7). Additionally, knocking down USP8 suppressed muscle satellite cell proliferation, possibly explaining that the relative mRNA level of USP8 was linearly related to muscle fibre density of Hu sheep. Overall, our research demonstrates that USP8 plays a role in proliferation and early differentiation of skeletal muscle satellite cells.

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Majumdar, Parinita, Premananda Karidas, Imran Siddiqi, and Utpal Nath. "The Ubiquitin-Specific Protease TNI/UBP14 Functions in Ubiquitin Recycling and Affects Auxin Response." Plant Physiology 184, no. 3 (August 28, 2020): 1499–513. http://dx.doi.org/10.1104/pp.20.00689.

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Tencer, Adam H., Qin Liang, and Zhihao Zhuang. "Divergence in Ubiquitin Interaction and Catalysis among the Ubiquitin-Specific Protease Family Deubiquitinating Enzymes." Biochemistry 55, no. 33 (August 8, 2016): 4708–19. http://dx.doi.org/10.1021/acs.biochem.6b00033.

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Gredmark, Sara, Christian Schlieker, Victor Quesada, Eric Spooner, and Hidde L. Ploegh. "A Functional Ubiquitin-Specific Protease Embedded in the Large Tegument Protein (ORF64) of Murine Gammaherpesvirus 68 Is Active during the Course of Infection." Journal of Virology 81, no. 19 (July 18, 2007): 10300–10309. http://dx.doi.org/10.1128/jvi.01149-07.

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ABSTRACT All herpesviruses contain a ubiquitin (Ub)-specific cysteine protease domain embedded within their large tegument protein, based on homology with the corresponding sequences of UL36 from herpes simplex virus type 1 and M48 from murine cytomegalovirus. This type of activity has yet to be demonstrated for cells infected with a gammaherpesvirus. By activity-based profiling, we show that the large tegument protein of murine gammaherpesvirus (MHV-68) ORF64 (273 kDa) is a functional deubiquitinating protease, as assessed by tandem mass spectrometry of adducts in extracts from MHV-68-infected cells that had been labeled with ubiquitin vinylmethylester, a ubiquitin-based active site-directed probe. The recombinantly expressed amino-terminal segment of ORF64 displays deubiquitinating activity toward Ub C-terminal 7-amido-4-methylcoumarin in vitro. The findings reported here for MHV-68 ORF64 extend those made for the alpha- and betaherpesvirus families and are consistent with an important, conserved enzymatic function of the tegument protein.

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Shen, Lin Nan, Changjiang Dong, Huanting Liu, James H. Naismith, and Ronald T. Hay. "The structure of SENP1–SUMO-2 complex suggests a structural basis for discrimination between SUMO paralogues during processing." Biochemical Journal 397, no. 2 (June 28, 2006): 279–88. http://dx.doi.org/10.1042/bj20052030.

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The SUMO (small ubiquitin-like modifier)-specific protease SENP1 (sentrin-specific protease 1) can process the three forms of SUMO to their mature forms and deconjugate SUMO from modified substrates. It has been demonstrated previously that SENP1 processed SUMO-1 more efficiently than SUMO-2, but displayed little difference in its ability to deconjugate the different SUMO paralogues from modified substrates. To determine the basis for this substrate specificity, we have determined the crystal structure of SENP1 in isolation and in a transition-state complex with SUMO-2. The interface between SUMO-2 and SENP1 has a relatively poor complementarity, and most of the recognition is determined by interaction between the conserved C-terminus of SUMO-2 and the cleft in the protease. Although SENP1 is rather similar in structure to the related protease SENP2, these proteases have different SUMO-processing activities. Electrostatic analysis of SENP1 in the region where the C-terminal peptide, removed during maturation, would project indicates that it is the electrostatic complementarity between this region of SENP1 and the C-terminal peptides of the various SUMO paralogues that mediates selectivity.

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Alegre, Kamela O., and David Reverter. "Swapping Small Ubiquitin-like Modifier (SUMO) Isoform Specificity of SUMO Proteases SENP6 and SENP7." Journal of Biological Chemistry 286, no. 41 (August 30, 2011): 36142–51. http://dx.doi.org/10.1074/jbc.m111.268847.

