Modulation of Extracellular ISG15 Signaling by Pathogens and Viral Effector Proteins

Swaim, Caleb D.; Canadeo, Larissa A.; Monte, Kristen; Khanna, Swati; Lenschow, Deborah J.; Huibregtse, Jon M.

doi:10.1016/j.celrep.2020.107772

Cited by 78 publications

(95 citation statements)

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“…Therapeutic inhibition of PLpro would therefore be predicted to have two antiviral effects: restoration of the antiviral effect of ISGylation and inhibition of viral replication by blocking polyprotein cleavage. Further, de-ISGylation by PLpro, through generation of free (unconjugated) ISG15, enhances the secretion and extracellular signaling function of ISG15, which in turn promotes pro-inflammatory cytokine production from cells of the immune system ( 7 ). Therefore, a potential third effect of inhibiting PLpro would be a decrease in pro-inflammatory “cytokine storms” associated with COVID-19 ( 8 ).…”

Section: Main Textmentioning

confidence: 99%

“…In addition to its function as a ubiquitin-like modifier, free (unconjugated) ISG15 exits cells and signals to LFA-1-expressing cells of the immune system ( e.g ., NK cells, T cells) to release specific cytokines, including IFN-γ and IL-10 ( 7, 12 ), while ISG15 conjugates are retained in cells. Therefore, a secondary effect of PLpro-mediated de-ISGylation is the enhanced secretion of ISG15 ( 7 ). We determined whether 6-TG would block this effect.…”

Section: Main Textmentioning

confidence: 99%

“…PBMCs were placed in the upper chamber, separated from the lower chamber by a 0.4 micron membrane. In this system, ISG15 released from cells in the lower chamber signals to LFA-1-expressing cells in the upper chamber, and cytokine release in the upper chamber is measured by ELISA ( 7 ). Fig.…”

Section: Main Textmentioning

confidence: 99%

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6-Thioguanine blocks SARS-CoV-2 replication by inhibition of PLpro protease activities

Swaim

Perng

Zhao

et al. 2020

Preprint

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AbstractA recently emerged betacoronavirus, SARS-CoV-2, has led to a global health crisis that calls for the identification of effective therapeutics for COVID-19 disease. Coronavirus papain-like protease (PLpro) is an attractive drug target as it is essential for viral polyprotein cleavage and for deconjugation of ISG15, an antiviral ubiquitin-like protein. We show here that 6-Thioguanine (6-TG) inhibits SARS-CoV-2 PLpro-catalyzed viral polyprotein cleavage and ISG15 deconjugation in cells and inhibits replication of SARS-CoV-2 in Vero-E6 cells and Calu3 cells at submicromolar levels. As a well-characterized FDA-approved orally delivered drug, 6-TG represents a promising therapeutic for COVID-19 and other emerging coronaviruses.One Sentence SummaryA repurposed drug that targets an essential enzymatic activity of SARS-CoV-2 represents a promising COVID-19 therapeutic.

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Section: Main Textmentioning

confidence: 99%

Section: Main Textmentioning

confidence: 99%

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6-Thioguanine blocks SARS-CoV-2 replication by inhibition of PLpro protease activities

Swaim

Perng

Zhao

et al. 2020

Preprint

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“…However, it had been described that intracellular ISGylation inhibits the secretion of ISG15, so it seems that that they have separated pathways, one inhibiting the other. Novel de-ISGylases can reverse intracellular ISGylation improving the extracellular ISG15 secretion (66). Consequently, is more plausible that ISG15 coming from lupus NETs is in a free, extracellularly, cytokine-like form accounting, at least in part, for durable pro-in ammatory responses.…”

Section: Discussionmentioning

confidence: 99%

Conformational changes in myeloperoxidase induced by ubiquitin and NETs containing free ISG15 from Systemic Lupus Erythematosus patients promotes a pro-inflammatory cytokine response in CD4+ T cells.

