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1.
J Clin Invest ; 133(9)2023 05 01.
Article in English | MEDLINE | ID: covidwho-2320676

ABSTRACT

Inflammation promotes adverse ventricular remodeling, a common antecedent of heart failure. Here, we set out to determine how inflammatory cells affect cardiomyocytes in the remodeling heart. Pathogenic cardiac macrophages induced an IFN response in cardiomyocytes, characterized by upregulation of the ubiquitin-like protein IFN-stimulated gene 15 (ISG15), which posttranslationally modifies its targets through a process termed ISGylation. Cardiac ISG15 is controlled by type I IFN signaling, and ISG15 or ISGylation is upregulated in mice with transverse aortic constriction or infused with angiotensin II; rats with uninephrectomy and DOCA-salt, or pulmonary artery banding; cardiomyocytes exposed to IFNs or CD4+ T cell-conditioned medium; and ventricular tissue of humans with nonischemic cardiomyopathy. By nanoscale liquid chromatography-tandem mass spectrometry, we identified the myofibrillar protein filamin-C as an ISGylation target. ISG15 deficiency preserved cardiac function in mice with transverse aortic constriction and led to improved recovery of mouse hearts ex vivo. Metabolomics revealed that ISG15 regulates cardiac amino acid metabolism, whereas ISG15 deficiency prevented misfolded filamin-C accumulation and induced cardiomyocyte autophagy. In sum, ISG15 upregulation is a feature of pathological ventricular remodeling, and protein ISGylation is an inflammation-induced posttranslational modification that may contribute to heart failure development by altering cardiomyocyte protein turnover.


Subject(s)
Cytokines , Heart Failure , Humans , Rats , Mice , Animals , Cytokines/genetics , Cytokines/metabolism , Filamins , Ventricular Remodeling/genetics , Heart Failure/metabolism , Inflammation , Ubiquitins/genetics
2.
Eur J Intern Med ; 105: 1-7, 2022 11.
Article in English | MEDLINE | ID: covidwho-2309780

ABSTRACT

Vaccine-induced immune thrombocytopenia and thrombosis (VITT) is a rare syndrome characterized by high-titer anti-platelet factor 4 (PF4) antibodies, thrombocytopenia and arterial and venous thrombosis in unusual sites, as cerebral venous sinuses and splanchnic veins. VITT has been described to occur almost exclusively after administration of ChAdOx1 nCoV-19 and Ad26.COV2.S adenovirus vector- based COVID-19 vaccines. Clinical and laboratory features of VITT resemble those of heparin-induced thrombocytopenia (HIT). It has been hypothesized that negatively charged polyadenylated hexone proteins of the AdV vectors could act as heparin to induce the conformational changes of PF4 molecule that lead to the formation of anti-PF4/polyanion antibodies. The anti-PF4 immune response in VITT is fostered by the presence of a proinflammatory milieu, elicited by some impurities found in ChAdOx1 nCoV-19 vaccine, as well as by soluble spike protein resulting from alternative splice events. Anti-PF4 antibodies bind PF4, forming immune complexes which activate platelets, monocytes and granulocytes, resulting in the VITT's immunothrombosis. The reason why only a tiny minority of patents receiving AdV-based COVID-19 vaccines develop VITT is still unknown. It has been hypothesized that individual intrinsic factors, either acquired (i.e., pre-priming of B cells to produce anti-PF4 antibodies by previous contacts with bacteria or viruses) or inherited (i.e., differences in platelet T-cell ubiquitin ligand-2 [TULA-2] expression) can predispose a few subjects to develop VITT. A better knowledge of the mechanistic basis of VITT is essential to improve the safety and the effectiveness of future vaccines and gene therapies using adenovirus vectors.


Subject(s)
COVID-19 , Purpura, Thrombocytopenic, Idiopathic , Thrombocytopenia , Thrombosis , Vaccines , Humans , Antigen-Antibody Complex , COVID-19 Vaccines/adverse effects , Ad26COVS1 , ChAdOx1 nCoV-19 , Ligands , Spike Glycoprotein, Coronavirus , COVID-19/prevention & control , Platelet Factor 4/genetics , Platelet Factor 4/metabolism , Heparin/adverse effects , Thrombocytopenia/chemically induced , Vaccines/adverse effects , Purpura, Thrombocytopenic, Idiopathic/chemically induced , Ubiquitins
3.
Virology ; 582: 114-127, 2023 05.
Article in English | MEDLINE | ID: covidwho-2298993

ABSTRACT

Coronavirus infection induces a variety of cellular antiviral responses either dependent on or independent of type I interferons (IFNs). Our previous studies using Affymetrix microarray and transcriptomic analysis revealed the differential induction of three IFN-stimulated genes (ISGs), IRF1, ISG15 and ISG20, by gammacoronavirus infectious bronchitis virus (IBV) infection of IFN-deficient Vero cells and IFN-competent, p53-defcient H1299 cells, respectively. In this report, the induction kinetics and anti-IBV functions of these ISGs as well as mechanisms underlying their differential induction are characterized. The results confirmed that these three ISGs were indeed differentially induced in H1299 and Vero cells infected with IBV, significantly more upregulation of IRF1, ISG15 and ISG20 was elicited in IBV-infected Vero cells than that in H1299 cells. Induction of these ISGs was also detected in cells infected with human coronavirus-OC43 (HCoV-OC43) and porcine epidemic diarrhea virus (PEDV), respectively. Manipulation of their expression by overexpression, knockdown and/or knockout demonstrated that IRF1 played an active role in suppressing IBV replication, mainly through the activation of the IFN pathway. However, a minor, if any, role in inhibiting IBV replication was played by ISG15 and ISG20. Furthermore, p53, but not IRF1, was implicated in regulating the IBV infection-induced upregulation of ISG15 and ISG20. This study provides new information on the mechanisms underlying the induction of these ISGs and their contributions to the host cell antiviral response during IBV infection.


