Deubiquitinating Activity Research Articles

Discovery of novel papain-like protease inhibitors for potential treatment of COVID-19

The recent emergence of different SARS-CoV-2 variants creates an urgent need to develop more effective therapeutic agents to prevent COVID-19 outbreaks. Among SARS-CoV-2 essential proteases is papain-like protease (SARS-CoV-2 PLpro), which plays multiple roles in regulating SARS-CoV-2 viral spread and innate immunity such as deubiquitinating and deISG15ylating (interferon-induced gene 15) activities. Many studies are currently focused on targeting this protease to tackle SARS-CoV-2 infection. In this context, we performed a phenotypic screening using an in-house pilot compounds collection possessing a diverse skeleta against SARS-CoV-2 PLpro. This screen identified SIMR3030 as a potent inhibitor of SARS-CoV-2. SIMR3030 has been shown to exhibit deubiquitinating activity and inhibition of SARS-CoV-2 specific gene expression (ORF1b and Spike) in infected host cells and possessing virucidal activity. Moreover, SIMR3030 was demonstrated to inhibit the expression of inflammatory markers, including IFN-α, IL-6, and OAS1, which are reported to mediate the development of cytokine storms and aggressive immune responses. In vitro absorption, distribution, metabolism, and excretion (ADME) assessment of the drug-likeness properties of SIMR3030 demonstrated good microsomal stability in liver microsomes. Furthermore, SIMR3030 demonstrated very low potency as an inhibitor of CYP450, CYP3A4, CYP2D6 and CYP2C9 which rules out any potential drug-drug interactions. In addition, SIMR3030 showed moderate permeability in Caco2-cells. Critically, SIMR3030 has maintained a high in vivo safety profile at different concentrations. Molecular modeling studies of SIMR3030 in the active sites of SARS-CoV-2 and MERS-CoV PLpro were performed to shed light on the binding modes of this inhibitor. This study demonstrates that SIMR3030 is a potent inhibitor of SARS-CoV-2 PLpro that forms the foundation for developing new drugs to tackle the COVID-19 pandemic and may pave the way for the development of novel therapeutics for a possible future outbreak of new SARS-CoV-2 variants or other Coronavirus species.

The Papain-Like Protease of Porcine Reproductive and Respiratory Syndrome Virus Impedes STING Translocation from the Endoplasmic Reticulum to the Golgi Apparatus by Deubiquitinating STIM1.

Stimulator of interferon (IFN) genes (STING) was recently pinpointed as an antiviral innate immune factor during the infection of RNA viruses. Porcine reproductive and respiratory syndrome virus (PRRSV), the swine arterivirus, is an enveloped RNA virus which has evolved many strategies to evade innate immunity. To date, the interactive network between PRRSV and STING remains to be fully established. Herein, we report that STING suppresses PRRSV replication through type I interferon signaling. However, PRRSV impedes STING trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus, leading to the decreased phosphorylation of TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3). Furthermore, PRRSV nonstructural protein 2 (Nsp2) colocalizes with STING, blocks STING translocation, and disrupts the STING-TBK1-IRF3 complex. Mechanistically, PRRSV Nsp2 retains STING at the ER by increasing the level of Ca2+ sensor stromal interaction molecule 1 (STIM1) protein. Functional analysis reveals that PRRSV Nsp2 deubiquitinates STIM1 by virtue of its papain-like protease 2 (PLP2) deubiquitinating (DUB) activity. Finally, we demonstrate that loss of STIM1 is associated with an elevated IFN response and restricts PRRSV replication. This work delineates the relationship between PRRSV infection and STING signaling and the importance of papain-like proteases (PLPs) in interfering in this axis. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the family Arteriviridae, is responsible for reproductive disorders in pregnant sows and respiratory problems in piglets, resulting in huge losses in the swine industry worldwide. Of note, PRRSV infection causes immunosuppression, of which the mechanism is not completely understood. Here, we demonstrate for the first time that STING, a protein typically associated with the antiviral response in DNA viruses, plays a critical role in controlling PRRSV infection. However, PRRSV utilizes its encoded protein Nsp2 to inhibit STING activity by blocking its translocation from the ER to the Golgi apparatus. In particular, Nsp2 retains STING at the ER by interacting with and further deubiquitinating STIM1. For this process, the activity of the viral PLP2 DUB enzyme is indispensable. The study describes a novel mechanism by which PLP2 plays a critical role in suppressing the innate immune response against arteriviruses and potentially other viruses that encode similar proteases.

