Interferon-stimulated Gene 15 Protein Research Articles

ISG15 mediates the function of extracellular vesicles in promoting ovarian cancer progression and metastasis

AbstractThe interferon stimulated gene 15 (ISG15), a ubiquitin like protein and its conjugates have been implicated in various human malignancies. However, its role in ovarian cancer progression and metastasis is largely unknown. In high grade serous ovarian cancer (HGSOC), ascites is the major contributor to peritoneal metastasis. In this study, we identified significantly elevated ISG15 protein expression in HGSOC patient ascites, ascites derived primary ovarian cancer cells (POCCs), POCC small extracellular vesicles (sEVs) as well as metastatic tissue. Our results demonstrates that ISG15 increases exocytosis in ascites‐derived POCCs by decreasing the endosome‐lysosomal fusion, indicating a key role in sEV secretion. Further, knockdown (KD) of ISG15 resulted in a significant decrease in vesicles secretion from HGSOC cells and in vivo mouse models, leading to reduced HGSOC cell migration and invasion. Furthermore, our pre‐clinical mouse model studies revealed the influence of vesicular ISG15 on disease progression and metastasis. In addition, knockdown of ISG15 or using the ISG15 inhibitor, DAP5, in combination therapy with carboplatin showed to improve the platinum sensitivity in‐vitro and reduce tumour burden in‐vivo. We also found that ISG15 expression within sEV represents a promising prognostic marker for HGSOC patients. Our findings suggest that ISG15 is a potential therapeutic target for inhibiting progression and metastasis in HGSOC and that vesicular ISG15 expression could be a promising biomarker in the clinical management of ovarian cancer.Significance: High‐grade serous ovarian cancer (HGSOC) has high morbidity and mortality rates, but its progression and metastasis are still poorly understood, and there is an urgent need for early detection and targeted therapies. Our study presents novel findings that implicate ISG15‐mediated vesicular proteins in the advancement and spread of HGSOC. These results offer pre‐clinical evidence of potential new molecular targets, prognostic markers and therapeutic strategies for HGSOC that could ultimately enhance patient survival.

The role of interferon-stimulated gene 15 in the occurence and progression of cervical squamous cell carcinoma.

To identify molecular markers for early diagnosis and new targets for treatment of cervical squamous cell carcinoma. Our study involved 52 carcinoma tissues that were confirmed pathologically as cervical squamous cell carcinoma (CSCC) at the Fourth Hospital of Hebei Medical University in 2021. We obtained 36 control specimens from patients who had undergone hysterectomy for benign uterine diseases in 2021, with no cervical lesions as confirmed by pathology. Total RNA was extracted from all the samples. Reverse transcription and quantitative real-time PCR were performed. Immunohistochemical staining for interferon-stimulated gene 15 (ISG15) protein was performed. Descriptive analyses including mean and standard deviation were used to compare different groups. For data that do not conform to normal distribution, we use Wilcox rank sum test to make statistics to compare different groups with the median and interquartile. Mann Whitney U test was used to compare non-parametric continuous data, and categorical variables were analyzed using chi-square test. Receiver operating characteristic (ROC) curve was used to evaluate the possibility of using ISG15 as a new biomarker for cervical squamous cell carcinoma. Compared with normal cervical tissues, mRNA expression of ISG15 in cervical cancer tissues was significantly lower (P<0.01); mRNA expression was significantly lower in patients with nerve invasion (P<0.05). Difference in ISG15 protein expression was statistically significant (no expression/low expression) in the cancer samples compared to normal tissues (P<0.01). The area under ROC curve was 0.810 (P<0.001) and the sensitivity and specificity were 75% and 54%, respectively. Spearman's correlation analysis showed that ISG15 mRNA was positively correlated with protein expression (r=0.358, P=0.001). Deficiency of ISG15 may be associated with the occurrence and progression of CSCC. It could be used as a potential tumor marker in research and treatment of CSCC.

The role of interferon-stimulated gene 15 in the occurence and progression of cervical squamous cell carcinoma.