Subject(s)
Coronavirus Infections , Gammacoronavirus , Infectious bronchitis virus , Animals , Humans , Antiviral Agents/pharmacology , Chlorocebus aethiops , Coronavirus Infections/veterinary , Cytokines/genetics , Exoribonucleases , Infectious bronchitis virus/genetics , Swine , Tumor Suppressor Protein p53 , Ubiquitins , Vero Cells
4.
Nat Commun ; 14(1): 2366, 2023 04 25.
Article in English | MEDLINE | ID: covidwho-2305876

ABSTRACT

The Papain-like protease (PLpro) is a domain of a multi-functional, non-structural protein 3 of coronaviruses. PLpro cleaves viral polyproteins and posttranslational conjugates with poly-ubiquitin and protective ISG15, composed of two ubiquitin-like (UBL) domains. Across coronaviruses, PLpro showed divergent selectivity for recognition and cleavage of posttranslational conjugates despite sequence conservation. We show that SARS-CoV-2 PLpro binds human ISG15 and K48-linked di-ubiquitin (K48-Ub2) with nanomolar affinity and detect alternate weaker-binding modes. Crystal structures of untethered PLpro complexes with ISG15 and K48-Ub2 combined with solution NMR and cross-linking mass spectrometry revealed how the two domains of ISG15 or K48-Ub2 are differently utilized in interactions with PLpro. Analysis of protein interface energetics predicted differential binding stabilities of the two UBL/Ub domains that were validated experimentally. We emphasize how substrate recognition can be tuned to cleave specifically ISG15 or K48-Ub2 modifications while retaining capacity to cleave mono-Ub conjugates. These results highlight alternative druggable surfaces that would inhibit PLpro function.


Subject(s)
COVID-19 , SARS-CoV-2 , Ubiquitin , Humans , Cytokines/metabolism , Papain/metabolism , Peptide Hydrolases/metabolism , SARS-CoV-2/metabolism , Ubiquitin/metabolism , Ubiquitins/metabolism
5.
J Virol ; 96(5): e0088921, 2022 03 09.
Article in English | MEDLINE | ID: covidwho-2223570

ABSTRACT

Porcine epidemic diarrhea virus (PEDV) causes a porcine disease associated with swine epidemic diarrhea. Different antagonistic strategies have been identified, and the mechanism by which PEDV infection impairs the production of interferon (IFN) and delays the activation of the IFN response to escape host innate immunity has been determined, but the pathogenic mechanisms of PEDV infection remain enigmatic. Our preliminary results revealed that endogenous F-box and WD repeat domain-containing 7 (FBXW7) protein, the substrate recognition component of the SCF-type E3 ubiquitin ligase, is downregulated in PEDV-infected Vero E6 cells, according to the results from an isobaric tags for relative and absolute quantification (iTRAQ) analysis. Overexpression of FBXW7 in target cells makes them more resistant to PEDV infection, whereas ablation of FBXW7 expression by small interfering RNA (siRNA) significantly promotes PEDV infection. In addition, FBXW7 was verified as an innate antiviral factor capable of enhancing the expression of RIG-I and TBK1, and it was found to induce interferon-stimulated genes (ISGs), which led to an elevated antiviral state of the host cells. Moreover, we revealed that PEDV nonstructural protein 2 (nsp2) interacts with FBXW7 and targets FBXW7 for degradation through the K48-linked ubiquitin-proteasome pathway. Consistent with the results proven in vitro, FBXW7 reduction was also confirmed in different intestinal tissues from PEDV-infected specific-pathogen-free (SPF) pigs. Taken together, the data indicated that PEDV has evolved with a distinct antagonistic strategy to circumvent the host antiviral response by targeting the ubiquitin-proteasome-mediated degradation of FBXW7. Our findings provide novel insights into PEDV infection and pathogenesis. IMPORTANCE To counteract the host antiviral defenses, most viruses, including coronaviruses, have evolved with diverse strategies to dampen host IFN-mediated antiviral response, by interfering with or evading specific host regulators at multiple steps of this response. In this study, a novel antagonistic strategy was revealed showing that PEDV infection could circumvent the host innate response by targeted degradation of endogenous FBXW7 in target cells, a process that was verified to be a positive modulator for the host innate immune system. Degradation of FBXW7 hampers host innate antiviral activation and facilitates PEDV replication. Our findings reveal a new mechanism exploited by PEDV to suppress the host antiviral response.