USP35 dimer prevents its degradation by E3 ligase CHIP through auto-deubiquitinating activity.

Recently, a number of reports on the importance of USP35 in cancer have been published. However, very little is known about the exact mechanism by which USP35 activity is regulated. Here, we show the possible regulation of USP35 activity and the structural specificity affecting its function by analyzing various fragments of USP35. Interestingly, the catalytic domain of USP35 alone does not exhibit deubiquitinating activity; in contrast, the C-terminal domain and insertion region in the catalytic domain is required for full USP35 activity. Additionally, through its C-terminal domain, USP35 forms a homodimer that prevents USP35 degradation. CHIP bound to HSP90 interacts with and ubiquitinates USP35. However, when fully functional USP35 undergoes auto-deubiquitination, which attenuates CHIP-mediated ubiquitination. Finally, USP35 dimer is required for deubiquitination of the substrate Aurora B and regulation of faithful mitotic progression. The properties of USP35 identified in this study are a unique homodimer structure, regulation of deubiquitinating activity through this, and utilization of a novel E3 ligase involved in USP35 auto-deubiquitination, which adds another complexity to the regulation of deubiquitinating enzymes.

The SAGA HAT module is tethered by its SWIRM domain and modulates activity of the SAGA DUB module

The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is a transcriptional co-activator that both acetylates and deubiquitinates histones. The histone acetyltransferase (HAT) subunit, Gcn5, is part of a subcomplex of SAGA called the HAT module. A minimal HAT module complex containing Gcn5 bound to Ada2 and Ada3 is required for full Gcn5 activity on nucleosomes. Deletion studies have suggested that the Ada2 SWIRM domain plays a role in tethering the HAT module to the remainder of SAGA. While recent cryo-EM studies have resolved the structure of the core of the SAGA complex, the HAT module subunits and molecular details of its interactions with the SAGA core could not be resolved. Here we show that the SWIRM domain is required for incorporation of the HAT module into the yeast SAGA complex, but not the ADA complex, a distinct six-protein acetyltransferase complex that includes the SAGA HAT module proteins. In the isolated Gcn5/Ada2/Ada3 HAT module, deletion of the SWIRM domain modestly increased activity but had negligible effect on nucleosome binding. Loss of the HAT module due to deletion of the SWIRM domain decreases the H2B deubiquitinating activity of SAGA, indicating a role for the HAT module in regulating SAGA DUB module activity. A model of the HAT module created with Alphafold Multimer provides insights into the structural basis for our biochemical data, as well as prior deletion studies.

Depleting the 19S proteasome regulatory PSMD1 subunit as a cancer therapy strategy.

Proteasome inhibitors are in use in treating certain types of cancers. These drugs inhibit the catalytic activity of the 20S proteasome, shared by all the different proteasome complexes. Inhibitors of the 26S-associated deubiquitinating activity explicitly inhibit the 26S proteasomal degradation of ubiquitinylated substrates. We have previously reported an alternative strategy that is based on reducing the 26S/20S ratio by depleting PSMD1, 6, and 11, the subunits of the 19S proteasome regulatory complex. Given the addiction of the many cancer types to a high 26S/20S ratio, the depletion strategy is highly effective in killing many aggressive cancer cell lines but not mouse and human immortalized and normal cells. We used two aggressive cell lines, MDA-MB-231, a triple-negative breast tumor cell line, and OVCAR8, a high-grade ovary adenocarcinoma. Cell culture, mouse MDA-MB-231, OVCAR8 xenografts, and patient-derived ovarian cancer xenograft (PDX) models were transduced with lentivectors expressing PSMD1 shRNA. Tumor size was measured to follow treatment efficacy. Using different experimental strategies of expressing shRNA, we found that PSMD1 depletion, either by expressing PSMD1 shRNA in an inducible manner or in a constitutive manner, robustly inhibited MDA-MB-231, and OVCAR8 xenograft tumor growth. Furthermore, the PSMD1 depletion strategy compromised the growth of the PDX of primary ovarian cancer. Our results suggest that reducing the 26S/20S ratio might be a valuable strategy for treating drug-resistant aggressive types of cancers.