To identify molecular markers for early diagnosis and new targets for treatment of cervical squamous cell carcinoma. Our study involved 52 carcinoma tissues that were confirmed pathologically as cervical squamous cell carcinoma (CSCC) at the Fourth Hospital of Hebei Medical University in 2021. We obtained 36 control specimens from patients who had undergone hysterectomy for benign uterine diseases in 2021, with no cervical lesions as confirmed by pathology. Total RNA was extracted from all the samples. Reverse transcription and quantitative real-time PCR were performed. Immunohistochemical staining for interferon-stimulated gene 15 (ISG15) protein was performed. Descriptive analyses including mean and standard deviation were used to compare different groups. For data that do not conform to normal distribution, we use Wilcox rank sum test to make statistics to compare different groups with the median and interquartile. Mann Whitney U test was used to compare non-parametric continuous data, and categorical variables were analyzed using chi-square test. Receiver operating characteristic (ROC) curve was used to evaluate the possibility of using ISG15 as a new biomarker for cervical squamous cell carcinoma. Compared with normal cervical tissues, mRNA expression of ISG15 in cervical cancer tissues was significantly lower (P<0.01); mRNA expression was significantly lower in patients with nerve invasion (P<0.05). Difference in ISG15 protein expression was statistically significant (no expression/low expression) in the cancer samples compared to normal tissues (P<0.01). The area under ROC curve was 0.810 (P<0.001) and the sensitivity and specificity were 75% and 54%, respectively. Spearman's correlation analysis showed that ISG15 mRNA was positively correlated with protein expression (r=0.358, P=0.001). Deficiency of ISG15 may be associated with the occurrence and progression of CSCC. It could be used as a potential tumor marker in research and treatment of CSCC.

Abstract 11283: Induction of Interferon-Stimulated Gene 15 by Pressure Overload Facilitates Adverse Ventricular Remodeling

Introduction: Inflammation promotes adverse ventricular remodeling, a common antecedent of heart failure. Here, we set out to determine how cardiomyocytes respond to accumulating inflammatory cells within the remodeling heart and how this response, in turn, affects heart function. Methods: Experiments were performed in mouse and rat models of pressure overload, cultured mouse and human cardiomyocytes and human heart failure tissue. Results: CCR2+ monocyte-derived macrophages from Ccr2 gfp/+ mice accumulated early in mouse hearts after transverse aortic constriction (TAC) and Ccr2 knockout attenuated cardiac function decline (ejection fraction, fractional shortening, cardiac output and stroke volume). Cardiomyocyte RNA sequencing revealed that the secretome of CCR2+ cells isolated from TAC hearts induced an interferon response in cardiomyocytes, characterized by upregulation of interferon-stimulated gene 15 (ISG15), which post-translationally modifies actively translated proteins through a process termed ISGylation. ISG15 upregulation or protein ISGylation were also observed in the hearts of mice infused with angiotensin II, uninephrectomized rats treated with DOCA-salt, rats after pulmonary artery banding; mouse and human cardiomyocytes exposed to recombinant interferons; and ventricular tissue of humans with non-ischemic cardiomyopathy. ISG15 accumulated at, or close to, cardiomyocyte intercalated discs and Nano LC-MS/MS, co-immunoprecipitation and co-localization experiments identified the myofibrillar protein filamin-C to be an ISGylation target at these sites. Lastly, knockout of Isg15 attenuated cardiac function decline in mice following TAC, accompanied by a transcriptional profile indicative of preserved cardiac energetics. Conclusions: Collectively, these studies reveal that ISG15 induction is a characteristic of non-ischemic ventricular remodeling and they identify protein ISGylation as an inflammation-induced post-translational modification that may contribute to heart failure development. Strategies that prevent the ISGylation of cardiomyocyte proteins occurring during ventricular remodeling may improve heart failure outcomes.

Expedient Synthesis of Ubiquitin‐like Protein ISG15 Tools through Chemo‐Enzymatic Ligation Catalyzed by a Viral Protease Lbpro

AbstractUbiquitin (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.

Expedient Synthesis of Ubiquitin‐like Protein ISG15 Tools through Chemo‐Enzymatic Ligation Catalyzed by a Viral Protease Lbpro

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.

ISG15 and ISGylation in Human Diseases.