Subject(s)
Coronavirus Infections/veterinary , F-Box-WD Repeat-Containing Protein 7/metabolism , Immune Evasion , Immunity, Innate , Porcine epidemic diarrhea virus/immunology , Swine Diseases/immunology , Animals , Antiviral Agents/immunology , Chlorocebus aethiops , Coronavirus Infections/immunology , Coronavirus Infections/prevention & control , Coronavirus Infections/virology , Interferon Type I/metabolism , Proteasome Endopeptidase Complex/metabolism , Signal Transduction/immunology , Swine , Swine Diseases/prevention & control , Swine Diseases/virology , Ubiquitins/metabolism , Vero Cells
6.
J Virol ; 97(1): e0161422, 2023 01 31.
Article in English | MEDLINE | ID: covidwho-2223572

ABSTRACT

Porcine epidemic diarrhea (PED) indicates the disease of the acute and highly contagious intestinal infection due to porcine epidemic diarrhea virus (PEDV), with the characteristics of watery diarrhea, vomiting, and dehydration. One of the reasons for diarrhea and death of piglets is PEDV, which leads to 100% mortality in neonatal piglets. Therefore, it is necessary to explore the interaction between virus and host to prevent and control PEDV. This study indicated that the host protein, pre-mRNA processing factor 19 (PRPF19), could be controlled by the signal transducer as well as activator of transcription 1 (STAT1). Thus, PEDV replication could be hindered through selective autophagy. Moreover, PRPF19 was found to recruit the E3 ubiquitin ligase MARCH8 to the N protein for ubiquitination. For the purpose of degradation, the ubiquitin N protein is acknowledged by the cargo receptor NDP52 and transported to autolysosomes, thus inhibiting virus proliferation. To conclude, a unique antiviral mechanism of PRPF19-mediated virus restriction was shown. Moreover, a view of the innate immune response and protein degradation against PEDV replication was provided in this study. IMPORTANCE The highly virulent porcine epidemic diarrhea virus (PEDV) emerged in 2010, and causes high mortality rates in newborn pigs. There are no effective and safe vaccines against the highly virulent PEDV. This virus has caused devastating economic losses in the pork industry worldwide. Studying the relationship between virus and host antiviral factors is important to develop the new antiviral strategies. This study identified the pre-mRNA processing factor 19 (PRPF19) as a novel antiviral protein in PEDV replication and revealed its viral restriction mechanisms for the first time. PRPF19 recruited the E3 ubiquitin ligase MARCH8 to the PEDV N protein for ubiquitination, and the ubiquitin N protein was acknowledged by the cargo receptor NDP52 and transported to autolysosomes for degradation. Our findings provide new insights in host antiviral factors PRPF19 that regulate the selective autophagy protein degradation pathway to inhibit PEDV replication.


Subject(s)
Capsid Proteins , Coronavirus Infections , Porcine epidemic diarrhea virus , Swine Diseases , Animals , Capsid Proteins/metabolism , Coronavirus Infections/immunology , Coronavirus Infections/veterinary , Coronavirus Infections/virology , Porcine epidemic diarrhea virus/physiology , Swine , Swine Diseases/immunology , Swine Diseases/virology , Ubiquitin-Protein Ligases/metabolism , Ubiquitins , Virus Replication/genetics , Nuclear Proteins/metabolism , Autophagy
7.
Semin Cell Dev Biol ; 132: 16-26, 2022 Dec.
Article in English | MEDLINE | ID: covidwho-2211427

ABSTRACT

Ubiquitin-like proteins (Ubls) share some features with ubiquitin (Ub) such as their globular 3D structure and the ability to attach covalently to other proteins. Interferon Stimulated Gene 15 (ISG15) is an abundant Ubl that similar to Ub, marks many hundreds of cellular proteins, altering their fate. In contrast to Ub, , ISG15 requires interferon (IFN) induction to conjugate efficiently to other proteins. Moreover, despite the multitude of E3 ligases for Ub-modified targets, a single E3 ligase termed HERC5 (in humans) is responsible for the bulk of ISG15 conjugation. Targets include both viral and cellular proteins spanning an array of cellular compartments and metabolic pathways. So far, no common structural or biochemical feature has been attributed to these diverse substrates, raising questions about how and why they are selected. Conjugation of ISG15 mitigates some viral and bacterial infections and is linked to a lower viral load pointing to the role of ISG15 in the cellular immune response. In an apparent attempt to evade the immune response, some viruses try to interfere with the ISG15 pathway. For example, deconjugation of ISG15 appears to be an approach taken by coronaviruses to interfere with ISG15 conjugates. Specifically, coronaviruses such as SARS-CoV, MERS-CoV, and SARS-CoV-2, encode papain-like proteases (PL1pro) that bear striking structural and catalytic similarities to the catalytic core domain of eukaryotic deubiquitinating enzymes of the Ubiquitin-Specific Protease (USP) sub-family. The cleavage specificity of these PLpro enzymes is for flexible polypeptides containing a consensus sequence (R/K)LXGG, enabling them to function on two seemingly unrelated categories of substrates: (i) the viral polyprotein 1 (PP1a, PP1ab) and (ii) Ub- or ISG15-conjugates. As a result, PLpro enzymes process the viral polyprotein 1 into an array of functional proteins for viral replication (termed non-structural proteins; NSPs), and it can remove Ub or ISG15 units from conjugates. However, by de-conjugating ISG15, the virus also creates free ISG15, which in turn may affect the immune response in two opposite pathways: free ISG15 negatively regulates IFN signaling in humans by binding non-catalytically to USP18, yet at the same time free ISG15 can be secreted from the cell and induce the IFN pathway of the neighboring cells. A deeper understanding of this protein-modification pathway and the mechanisms of the enzymes that counteract it will bring about effective clinical strategies related to viral and bacterial infections.