A ubiquitin-specific protease functions in regulating cell death and immune responses in rice.

Ubiquitin-specific proteases (UBPs) process deubiquitination in eukaryotic organisms and are widely involved in plant development and responses to environmental stress. However, their role in cell death and plant immunity remains largely unknown. Here, we identified a rice lesion mimic mutant (LMM) and cloned its causative gene, LMM22. Both dysfunction and overexpression of LMM22 gave rise to the hypersensitive response-like cell death, reactive oxygen species bursts, and activated defence responses. LMM22 encodes an active UBP that is localised to the endoplasmic reticulum (ER) and displays a constitutive expression pattern in rice. LMM22 interacts with SPOTTED LEAF 35 (SPL35), a coupling of ubiquitin conjugation to ER degradation domain-containing protein that is known to participate in ubiquitination and the regulation of cell death and disease response in rice. Additional analyses suggest that LMM22 can positively regulate and stabilise the abundance of SPL35 protein likely through its deubiquitination activity. These data therefore improve our understanding of the function of UBP in rice innate immune responses by demonstrating that LMM22 functions as a critical regulator of SPL35 in cell death and disease resistance.

Opposing USP19 splice variants in TGF-β signaling and TGF-β-induced epithelial–mesenchymal transition of breast cancer cells

Ubiquitin-specific protease (USP)19 is a deubiquitinating enzyme that regulates the stability and function of multiple proteins, thereby controlling various biological responses. The alternative splicing of USP19 results in the expression of two major encoded variants that are localized to the endoplasmic reticulum (ER) (USP19-ER) and cytoplasm (USP19-CY). The importance of alternative splicing for the function of USP19 remains unclear. Here, we demonstrated that USP19-CY promotes TGF-β signaling by directly interacting with TGF-β type I receptor (TβRI) and protecting it from degradation at the plasma membrane. In contrast, USP19-ER binds to and sequesters TβRI in the ER. By decreasing cell surface TβRI levels, USP19-ER inhibits TGF-β/SMAD signaling in a deubiquitination-independent manner. Moreover, USP19-ER inhibits TGF-β-induced epithelial–mesenchymal transition (EMT), whereas USP19-CY enhances EMT, as well as the migration and extravasation of breast cancer cells. Furthermore, USP19-CY expression is correlated with poor prognosis and is higher in breast cancer tissues than in adjacent normal tissues. Notably, the splicing modulator herboxidiene inhibits USP19-CY, increases USP19-ER expression and suppresses breast cancer cell migration. Targeting USP19 splicing or its deubiquitinating activity may have potential therapeutic effects on breast cancer.