Carrillo-Vázquez

Jardón-Valadez

Torres-Ruíz

et al. 2020

Preprint

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Background: Neutrophil extracellular traps (NETs) from patients with Systemic Lupus Erythematosus (SLE) are characterized by lower ubiquitylation and myeloperoxidase (MPO) as a substrate. The structural and functional effect of such modification and if there are additional post-translational modifications (PTMs) are unknown. Methods: To assess the expression and functional role of PTMs in NETs of patients with SLE; reactivation, proliferation and cytokine production was evaluated by flow cytometry using co-cultures with dendritic cells (DC) and CD4+ from SLE patients and healthy controls. The impact of ubiquitylation on MPO was assessed by molecular dynamics. The expression of ISG15 in NETs was evaluated by immunofluorescence and Western Blot. Results: Fifteen patients with SLE and 10 healthy controls were included. In the co-cultures of CD4+ lymphocytes with DC stimulated with ubiquitylated MPO or recombinant MPO, a higher expression of IFNγ and IL17A was found in CD4+ from SLE patients (p<0.05). Furthermore, with DC stimulated with ubiquitylated MPO a trend towards increased expression of CD25 and Ki67 was found in lupus CD4+ lymphocytes, while the opposite was documented in controls (p<0.05). Through molecular dynamics we found the K129-K488-K505 residues of MPO as susceptible to ubiquitylation. Ubiquitylation affects the hydration status of the HEME group depending on the residue to which it is conjugated. R239 was found near by the HEME group when the ubiquitin was in K488-K505. In addition, we found greater expression of ISG15 in the SLE NETs vs controls (p<0.05), colocalization with H2B (r=0.78) only in SLE samples and increased production of IFNγ in PBMCs stimulated with lupus NETs compared to healthy controls NETs. Conclusion: The ubiquitylated MPO has a differential effect on the induction of reactivation of CD4+ lymphocytes in patients with SLE, which may be related to structural changes by ubiquitylation at the catalytic site of MPO. Besides a lower ubiquitylation pattern, NETs of patients with SLE are characterized by the expression of ISG15, and the induction of IFNγ by Th1 cells.

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“…SARS-CoV PLpro is a cysteine protease with multiple major functions, including processing of the viral polyprotein chain for viral protein maturation, dysregulating host inflammation responses through deubiquination and impairing the host type I interferon antiviral immune responses by removing ISG15 modifications from host proteins (16)(17)(18). SARS-CoV-2 PLpro (MW = 35.6 kDa) contains two domains including an N-terminal Ubiquitin-like domain and a C-terminal ubiquitin specific protease (USP) domain with implicated catalytic functions of cleaving ubiquitin (Ub) or ISG15 modifications from host proteins (19).…”

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confidence: 99%

Structural basis for the inhibition of the papain-like protease of SARS-CoV-2 by small molecules

Huang

Tang

et al. 2020

Preprint

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SARS-CoV-2 is the pathogen responsible for the COVID-19 pandemic. The SARS-CoV-2 papain-like cysteine protease has been implicated in virus maturation, dysregulation of host inflammation and antiviral immune responses. We showed that PLpro preferably cleaves the K48-ubiquitin linkage while also being capable of cleaving ISG15 modification. The multiple functions of PLpro render it a promising drug target. Therefore, we screened an FDA-approved drug library and also examined available inhibitors against PLpro. Inhibitor GRL0617 showed a promising IC50 of 2.1 μM. The co-crystal structure of SARS-CoV-2 PLpro-C111S in complex with GRL0617 suggests that GRL0617 is a non-covalent inhibitor. NMR data indicate that GRL0617 blocks the binding of ISG15 to PLpro. The antiviral activity of GRL0617 reveal that PLpro is a promising drug target for therapeutically treating COVID-19.

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Modulation of Extracellular ISG15 Signaling by Pathogens and Viral Effector Proteins

Cited by 78 publications

References 54 publications

6-Thioguanine blocks SARS-CoV-2 replication by inhibition of PLpro protease activities

6-Thioguanine blocks SARS-CoV-2 replication by inhibition of PLpro protease activities

Conformational changes in myeloperoxidase induced by ubiquitin and NETs containing free ISG15 from Systemic Lupus Erythematosus patients promotes a pro-inflammatory cytokine response in CD4+ T cells.

Structural basis for the inhibition of the papain-like protease of SARS-CoV-2 by small molecules

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