Curcumin partly prevents ISG15 activation via ubiquitin-activating enzyme E1-like protein and decreases ISGylation.

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.

Free ISG15 and Protein ISGylation Emerging in SARS-CoV-2 Infection.

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.

Targeting the Ubiquitylation and ISGylation Machinery for the Treatment of COVID-19.

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.

ISGylation is increased in the peripheral blood mononuclear cells derived from symptomatic COVID-19 patients.

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.

ISGylation in Innate Antiviral Immunity and Pathogen Defense Responses: A Review.

The interferon-stimulating gene 15 (ISG15) protein is a ubiquitin-like protein induced by interferons or pathogens. ISG15 can exist in free form or covalently bind to the target protein through an enzymatic cascade reaction, which is called ISGylation. ISGylation has been found to play an important role in the innate immune responses induced by type I interferon, and is, thus, critical for the defense of host cells against RNA, DNA, and retroviruses. Through covalent binding with the host and viral target proteins, ISG15 inhibits the release of viral particles, hinder viral replication, and regulates the incubation period of viruses, thereby exerting strong antiviral effects. The SARS-CoV-2 papain-like protease, a virus-encoded deubiquitinating enzyme, has demonstrated activity on both ubiquitin and ISG15 chain conjugations, thus playing a suppressive role against the host antiviral innate immune response. Here we review the recent research progress in understanding ISG15-type ubiquitin-like modifications, with an emphasis on the underlying molecular mechanisms. We provide comprehensive references for further studies on the role of ISG15 in antiviral immunity, which may enable development of new antiviral drugs.

HERC5 and the ISGylation Pathway: Critical Modulators of the Antiviral Immune Response.

Mammalian cells have developed an elaborate network of immunoproteins that serve to identify and combat viral pathogens. Interferon-stimulated gene 15 (ISG15) is a 15.2 kDa tandem ubiquitin-like protein (UBL) that is used by specific E1-E2-E3 ubiquitin cascade enzymes to interfere with the activity of viral proteins. Recent biochemical studies have demonstrated how the E3 ligase HECT and RCC1-containing protein 5 (HERC5) regulates ISG15 signaling in response to hepatitis C (HCV), influenza-A (IAV), human immunodeficiency virus (HIV), SARS-CoV-2 and other viral infections. Taken together, the potent antiviral activity displayed by HERC5 and ISG15 make them promising drug targets for the development of novel antiviral therapeutics that can augment the host antiviral response. In this review, we examine the emerging role of ISG15 in antiviral immunity with a particular focus on how HERC5 orchestrates the specific and timely ISGylation of viral proteins in response to infection.

EGCG, a Green Tea Catechin, as a Potential Therapeutic Agent for Symptomatic and Asymptomatic SARS-CoV-2 Infection.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has emerged to be the greatest threat to humanity in the modern world and has claimed nearly 2.2 million lives worldwide. The United States alone accounts for more than one fourth of 100 million COVID-19 cases across the globe. Although vaccination against SARS-CoV-2 has begun, its efficacy in preventing a new or repeat COVID-19 infection in immunized individuals is yet to be determined. Calls for repurposing of existing, approved, drugs that target the inflammatory condition in COVID-19 are growing. Our initial gene ontology analysis predicts a similarity between SARS-CoV-2 induced inflammatory and immune dysregulation and the pathophysiology of rheumatoid arthritis. Interestingly, many of the drugs related to rheumatoid arthritis have been found to be lifesaving and contribute to lower COVID-19 morbidity. We also performed in silico investigation of binding of epigallocatechin gallate (EGCG), a well-known catechin, and other catechins on viral proteins and identified papain-like protease protein (PLPro) as a binding partner. Catechins bind to the S1 ubiquitin-binding site of PLPro, which might inhibit its protease function and abrogate SARS-CoV-2 inhibitory function on ubiquitin proteasome system and interferon stimulated gene system. In the realms of addressing inflammation and how to effectively target SARS-CoV-2 mediated respiratory distress syndrome, we review in this article the available knowledge on the strategic placement of EGCG in curbing inflammatory signals and how it may serve as a broad spectrum therapeutic in asymptomatic and symptomatic COVID-19 patients.