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SUMO proteases can regulate the amounts of SUMO-conjugated proteins in the cell by cleaving off the isopeptidic bond between SUMO and the target protein. Of the six members that constitute the human SENP/ULP protease family, SENP6 and SENP7 are the most divergent members in their conserved catalytic domain. The SENP6 and SENP7 subclass displays a clear proteolytic cleavage preference for SUMO2/3 isoforms. To investigate the structural determinants for such isoform specificity, we have identified a unique sequence insertion in the SENP6 and SENP7 subclass that is essential for their proteolytic activity and that forms a more extensive interface with SUMO during the proteolytic reaction. Furthermore, we have identified a region in the SUMO surface determinant for the SUMO2/3 isoform specificity of SENP6 and SENP7. Double point amino acid mutagenesis on the SUMO surface allows us to swap the specificity of SENP6 and SENP7 between the two SUMO isoforms. Structure-based comparisons combined with biochemical and mutagenesis analysis have revealed Loop 1 insertion in SENP6 and SENP7 as a platform to discriminate between SUMO1 and SUMO2/3 isoforms in this subclass of the SUMO protease family.

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Liu, Xiaoguang, Kaluvu Balaraman, Ciarán C. Lynch, Michaeline Hebron, Christian Wolf, and Charbel Moussa. "Novel Ubiquitin Specific Protease-13 Inhibitors Alleviate Neurodegenerative Pathology." Metabolites 11, no. 9 (September 15, 2021): 622. http://dx.doi.org/10.3390/metabo11090622.

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Ubiquitin Specific Protease-13 (USP13) promotes protein de-ubiquitination and is poorly understood in neurodegeneration. USP13 is upregulated in Alzheimer’s disease (AD) and Parkinson’s disease (PD), and USP13 knockdown via shRNA reduces neurotoxic proteins and increases proteasome activity in models of neurodegeneration. We synthesized novel analogues of spautin-1 which is a non-specific USP13 inhibitor but unable to penetrate the brain. Our synthesized small molecule compounds are able to enter the brain, more potently inhibit USP13, and significantly reduce alpha-synuclein levels in vivo and in vitro. USP13 inhibition in transgenic mutant alpha-synuclein (A53T) mice increased the ubiquitination of alpha-synuclein and reduced its protein levels. The data suggest that novel USP13 inhibitors improve neurodegenerative pathology via antagonism of de-ubiquitination, thus alleviating neurotoxic protein burden in neurodegenerative diseases.

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Sun, Jian, Qianwen Hu, Hong Peng, Cheng Peng, Liheng Zhou, Jinsong Lu, and Chuanxin Huang. "The ubiquitin-specific protease USP8 deubiquitinates and stabilizes Cx43." Journal of Biological Chemistry 293, no. 21 (April 6, 2018): 8275–84. http://dx.doi.org/10.1074/jbc.ra117.001315.

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Connexin-43 (Cx43, also known as GJA1) is the most ubiquitously expressed connexin isoform in mammalian tissues. It forms intercellular gap junction (GJ) channels, enabling adjacent cells to communicate both electrically and metabolically. Cx43 is a short-lived protein which can be quickly degraded by the ubiquitin-dependent proteasomal, endolysosomal, and autophagosomal pathways. Here, we report that the ubiquitin-specific peptidase 8 (USP8) interacts with and deubiquitinates Cx43. USP8 reduces both multiple monoubiquitination and polyubiquitination of Cx43 to prevent autophagy-mediated degradation. Consistently, knockdown of USP8 results in decreased Cx43 protein levels in cultured cells and suppresses intercellular communication, revealed by the dye transfer assay. In human breast cancer specimens, the expression levels of USP8 and Cx43 proteins are positively correlated. Taken together, these results identified USP8 as a crucial and bona fide deubiquitinating enzyme involved in autophagy-mediated degradation of Cx43.

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Ha, Byung Hak, Young Joo Jeon, Sang Chul Shin, Kanako Tatsumi, Masaaki Komatsu, Keiji Tanaka, Christopher M. Watson, Gillian Wallis, Chin Ha Chung, and Eunice EunKyeong Kim. "Structure of Ubiquitin-fold Modifier 1-specific Protease UfSP2." Journal of Biological Chemistry 286, no. 12 (January 12, 2011): 10248–57. http://dx.doi.org/10.1074/jbc.m110.172171.