Subject(s)
COVID-19 , Interferons , Humans , Peptide Hydrolases/metabolism , SARS-CoV-2 , Ubiquitin/metabolism , Antiviral Agents , Polyproteins , Immunity , Cytokines/metabolism , Ubiquitins/genetics , Ubiquitin Thiolesterase
8.
Methods Mol Biol ; 2591: 171-188, 2023.
Article in English | MEDLINE | ID: covidwho-2103726

ABSTRACT

Both severe acute respiratory syndrome coronavirus 1 and 2 (SARS-CoV-1 and SARS-CoV-2) encode a papain-like protease (PLpro), which plays a vital role in viral propagation. PLpro accomplishes this function by processing the viral polyproteins essential for viral replication and removing the small proteins, ubiquitin and ISG15 from the host's key immune signaling proteins, thereby preventing the host's innate immune response. Although PLpro from both SARS-CoV-1 and SARS-CoV-2 are structurally highly similar (83% sequence identity), they exhibit functional variability. Hence, to further elucidate the mechanism and aid in drug discovery efforts, the biochemical and kinetic characterization of PLpro is needed. This chapter describes step-by-step experimental procedures for evaluating PLpro activity in vitro using activity-based probes (ABPs) along with fluorescence-based substrates. Herein we describe a step-by-step experimental procedure to assess the activity of PLpro in vitro using a suite of activity-based probes (ABPs) and fluorescent substrates and how they can be applied as fast and yet sensitive methods to calculate kinetic parameters.


Subject(s)
COVID-19 , Ubiquitin , Humans , Ubiquitin/metabolism , SARS-CoV-2/genetics , Coronavirus Papain-Like Proteases , Papain , Peptide Hydrolases/metabolism , Ubiquitins/metabolism , Cytokines/metabolism
9.
Biochem Biophys Res Commun ; 625: 94-101, 2022 10 15.
Article in English | MEDLINE | ID: covidwho-2035787

ABSTRACT

The expression of the ubiquitin-like molecule interferon-stimulated gene 15 kDa (ISG15) and post-translational protein modification by ISG15 (ISGylation) are strongly activated by interferons or pathogen infection, suggesting that ISG15 and ISGylation play an important role in innate immune responses. More than 400 proteins have been found to be ISGylated. ISG15 is removed from substrates by interferon-induced ubiquitin-specific peptidase 18 or severe acute respiratory syndrome coronavirus 2‒derived papain-like protease. Therefore, maintaining strong ISGylation may help prevent the spread of coronavirus disease 2019 (COVID-19). However, it is unknown whether nutrients or chemicals affect ISGylation level. Curcumin is the major constituent of turmeric and functions as an immunomodulator. Here, we investigated the effect of curcumin on ISGylation. MCF10A and A549 cells were treated with interferon α and curcumin after which the expression levels of various proteins were determined. The effect of curcumin on ubiquitylation was also determined. Curcumin treatment was found to reduce ISGylation in a dose-dependent manner. The findings suggested that curcumin partly prevents disulfide bond-mediated ISG15 dimerization directly or indirectly, thereby increasing monomer ISG15 levels. Reduced ISGylation may also occur via the prevention of ISG15 activation by ubiquitin-activating enzyme E1-like protein. In conclusion, curcumin treatment was found to reduce ISGylation, suggesting that it may contribute to severe COVID-19. This is the first study to report a relationship between ISGylation and a food component.


Subject(s)
COVID-19 , Curcumin , Antiviral Agents/pharmacology , Autophagy-Related Protein 7 , Curcumin/pharmacology , Cytokines/metabolism , Humans , Interferon-alpha , Ubiquitin-Activating Enzymes/genetics , Ubiquitins/metabolism
10.
J Virol ; 96(17): e0074122, 2022 09 14.
Article in English | MEDLINE | ID: covidwho-1992937