Ubiquitin receptor PSMD4/Rpn10 is a novel therapeutic target in multiple myeloma

AbstractPSMD4/Rpn10 is a subunit of the 19S proteasome unit that is involved with feeding target proteins into the catalytic machinery of the 26S proteasome. Because proteasome inhibition is a common therapeutic strategy in multiple myeloma (MM), we investigated Rpn10 and found that it is highly expressed in MM cells compared with normal plasma cells. Rpn10 levels inversely correlated with overall survival in patients with MM. Inducible knockout or knockdown of Rpn10 decreased MM cell viability both in vitro and in vivo by triggering the accumulation of polyubiquitinated proteins, cell cycle arrest, and apoptosis associated with the activation of caspases and unfolded protein response-related pathways. Proteomic analysis revealed that inhibiting Rpn10 increased autophagy, antigen presentation, and the activation of CD4+ T and natural killer cells. We developed an in vitro AlphaScreen binding assay for high-throughput screening and identified a novel Rpn10 inhibitor, SB699551 (SB). Treating MM cell lines, leukemic cell lines, and primary cells from patients with MM with SB decreased cell viability without affecting the viability of normal peripheral blood mononuclear cells. SB inhibited the proliferation of MM cells even in the presence of the tumor-promoting bone marrow milieu and overcame proteasome inhibitor (PI) resistance without blocking the 20S proteasome catalytic function or the 19S deubiquitinating activity. Rpn10 blockade by SB triggered MM cell death via similar pathways as the genetic strategy. In MM xenograft models, SB was well tolerated, inhibited tumor growth, and prolonged survival. Our data suggest that inhibiting Rpn10 will enhance cytotoxicity and overcome PI resistance in MM, providing the basis for further optimization studies of Rpn10 inhibitors for clinical application.

Deubiquitinating enzyme Josephin-2 stabilizes PHGDH to promote a cancer stem cell phenotype in hepatocellular carcinoma.

Deubiquitinating enzymes (DUBs) have been shown to be possible targets for hepatocellular carcinoma (HCC) treatment. This study was designed to reveal the effect and underlying mechanism of Josephin-2, a relatively newly defined DUB, in HCC progression. SNU-387 and PLC/PRF/5 cells were used for in vitro functional assays. The levels of Josephin-2 and phosphoglycerate dehydrogenase (PHGDH) were determined using RT-qPCR and western blotting. Cell proliferation, migration and invasion were assessed by CCK-8, colony formation and Transwell. Spheroid-forming assay was performed to assess the cancer stem cell (CSC)-phenotype of HCC cells. A xenograft mice model was applied to verify the effect of Josephin-2 on HCC cell growth in vivo. Herein, we showed that Josephin-2 expression was negatively correlated with HCC patient survival in data from the online database. Cell experiments indicated that knockdown of Josephin-2 attenuated HCC cell malignant biological behaviors. Besides, Josephin-2 silencing also decreased the spheroid-formation while inhibited the expression of CSC biomarkers (CD133, OCT4, SOX2 and EpCAM) in HCC cells. Mechanistically, Josephin-2 had a deubiquitinating activity towards the regulation of PHGDH protein, the rate-limiting enzyme in the first step of serine biosynthesis pathway. Depletion of Josephin-2 enhanced the ubiquitination degradation of PHGDH and ultimately inhibited the proliferation and CSC-phenotype of HCC in vitro and in vivo. Our work uncovered the regulatory effects of Josephin-2 on PHGDH protein stability and profiled its contribution in HCC malignant progression, which might provide a potential therapeutic target for HCC.

BAP1 UCH‐domain enzymatic activity

AbstractPurpose: Pathogenic variants in BRCA‐associated protein 1 (BAP1) cause BAP1 tumour predisposition syndrome (BAP1‐TPDS) with an elevated risk of several cancers including uveal melanoma (UM). BAP1 is a deubiquitinating enzyme that needs to retain its enzymatic activity and be able to locate in the nucleus to function as a tumour suppressor. BAP1 loss of function variants are easily interpreted as pathogenic, but many missense variants remain as of unknown significance (VUS). We studied the effect of UCH domain missense variants on the enzymatic activity of BAP1.Methods: We selected 22 rare missense variants within or near the UCH domain (aa1‐240). Nineteen have been identified in patients with UM (Walpole et al.) and three in the general population (according to gnomAD). The variants were cloned to a bacterial vector and expressed as a GST‐fusion protein. Finally, we assayed the purified fusion proteins for their ability to cleave ubiquitin.Results: Nine variants significantly reduced the deubiquitinating activity of BAP1. Seven mutants with variants found in patients with familial BAP1‐TPDS, retained <20% of their activity. Three variants retained about 50% of activity, and the function of 12 variants was interpreted as normal (70%–120%).Conclusions: Functional studies are needed for accurate classification of BAP1 missense variants. Although BAP1 variants are dominant, penetrance might be affected by variants effect on enzymatic activity and patients with pathogenic variants might not exhibit familial BAP1 ‐TPDS. Some pathogenic variants that have been suggested to be pathogenic retained enzymatic activity, casting doubt on their classification. If enzymatic activity is retained, re‐classification should be considered or, when found in a family with clear BAP1‐TPDS, further studies need to be conducted.References.1. Walpole S, Pritchard AL, Cebulla CM, et al. Comprehensive Study of the Clinical Phenotype of Germline BAP1 Variant‐Carrying Families Worldwide. JNCI: Journal of the National Cancer Institute 2018; 110(12): 1328–1341.