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Ubiquitin-fold modifier 1 (Ufm1)-specific protease 2 (UfSP2) is a cysteine protease that is responsible for the release of Ufm1 from Ufm1-conjugated cellular proteins, as well as for the generation of mature Ufm1 from its precursor. The 2.6 Å resolution crystal structure of mouse UfSP2 reveals that it is composed of two domains. The C-terminal catalytic domain is similar to UfSP1 with Cys294, Asp418, His420, Tyr282, and a regulatory loop participating in catalysis. The novel N-terminal domain shows a unique structure and plays a role in the recognition of its cellular substrate C20orf116 and thus in the recruitment of UfSP2 to the endoplasmic reticulum, where C20orf116 predominantly localizes. Mutagenesis studies were carried out to provide the structural basis for understanding the loss of catalytic activity observed in a recently identified UfSP2 mutation that is associated with an autosomal dominant form of hip dysplasia.

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Faus, Hortensia, Hellmuth-Alexander Meyer, Martina Huber, Inke Bahr, and Bernard Haendler. "The ubiquitin-specific protease USP10 modulates androgen receptor function." Molecular and Cellular Endocrinology 245, no. 1-2 (December 2005): 138–46. http://dx.doi.org/10.1016/j.mce.2005.11.011.

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Deveraux, Q., V. Ustrell, C. Pickart, and M. Rechsteiner. "A 26 S protease subunit that binds ubiquitin conjugates." Journal of Biological Chemistry 269, no. 10 (March 1994): 7059–61. http://dx.doi.org/10.1016/s0021-9258(17)37244-7.

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Zhu, Lihong, Wei Bi, Dan Lu, Chanjuan Zhang, Xiaoming Shu, Huadong Wang, Renbing Qi, Qiaoyun Shi, and Daxiang Lu. "Regulation of ubiquitin-specific processing protease 8 suppresses neuroinflammation." Molecular and Cellular Neuroscience 64 (January 2015): 74–83. http://dx.doi.org/10.1016/j.mcn.2014.05.004.

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Leo, Manuela, Giulia Fanelli, Serena Di Vito, Barbara Traversetti, Mariafrancesca La Greca, Raffaele A. Palladino, Arianna Montanari, Silvia Francisci, and Patrizia Filetici. "Ubiquitin protease Ubp8 is necessary for S. cerevisiae respiration." Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1865, no. 10 (October 2018): 1491–500. http://dx.doi.org/10.1016/j.bbamcr.2018.07.025.

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Moon, Byeong Cheol, Man Soo Choi, Yun Hwan Kang, Min Chul Kim, Mi Sun Cheong, Chan Young Park, Jae Hyuk Yoo, et al. "Arabidopsis ubiquitin-specific protease 6 (AtUBP6) interacts with calmodulin." FEBS Letters 579, no. 18 (June 16, 2005): 3885–90. http://dx.doi.org/10.1016/j.febslet.2005.05.080.

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Zhou, Fangfang, Fang Li, Pengfei Fang, Tong Dai, Bing Yang, Hans van Dam, Junling Jia, Min Zheng, and Long Zhang. "Ubiquitin-Specific Protease 4 Antagonizes Osteoblast Differentiation Through Dishevelled." Journal of Bone and Mineral Research 31, no. 10 (May 20, 2016): 1888–98. http://dx.doi.org/10.1002/jbmr.2863.

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Agell, N., C. Ryan, and M. J. Schlesinger. "Partial purification and substrate specificity of a ubiquitin hydrolase from Saccharomyces cerevisiae." Biochemical Journal 273, no. 3 (February 1, 1991): 615–20. http://dx.doi.org/10.1042/bj2730615.

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A ubiquitin hydrolase that removes ubiquitin from a multi-ubiquitinated protein has been purified 600-fold from Saccharomyces cerevisiae. Four different ubiquitin-protein conjugates were assayed as substrates during the purification procedure. Enzymic activities that removed ubiquitin from ubiquitinated histone H2A, a ubiquitin-ubiquitin dimer and a ubiquitin-ribosomal fusion protein were separated during the purification from an activity that removed a single ubiquitin molecule linked by an isopeptide bond to a ubiquitinated protein. The size of the native enzyme was 160 kDa, based on its sedimentation in a sucrose gradient, and the subunit molecular mass was estimated to be 160 kDa, based on a profile of proteins eluted in different fractions by thiol-affinity chromatography. The partially purified hydrolase was not inhibited by a variety of protease inhibitors, except for thiol-blocking reagents. The natural substrate for this enzyme may be the polyubiquitin chain containing ubiquitin molecules bound to each other in isopeptide bonds, with one of them linked to a lysine residue of a protein targeted for intracellular proteolysis.

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Journal articles on the topic 'Ubiquitin protease'

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