ABSTRACT

Within the past 2 decades, three highly pathogenic human coronaviruses have emerged, namely, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The health threats and economic burden posed by these tremendously severe coronaviruses have paved the way for research on their etiology, pathogenesis, and treatment. Compared to SARS-CoV and SARS-CoV-2, MERS-CoV genome encoded fewer accessory proteins, among which the ORF4b protein had anti-immunity ability in both the cytoplasm and nucleus. Our work for the first time revealed that ORF4b protein was unstable in the host cells and could be degraded by the ubiquitin proteasome system. After extensive screenings, it was found that UBR5 (ubiquitin protein ligase E3 component N-recognin 5), a member of the HECT E3 ubiquitin ligases, specifically regulated the ubiquitination and degradation of ORF4b. Similar to ORF4b, UBR5 can also translocate into the nucleus through its nuclear localization signal, enabling it to regulate ORF4b stability in both the cytoplasm and nucleus. Through further experiments, lysine 36 was identified as the ubiquitination site on the ORF4b protein, and this residue was highly conserved in various MERS-CoV strains isolated from different regions. When UBR5 was knocked down, the ability of ORF4b to suppress innate immunity was enhanced and MERS-CoV replication was stronger. As an anti-MERS-CoV host protein, UBR5 targets and degrades ORF4b protein through the ubiquitin proteasome system, thereby attenuating the anti-immunity ability of ORF4b and ultimately inhibiting MERS-CoV immune escape, which is a novel antagonistic mechanism of the host against MERS-CoV infection. IMPORTANCE ORF4b was an accessory protein unique to MERS-CoV and was not present in SARS-CoV and SARS-CoV-2 which can also cause severe respiratory disease. Moreover, ORF4b inhibited the production of antiviral cytokines in both the cytoplasm and the nucleus, which was likely to be associated with the high lethality of MERS-CoV. However, whether the host proteins regulate the function of ORF4b is unknown. Our study first determined that UBR5, a host E3 ligase, was a potential host anti-MERS-CoV protein that could reduce the protein level of ORF4b and diminish its anti-immunity ability by inducing ubiquitination and degradation. Based on the discovery of ORF4b-UBR5, a critical molecular target, further increasing the degradation of ORF4b caused by UBR5 could provide a new strategy for the clinical development of drugs for MERS-CoV.


Subject(s)
Coronavirus Infections , Host Microbial Interactions , Middle East Respiratory Syndrome Coronavirus , Proteolysis , Ubiquitin-Protein Ligases , Ubiquitination , Viral Proteins , Coronavirus Infections/immunology , Coronavirus Infections/prevention & control , Coronavirus Infections/virology , Cytokines/immunology , Humans , Immunity, Innate , Middle East Respiratory Syndrome Coronavirus/immunology , Middle East Respiratory Syndrome Coronavirus/metabolism , Molecular Targeted Therapy , Proteasome Endopeptidase Complex/metabolism , Severe acute respiratory syndrome-related coronavirus , SARS-CoV-2 , Ubiquitin-Protein Ligases/metabolism , Ubiquitins/metabolism , Viral Proteins/chemistry , Viral Proteins/metabolism , Virus Replication
11.
Angew Chem Int Ed Engl ; 61(40): e202206205, 2022 10 04.
Article in English | MEDLINE | ID: covidwho-1990419

ABSTRACT

Ubiquitin (Ub)-like protein ISG15 (interferon-stimulated gene 15) regulates innate immunity and links with the evasion of host response by viruses such as SARS-CoV-2. Dissecting ISGylation pathways recently received increasing attention which can inform related disease interventions, but such studies necessitate the preparation and development of various ISG15 protein tools. Here, we find that the leader protease (Lbpro ) encoded by foot-and-mouth disease virus can promote ligation reactions between recombinant ISG15 and synthetic glycyl compounds, generating protein tools such as ISG15-propargylamide and ISG15-rhodamine110, which are needed for cellular proteomic studies of deISGylases, and the screening and evaluation of inhibitors against SARS-CoV-2 papain-like protease (PLpro). Furthermore, this strategy can be also used to load ISG15 onto the lysine of a synthetic peptide through an isopeptide bond, and prepare Ub and NEDD8 (ubiquitin-like protein Nedd8) protein tools.


Subject(s)
COVID-19 , Peptide Hydrolases , Animals , Catalysis , Cytokines/metabolism , Interferons , Lysine , NEDD8 Protein , Peptide Hydrolases/metabolism , Proteomics , SARS-CoV-2 , Ubiquitins/chemistry
12.
J Cell Biol ; 221(7)2022 07 04.
Article in English | MEDLINE | ID: covidwho-1956550

ABSTRACT

The process of membrane atg8ylation, defined herein as the conjugation of the ATG8 family of ubiquitin-like proteins to membrane lipids, is beginning to be appreciated in its broader manifestations, mechanisms, and functions. Classically, membrane atg8ylation with LC3B, one of six mammalian ATG8 family proteins, has been viewed as the hallmark of canonical autophagy, entailing the formation of characteristic double membranes in the cytoplasm. However, ATG8s are now well described as being conjugated to single membranes and, most recently, proteins. Here we propose that the atg8ylation is coopted by multiple downstream processes, one of which is canonical autophagy. We elaborate on these biological outputs, which impact metabolism, quality control, and immunity, emphasizing the context of inflammation and immunological effects. In conclusion, we propose that atg8ylation is a modification akin to ubiquitylation, and that it is utilized by different systems participating in membrane stress responses and membrane remodeling activities encompassing autophagy and beyond.