Ubiquitin-specific peptidase 14 maintains estrogen receptor α stability via its deubiquitination activity in endometrial cancer

USP14 deubiquitinates ERα to maintain its stability in ECEndometrial cancer (EC) is one of the common gynecological malignancies of which the incidence has been rising for decades. It is considered that continuously unopposed estrogen exposure is the main risk factor for EC initiation. Thus, exploring the modulation of estrogen/estrogen receptor α (ERα) signaling pathway in EC would be helpful to well understand the mechanism of EC development and find the potential target for EC therapy. Ubiquitin-specific peptidase 14 (USP14), a member of the proteasome-associated deubiquitinating enzyme family, plays a crucial role in a series of tumors. However, the function of USP14 in EC is still elusive. Here, our results have demonstrated that USP14 is highly expressed in EC tissues compared with that in normal endometrial tissues, and higher expression of USP14 is positively correlated with poor prognosis. Moreover, USP14 maintains ERα stability through its deubiquitination activity. Our results further demonstrate that USP14 depletion decreases the expression of ERα-regulated genes in EC-derived cell lines. Moreover, knockdown of USP14 or USP14-specific inhibitor treatment significantly suppresses cell growth and migration in EC cell lines or in mice. We further provide the evidence to show that the effect of USP14 on EC cell growth, if not all, at least is partially related to ERα pathway. Our study provides new sights for USP14 to be a potential therapeutic target for the treatment of EC, especially for EC patients with fertility preservation needs.

Insight into the physiological and pathological roles of USP44, a potential tumor target (Review).

Ubiquitin-specific peptidase 44 (USP44) is a member of the ubiquitin-specific proteases (USPs) family and its functions in various biological processes have been gradually elucidated in recent years. USP44 targets multiple downstream factors and regulates multiple mechanisms through its deubiquitination activity. Ubiquitination is, in essence, a process in which a single ubiquitin molecule or a multiubiquitin chain binds to a substrate protein to form an isopeptide bond. Deubiquitination is the catalyzing of the isopeptide bonds between ubiquitin and substrate proteins through deubiquitylating enzymes. These two processes serve an important role in the regulation of the expression, conformation, localization and function of substrate proteins by regulating their binding to ubiquitin. Based on existing research, this paper summarized the current state of knowledge about USP44. The physiological roles of USP44 in various cellular events and its pathophysiological roles in different cancer types are evaluated and the therapeutic potential of USP44 for cancer treatment is evaluated.

PUL36 Deubiquitinase Activity Augments Both the Initiation and the Progression of Lytic Herpes Simplex Virus Infection in IFN-Primed Cells.