Subject(s)
Autophagy , Ubiquitins , Animals , Autophagy/physiology , Autophagy-Related Protein 8 Family/genetics , Autophagy-Related Protein 8 Family/metabolism , Autophagy-Related Proteins/genetics , Autophagy-Related Proteins/metabolism , Mammals/metabolism , Microtubule-Associated Proteins/metabolism , Ubiquitination , Ubiquitins/genetics
13.
mBio ; 13(3): e0130022, 2022 06 28.
Article in English | MEDLINE | ID: covidwho-1874506

ABSTRACT

Ubiquitin signaling is essential for immunity to restrict pathogen proliferation. Due to its enormous impact on human health and the global economy, intensive efforts have been invested in studying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its interactions with hosts. However, the role of the ubiquitin network in pathogenicity has not yet been explored. Here, we found that ORF9b of SARS-CoV-2 is ubiquitinated on Lys-4 and Lys-40 by unknown E3 ubiquitin ligases and is degraded by the ubiquitin proteasomal system. Importantly, we identified USP29 as a host factor that prevents ORF9b ubiquitination and subsequent degradation. USP29 interacts with the carboxyl end of ORF9b and removes ubiquitin chains from the protein, thereby inhibiting type I interferon (IFN) induction and NF-κB activation. We also found that ORF9b stabilization by USP29 enhanced the virulence of VSV-eGFP and transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). Moreover, we observed that the mRNA level of USP29 in SARS-CoV-2 patients was higher than that in healthy people. Our findings provide important evidence indicating that targeting USP29 may effectively combat SARS-CoV-2 infection. IMPORTANCE Coronavirus disease 2019 (COVID-19) is a current global health threat caused by SARS-CoV-2. The innate immune response such as type I IFN (IFN-I) is the first line of host defense against viral infections, whereas SARS-CoV-2 proteins antagonize IFN-I production through distinct mechanisms. Among them, ORF9b inhibits the canonical IκB kinase alpha (IKKɑ)/ß/γ-NF-κB signaling and subsequent IFN production; therefore, discovering the regulation of ORF9b by the host might help develop a novel antiviral strategy. Posttranslational modification of proteins by ubiquitination regulates many biological processes, including viral infections. Here, we report that ORF9b is ubiquitinated and degraded through the proteasome pathway, whereas deubiquitinase USP29 deubiquitinates ORF9b and prevents its degradation, resulting in the enhancement of ORF9b-mediated inhibition of IFN-I and NF-κB activation and the enhancement of virulence of VSV-eGFP and SARS-CoV-2 trVLP.


Subject(s)
Biological Phenomena , COVID-19 , Coronavirus Nucleocapsid Proteins/metabolism , Deubiquitinating Enzymes , Humans , Immunity, Innate , NF-kappa B , Phosphoproteins/metabolism , Proteasome Endopeptidase Complex , SARS-CoV-2/genetics , Ubiquitin-Specific Proteases , Ubiquitins , Virulence
14.
Curr Drug Targets ; 23(7): 686-691, 2022.
Article in English | MEDLINE | ID: covidwho-1745211

ABSTRACT

Interferon-simulated gene 15 (ISG15) belongs to the family of ubiquitin-like proteins. ISG15 acts as a cytokine and modifies proteins through ISGylation. This posttranslational modification has been associated with antiviral and immune response pathways. In addition, it is known that the genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encodes proteases critical for viral replication. Consequently, these proteases are also central in the progression of coronavirus disease 2019 (COVID-19). Interestingly, the protease SARS-CoV-2-PLpro removes ISG15 from ISGylated proteins such as IRF3 and MDA5, affecting immune and antiviral defense from the host. Here, the implications of ISG15, ISGylation, and generation of SARS-CoV-2-PLpro inhibitors in SARS-CoV-2 infection are discussed.


Subject(s)
COVID-19 , Cytokines , Ubiquitins , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Cytokines/metabolism , Humans , Interferons , SARS-CoV-2 , Ubiquitins/metabolism
15.
Biomolecules ; 12(2)2022 02 12.
Article in English | MEDLINE | ID: covidwho-1686605

ABSTRACT

Ubiquitylation and ISGylation are protein post-translational modifications (PTMs) and two of the main events involved in the activation of pattern recognition receptor (PRRs) signals allowing the host defense response to viruses. As with similar viruses, SARS-CoV-2, the virus causing COVID-19, hijacks these pathways by removing ubiquitin and/or ISG15 from proteins using a protease called PLpro, but also by interacting with enzymes involved in ubiquitin/ISG15 machinery. These enable viral replication and avoidance of the host immune system. In this review, we highlight potential points of therapeutic intervention in ubiquitin/ISG15 pathways involved in key host-pathogen interactions, such as PLpro, USP18, TRIM25, CYLD, A20, and others that could be targeted for the treatment of COVID-19, and which may prove effective in combatting current and future vaccine-resistant variants of the disease.


Subject(s)
COVID-19 Drug Treatment , COVID-19/metabolism , Cytokines/metabolism , Ubiquitin/metabolism , Ubiquitination , Ubiquitins/metabolism , Animals , Humans , Protein Processing, Post-Translational/drug effects , SARS-CoV-2/drug effects
16.
Exp Biol Med (Maywood) ; 247(10): 842-847, 2022 05.
Article in English | MEDLINE | ID: covidwho-1673845

ABSTRACT

Cytokine-driven hyper inflammation has been identified as a critical factor behind poor outcomes in patients severely infected with SARS-CoV-2 virus. Notably, protein ISGylation, a protein conjugated form of Type 1 IFN-inducible ubiquitin-like protein ISG15 (Interferon-Stimulated Gene 15), induces cytokine storm (CS) and augments colonic inflammation in colitis-associated colon cancers in mouse models. However, whether ISGylation is increased and causally responsible for CS and hyper inflammation in symptomatic COVID-19 patients is unknown. Here, we measured ISGylation levels in peripheral blood mononuclear cells (PBMCs) from 10 symptomatic (SARS-CoV-2-positive with symptoms) and asymptomatic (SARS-CoV-2-positive with no symptoms) COVID-19 patients, and 4 uninfected individuals (SARS-CoV-2-negative), using WesTm assay. Strikingly, we note significant increases in protein ISGylation and MX-1 (myxovirus-resistance protein-1) protein levels, both induced by type-I IFN, in symptomatic but not in asymptomatic patients and uninfected individuals. Knowing that ISGylation augments CS and intestinal inflammation in colon cancers, we propose that increased ISGylation may be an underlying cause of CS and inflammation in symptomatic patients.


Subject(s)
COVID-19 , Ubiquitins , Animals , Cytokines/metabolism , Humans , Inflammation , Leukocytes, Mononuclear/metabolism , Mice , SARS-CoV-2 , Ubiquitins/metabolism
17.
Autophagy ; 18(10): 2350-2367, 2022 10.
Article in English | MEDLINE | ID: covidwho-1671990

ABSTRACT

Zaire ebolavirus (EBOV) causes a severe hemorrhagic fever in humans and non-human primates with high morbidity and mortality. EBOV infection is dependent on its structural glycoprotein (GP), but high levels of GP expression also trigger cell rounding, detachment, and downregulation of many surface molecules that is thought to contribute to its high pathogenicity. Thus, EBOV has evolved an RNA editing mechanism to reduce its GP expression and increase its fitness. We now report that the GP expression is also suppressed at the protein level in cells by protein disulfide isomerases (PDIs). Although PDIs promote oxidative protein folding by catalyzing correct disulfide formation in the endoplasmic reticulum (ER), PDIA3/ERp57 adversely triggered the GP misfolding by targeting GP cysteine residues and activated the unfolded protein response (UPR). Abnormally folded GP was targeted by ER-associated protein degradation (ERAD) machinery and, unexpectedly, was degraded via the macroautophagy/autophagy-lysosomal pathway, but not the proteasomal pathway. PDIA3 also decreased the GP expression from other ebolavirus species but increased the GP expression from Marburg virus (MARV), which is consistent with the observation that MARV-GP does not cause cell rounding and detachment, and MARV does not regulate its GP expression via RNA editing during infection. Furthermore, five other PDIs also had a similar inhibitory activity to EBOV-GP. Thus, PDIs negatively regulate ebolavirus glycoprotein expression, which balances the viral life cycle by maximizing their infection but minimizing their cellular effect. We suggest that ebolaviruses hijack the host protein folding and ERAD machinery to increase their fitness via reticulophagy during infection.Abbreviations: 3-MA: 3-methyladenine; 4-PBA: 4-phenylbutyrate; ACTB: ß-actin; ATF: activating transcription factor; ATG: autophagy-related; BafA1: bafilomycin A1; BDBV: Bundibugyo ebolavirus; CALR: calreticulin; CANX: calnexin; CHX: cycloheximide; CMA: chaperone-mediated autophagy; ConA: concanamycin A; CRISPR: clusters of regularly interspaced short palindromic repeats; Cas9: CRISPR-associated protein 9; dsRNA: double-stranded RNA; EBOV: Zaire ebolavirus; EDEM: ER degradation enhancing alpha-mannosidase like protein; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; Env: envelope glycoprotein; ER: endoplasmic reticulum; ERAD: ER-associated protein degradation; ERN1/IRE1: endoplasmic reticulum to nucleus signaling 1; GP: glycoprotein; HA: hemagglutinin; HDAC6: histone deacetylase 6; HMM: high-molecular-mass; HIV-1: human immunodeficiency virus type 1; HSPA5/BiP: heat shock protein family A (Hsp70) member 5; IAV: influenza A virus; IP: immunoprecipitation; KIF: kifenesine; Lac: lactacystin; LAMP: lysosomal associated membrane protein; MAN1B1/ERManI: mannosidase alpha class 1B member 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MARV: Marburg virus; MLD: mucin-like domain; NHK/SERPINA1: alpha1-antitrypsin variant null (Hong Kong); NTZ: nitazoxanide; PDI: protein disulfide isomerase; RAVV: Ravn virus; RESTV: Reston ebolavirus; SARS-CoV: severe acute respiratory syndrome coronavirus; SBOV: Sudan ebolavirus; sGP: soluble GP; SQSTM1/p62: sequestosome 1; ssGP: small soluble GP; TAFV: Taï Forest ebolavirus; TIZ: tizoxanide; TGN: thapsigargin; TLD: TXN (thioredoxin)-like domain; Ub: ubiquitin; UPR: unfolded protein response; VLP: virus-like particle; VSV: vesicular stomatitis virus; WB: Western blotting; WT: wild-type; XBP1: X-box binding protein 1.