The evolutionarily conserved, structural HSV-1 tegument protein pUL36 is essential for both virus entry and assembly. While its N-terminal deubiquitinase (DUB) activity is dispensable for infection in cell culture, it is required for efficient virus spread in vivo, as it acts as a potent viral immune evasin. Interferon (IFN) induces the expression of hundreds of antiviral factors, including many ubiquitin modulators, which HSV-1 needs to neutralize to efficiently initiate a productive infection. Herein, we discover two functions of the conserved pUL36 DUB during lytic replication in cell culture in an understudied but equally important scenario of HSV-1 infection in IFN-treated cells. Our data indicate that the pUL36 DUB contributes to overcoming the IFN-mediated suppression of productive infection in both the early and late phases of HSV-1 infection. We show that incoming tegument-derived pUL36 DUB activity contributes to the IFN resistance of HSV-1 in IFN-primed cells to efficiently initiate lytic virus replication. Subsequently, the de novo expressed DUB augmented the efficiency of virus replication and increased the output of infectious virus. Notably, the DUB defect was only apparent when IFN was applied prior to infection. Our data indicate that IFN-induced defense mechanisms exist and that they work to both neutralize infectivity early on and slow the progression of HSV-1 replication in the late stages of infection. Also, our data indicate that pUL36 DUB activity contributes to the disarming of these host responses. IMPORTANCE HSV-1 is a ubiquitous human pathogen that is responsible for common cold sores and may also cause life-threatening disease. pUL36 is an essential, conserved herpesvirus protein with N-terminal deubiquitinating (DUB) activity. The DUB is dispensable for HSV-1 replication in cell culture but represents an important viral immune evasin in vivo. IFN plays a pivotal role in HSV-1 infection and suppresses viral replication both in vitro and in vivo. Here, we show that DUB activity contributes to overcoming IFN-induced cellular resistance in order to more efficiently initiate lytic replication and produce infectious virions. As such, DUB activity in the incoming virions increases their infectivity, while the de novo synthesized DUB augments productive infection. Thus, the HSV-1 DUB antagonizes the activity of IFN-inducible effector proteins to facilitate productive infection at multiple levels. Our findings underscore the importance of using more challenging cell culture systems to fully understand virus protein functions.

A20 controls RANK-dependent osteoclast formation and bone physiology.

The anti-inflammatory protein A20 serves as a critical brake on NF-κB signaling and NF-κB-dependent inflammation. In humans, polymorphisms in or near the TNFAIP3/A20 gene have been associated with several inflammatory disorders, including rheumatoid arthritis (RA), and experimental studies in mice have demonstrated that myeloid-specific A20 deficiency causes the development of a severe polyarthritis resembling human RA. Myeloid A20 deficiency also promotes osteoclastogenesis in mice, suggesting a role for A20 in the regulation of osteoclast differentiation and bone formation. We show here that osteoclast-specific A20 knockout mice develop severe osteoporosis, but not inflammatory arthritis. In vitro, osteoclast precursor cells from A20 deficient mice are hyper-responsive to RANKL-induced osteoclastogenesis. Mechanistically, we show that A20 is recruited to the RANK receptor complex within minutes of ligand binding, where it restrains NF-κB activation independently of its deubiquitinating activity but through its zinc finger (ZnF) 4 and 7 ubiquitin-binding functions. Together, these data demonstrate that A20 acts as a regulator of RANK-induced NF-κB signaling to control osteoclast differentiation, assuring proper bone development and turnover.

Structural Insights into the Phosphorylation-Enhanced Deubiquitinating Activity of UCHL3 and Ubiquitin Chain Cleavage Preference Analysis.

Ubiquitin C-terminal hydrolase-L3 (UCHL3), an important member of the ubiquitin C-terminal hydrolase family, is involved in DNA repair and cancer development. UCHL3 can cleave only complexes of monoubiquitin and its conjugates, such as Ub-AMC, His, or small ubiquitin-like modifier, but not polyubiquitin chains. Phosphorylation of Ser75 promotes the cleavage activity of UCHL3 toward poly-ubiquitin chains in vivo, but biochemical evidence in vitro is still lacking. Here, we first analyzed the structure of simulated phosphorylated UCHL3S75E and the complex of UCHL3S75E with Ub-PA and preliminarily explained the structural mechanism of phosphorylation-enhanced UCHL3 deubiquitinating activity. Additionally, the cleavage activity of UCHL3 toward different types of synthesized poly-ubiquitin chains in vitro was tested. The results showed that purified UCHL3S75E enhanced the cleavage activity toward Ub-AMC compared to UCHL3WT. Meanwhile, UCHL3S75E and UCHL3WT did not show any cleavage activity for different types of di-ubiquitin and tri-ubiquitin chains. However, UCHL3 could hydrolyze the K48 tetra-ubiquitin chain, providing compelling in vitro evidence confirming previous in vivo results. Thus, this study shows that UCHL3 can hydrolyze and has a cleavage preference for polyubiquitin chains, which expands our understanding of the phosphorylation regulation of UCHL3 and lays a foundation for further elucidation of its physiological role.