Subject(s)
Autophagy , Ebolavirus , Actins/metabolism , Animals , CRISPR-Associated Protein 9/genetics , CRISPR-Associated Protein 9/metabolism , CRISPR-Associated Protein 9/pharmacology , Calnexin/metabolism , Calreticulin/genetics , Calreticulin/metabolism , Calreticulin/pharmacology , Cycloheximide , Cysteine/metabolism , Disulfides , Endoplasmic Reticulum/metabolism , Glycoproteins/metabolism , Heat-Shock Proteins/metabolism , Hemagglutinins/metabolism , Hemagglutinins/pharmacology , Histone Deacetylase 6/genetics , Intercellular Signaling Peptides and Proteins , Lysosome-Associated Membrane Glycoproteins/metabolism , Lysosomes/metabolism , Microtubule-Associated Proteins/metabolism , Mucins/genetics , Mucins/metabolism , Mucins/pharmacology , Prokaryotic Initiation Factor-2/genetics , Prokaryotic Initiation Factor-2/metabolism , Prokaryotic Initiation Factor-2/pharmacology , Protein Disulfide-Isomerases/genetics , Protein Disulfide-Isomerases/metabolism , RNA, Double-Stranded/metabolism , RNA, Double-Stranded/pharmacology , Sequestosome-1 Protein/metabolism , Thapsigargin/metabolism , Thapsigargin/pharmacology , Thioredoxins/genetics , Thioredoxins/metabolism , Thioredoxins/pharmacology , Ubiquitins/metabolism , X-Box Binding Protein 1/metabolism , alpha-Mannosidase/genetics , alpha-Mannosidase/metabolism , alpha-Mannosidase/pharmacology
18.
J Med Chem ; 65(1): 876-884, 2022 01 13.
Article in English | MEDLINE | ID: covidwho-1606194

ABSTRACT

Coronavirus disease 2019 (COVID-19) pandemic, a global health threat, was caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 papain-like cysteine protease (PLpro) was recognized as a promising drug target because of multiple functions in virus maturation and antiviral immune responses. Inhibitor GRL0617 occupied the interferon-stimulated gene 15 (ISG15) C-terminus-binding pocket and showed an effective antiviral inhibition. Here, we described a novel peptide-drug conjugate (PDC), in which GRL0617 was linked to a sulfonium-tethered peptide derived from PLpro-specific substrate LRGG. The EM-C and EC-M PDCs showed a promising in vitro IC50 of 7.40 ± 0.37 and 8.63 ± 0.55 µM, respectively. EC-M could covalently label PLpro active site C111 and display anti-ISGylation activities in cellular assays. The results represent the first attempt to design PDCs composed of stabilized peptide inhibitors and GRL0617 to inhibit PLpro. These novel PDCs provide promising opportunities for antiviral drug design.


Subject(s)
Aniline Compounds/chemistry , Antiviral Agents/metabolism , Benzamides/chemistry , Coronavirus Papain-Like Proteases/metabolism , Drug Design , Naphthalenes/chemistry , Peptides/chemistry , SARS-CoV-2/enzymology , Aniline Compounds/metabolism , Aniline Compounds/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Benzamides/metabolism , Benzamides/pharmacology , COVID-19/pathology , COVID-19/virology , Cell Line , Cell Survival/drug effects , Coronavirus Papain-Like Proteases/chemistry , Cytokines/chemistry , Drug Evaluation, Preclinical , Humans , Inhibitory Concentration 50 , Naphthalenes/metabolism , Naphthalenes/pharmacology , SARS-CoV-2/isolation & purification , Ubiquitins/chemistry , COVID-19 Drug Treatment
19.
J Chem Inf Model ; 61(12): 6038-6052, 2021 12 27.
Article in English | MEDLINE | ID: covidwho-1517585

ABSTRACT

The papain-like protease (PLpro) of the coronavirus (CoV) family plays an essential role in processing the viral polyprotein and immune evasion. Additional proteolytic activities of PLpro include deubiquitination and deISGylation, which can reverse the post-translational modification of cellular proteins conjugated with ubiquitin or (Ub) or Ub-like interferon-stimulated gene product 15 (ISG15). These activities regulate innate immune responses against viral infection. Thus, PLpro is a potential antiviral target. Here, we have described the structural and energetic basis of recognition of PLpro by the human ISG15 protein (hISG15) using atomistic molecular dynamics simulation across the CoV family, i.e., MERS-CoV (MCoV), SARS-CoV (SCoV), and SARS-CoV-2 (SCoV2). The cumulative simulation length for all trajectories was 32.0 µs. In the absence of the complete crystal structure of complexes, protein-protein docking was used. A mutation (R167E) was introduced across all three PLpro to study the effect of mutation on the protein-protein binding. Our study reveals that the apo-ISG15 protein remains closed while it adopts an open conformation when bound to PLpro, although the degree of openness varies across the CoV family. The binding free energy analysis suggests that hISG15 binds more strongly with SCoV2-PLpro compared to SCoV or MCoV. The intermolecular electrostatic interaction drives the hISG15-PLpro complexation. Our study showed that SCoV or MCoV-PLpro binds more strongly with the C-domain of hISG15, while SCoV2-PLpro binds more favorably the N-domain of hISG15. Overall, our study explains the molecular basis of differential deISGylating activities of PLpro among the CoV family and the specificity of SCoV2-PLpro toward hISG15.


Subject(s)
COVID-19 , Coronavirus Papain-Like Proteases , Antiviral Agents , Cytokines , Humans , Interferons , SARS-CoV-2 , Ubiquitins
20.
Nat Immunol ; 22(11): 1360-1362, 2021 11.
Article in English | MEDLINE | ID: covidwho-1475315
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