Discovery of a small-molecule inhibitor targeting the ovarian tumor domain of a novel Tamdy orthonairoviruse associated with human febrile illness.

Tick-borne orthonairoviruses have been characterized as a global health threat to humans and animals. Tacheng Tick virus 1 (TcTV-1) from this family was provided as evidence that is associated with the febrile illness syndrome. Here, we first identify and demonstrate that the ovarian tumor(OTU) domain of TcTV-1 has remarkable deubiquitinating activity both in vitro and in vivo. By solving the crystal structure of TcTV-1 OTU (tcOTU) domain and comparing it to that of human deubiquitinating enzymes, we found that overall structures of tcOTU and human OTU family are similar, but the residues involved in the catalytic pocket vary widely. Based on the tcOTU domain we screened 5090 bioactive compounds and found mecobalamin had a good effect on suppressing the deubiquitinating activity. The structural model of tcOTU and mecobalamin suggests that mecobalamin occupies the site of the substrate Ub, by blocking the substrate binding to the enzyme. Thus, our results showed OTU domain of TcTV-1 has a robust deubiquitinating activity and mecobalamin or its derivatives might be promising candidates for the treatment or prevention of disease caused by theTcTV-1 virus.

Deubiquitinase USP19 extends the residual enzymatic activity of phenylalanine hydroxylase variants

Phenylalanine hydroxylase (PAH) is a key enzyme in mammals that maintains the phenylalanine (Phe) concentration at an appropriate physiological level. Some genetic mutations in the PAH gene lead to destabilization of the PAH enzyme, leading to phenylketonuria (PKU). Destabilized PAH variants can have a certain amount of residual enzymatic activity that is sufficient for metabolism of Phe. However, accelerated degradation of those variants can lead to insufficient amounts of cellular PAH protein. The optimal protein level of PAH in cells is regulated by a balancing act between E3 ligases and deubiquitinating enzymes (DUBs). In this work, we analyzed the protein expression and stability of two PKU-linked PAH protein variants, R241C and R243Q, prevalent in the Asian population. We found that the tested PAH variants were highly ubiquitinated and thus targeted for rapid protein degradation. We demonstrated that USP19, a DUB that interacts with both PAH variants, plays a regulatory role by extending their half-lives. The deubiquitinating activity of USP19 prevents protein degradation and increases the abundance of both PAH protein variants. Thus, our study reveals a novel mechanism by which deubiquitinating activity of USP19 extends the residual enzymatic activity of PAH variants.

Papain-like protease of SARS-CoV-2 inhibits RLR signaling in a deubiquitination-dependent and deubiquitination-independent manner.

The newly emerged severe acute respiratory syndrome (SARS) coronavirus-2 (SARS-CoV-2) can result in dysregulated interferon (IFN) responses that contribute to disease severity. The papain-like protease of SARS-CoV-2 (SCoV2-PLpro) has been previously reported to attenuate IFN responses, but the underlying mechanism is not fully understood. In this study, we found that SCoV2-PLpro potently suppressed IFN production and signaling induced by Sendai virus as well as RIG-I-like receptor (RLR) signaling pathway components, including RIG-I, MAVS, TBK1, TRAF3, TRAF6, and IRF3. SCoV2-PLpro exhibited different specificity and efficiency than SARS-CoV PLpro, with the former exerting a greater inhibitory effect on the RIG-I- and TRAF3-mediated IFN response but a weaker effect on the MAVS-mediated IFN response. Furthermore, we showed that SCoV2-PLpro significantly reduced K63-ubiquitination of RIG-I, MAVS, TBK1, TRAF3, TRAF6, and IRF3 and K48-ubiquitination of IκBα, which are known critical for the innate immune signal transduction. The deubiquitinating (DUB) activity of SCoV2-PLpro required a catalytic residue cysteine 111 (C111) but not the UBL domain. Notably, by utilizing the DUB-defective C111 mutant, we demonstrated that SCoV2-PLpro targeted RLR signaling pathway regulators via deubiquitination-dependent and -independent mechanisms, with the inhibitory activities of RIG-I and TBK1 correlating with DUB function, whereas the antagonism effects on MAVS, TRAF3, TRAF6, and IRF3 independent on DUB activity. Overall, our results reveal that SCoV2-PLpro evolves differential IFN antagonism activity from SCoV1-PLpro and it targets multiple key RLR signaling pathway components via various mechanisms, providing insights into SARS-CoV-2 pathogenesis and clues for developing antiviral therapies for COVID-19.

Genotoxicity-Stimulated and CYLD-Driven Malignant Transformation.

Oxidative stress, which can cause DNA damage, can both activate TNF-R1 directly in the absence of TNF stimulation and phosphorylate c-Abl, thus promoting its cytoplasmic translocation. Persistent cytoplasmic localization of c-Abl has been associated with cellular transformation. c-Abl phosphorylates OTULIN at tyrosine 56, thereby disrupting its relationship with LUBAC. OTULIN-released LUBAC interacts with SPATA2 and is recruited to the TNF-R1sc, facilitating SPATA2-CYLD interaction. All these interactions are required for the activation of IKKβ to stimulate NF-κB transcriptional activity following genotoxic stress. IKKβ also induces the critical phosphorylation of CYLD at serine 568 to increase its deubiquitinating (DUB) activity required for the termination of signaling cascades. Contrary to the widespread belief that CYLD is an absolute tumor suppressor, CYLD initiates and terminates NF-κB activity by alternately using its oncoprotein and tumor suppressor activities, respectively. If IKKβ fails to achieve the DUB activity-inducing phosphorylation at serine 568, CYLD would operate in a sustained mode of oncogenic activity. The resulting dysregulated NF-κB activation and other accompanying pathologies will disrupt cellular homeostasis in favor of transformation.

A computational essential dynamics approach to investigate structural influences of ligand binding on Papain like protease from SARS-CoV-2

Papain like protease (PLpro) is a cysteine protease from the coronaviridae family of viruses. Coronaviruses possess a positive sense, single-strand RNA, leading to the translation of two viral polypeptides containing viral structural, non-structural and accessory proteins. PLpro is responsible for the cleavage of nsp1–3 from the viral polypeptide. PLpro also possesses deubiquitinating and deISGlyating activity, which sequesters the virus from the host's immune system. This indispensable attribute of PLpro makes it a protein of interest as a drug target. The present study aims to analyze the structural influences of ligand binding on PLpro. First, PLpro was screened against the ZINC-in-trials library, from which four lead compounds were identified based on estimated binding affinity and interaction patterns. Next, based on molecular docking results, ZINC000000596945, ZINC000064033452 and VIR251 (control molecule) were subjected to molecular dynamics simulation. The study evaluated global and essential dynamics analyses utilising principal component analyses, dynamic cross-correlation matrix, free energy landscape and time-dependant essential dynamics to predict the structural changes observed in PLpro upon ligand binding in a simulated environment. The MM/PBSA-based binding free energy calculations of the two selected molecules, ZINC000000596945 (−41.23 ± 3.70 kcal/mol) and ZINC000064033452 (−25.10 ± 2.65 kcal/mol), displayed significant values which delineate them as potential inhibitors of PLpro from SARS-CoV-2.