Type I Interferons (IFNs) induce the expression of >500 genes, which are collectively called ISGs (IFN-stimulated genes). One of the earliest ISGs induced by IFNs is ISG15 (Interferon-Stimulated Gene 15). Free ISG15 protein synthesized from the ISG15 gene is post-translationally conjugated to cellular proteins and is also secreted by cells into the extracellular milieu. ISG15 comprises two ubiquitin-like domains (UBL1 and UBL2), each of which bears a striking similarity to ubiquitin, accounting for its earlier name ubiquitin cross-reactive protein (UCRP). Like ubiquitin, ISG15 harbors a characteristic β-grasp fold in both UBL domains. UBL2 domain has a conserved C-terminal Gly-Gly motif through which cellular proteins are appended via an enzymatic cascade similar to ubiquitylation called ISGylation. ISG15 protein is minimally expressed under physiological conditions. However, its IFN-dependent expression is aberrantly elevated or compromised in various human diseases, including multiple types of cancer, neurodegenerative disorders (Ataxia Telangiectasia and Amyotrophic Lateral Sclerosis), inflammatory diseases (Mendelian Susceptibility to Mycobacterial Disease (MSMD), bacteriopathy and viropathy), and in the lumbar spinal cords of veterans exposed to Traumatic Brain Injury (TBI). ISG15 and ISGylation have both inhibitory and/or stimulatory roles in the etiology and pathogenesis of human diseases. Thus, ISG15 is considered a “double-edged sword” for human diseases in which its expression is elevated. Because of the roles of ISG15 and ISGylation in cancer cell proliferation, migration, and metastasis, conferring anti-cancer drug sensitivity to tumor cells, and its elevated expression in cancer, neurodegenerative disorders, and veterans exposed to TBI, both ISG15 and ISGylation are now considered diagnostic/prognostic biomarkers and therapeutic targets for these ailments. In the current review, we shall cover the exciting journey of ISG15, spanning three decades from the bench to the bedside.

The prognostic significance of interferon-stimulated gene 15 (ISG15) in invasive breast cancer

BackgroundLymphovascular invasion (LVI) is a prognostic factor in early-stage invasive breast cancer (BC). Through bioinformatics, data analyses of multiple BC cohorts revealed the positive association between interferon-stimulated gene 15 (ISG15) LVI status. Thus, we explored the prognostic significance of ISG15 in BC.MethodsThe prognostic significance of ISG15 mRNA was assessed in METABRIC (n = 1980), TCGA (n = 854) and Kaplan–Meier Plotter (n = 3951). ISG15 protein was evaluated using immunohistochemistry (n = 859) in early-stage invasive BC patients with long-term follow-up. The associations between ISG15 expression and clinicopathological features, expression of immune cell markers and patient outcome data were evaluated.ResultsHigh mRNA and protein ISG15 expression were associated with LVI, higher histological grade, larger tumour size, hormonal receptor negativity, HER2 positivity, p53 and Ki67. High ISG15 protein expression was associated with HER2-enriched BC subtypes and immune markers (CD8, FOXP3 and CD68). High ISG15 mRNA and ISG15 expressions were associated with poor patient outcome. Cox proportional multivariate analysis revealed that the elevated ISG15 expression was an independent prognostic factor of shorter BC-specific survival.ConclusionThis study provides evidence for the role of ISG15 in LVI development and BC prognosis. Further functional studies in BC are warranted to evaluate the therapeutic potential of ISG15.

Mechanism and inhibition of the papain-like protease, PLpro, of SARS-CoV-2.

The SARS‐CoV‐2 coronavirus encodes an essential papain‐like protease domain as part of its non‐structural protein (nsp)‐3, namely SARS2 PLpro, that cleaves the viral polyprotein, but also removes ubiquitin‐like ISG15 protein modifications as well as, with lower activity, Lys48‐linked polyubiquitin. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin‐binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. To identify PLpro inhibitors in a repurposing approach, screening of 3,727 unique approved drugs and clinical compounds against SARS2 PLpro identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, non‐covalent small molecule SARS PLpro inhibitors also target SARS2 PLpro, prevent self‐processing of nsp3 in cells and display high potency and excellent antiviral activity in a SARS‐CoV‐2 infection model.

Inhibition of UBE2L6 attenuates ISGylation and impedes ATRA-induced differentiation of leukemic cells.

Ubiquitin/ISG15‐conjugating enzyme E2L6 (UBE2L6) is a critical enzyme in ISGylation, a post‐translational protein modification that conjugates the ubiquitin‐like modifier, interferon‐stimulated gene 15 (ISG15), to target substrates. Previous gene expression studies in acute promyelocytic leukemia (APL) cells showed that all‐trans‐retinoic acid (ATRA) altered the expression of many genes, including UBE2L6 (200‐fold) and other members of the ISGylation pathway. Through gene expression analyses in a cohort of 98 acute myeloid leukemia (AML) patient samples and in primary neutrophils from healthy donors, we found that UBE2L6 gene expression is reduced in primary AML cells compared with normal mature granulocytes. To assess whether UBE2L6 expression is important for leukemic cell differentiation—two cell line models were employed: the human APL cell line NB4 and its ATRA‐resistant NB4R counterpart, as well as the ATRA‐sensitive human AML HL60 cells along with their ATRA‐resistant subclone—HL60R. ATRA strongly induced UBE2L6 in NB4 APL cells and in ATRA‐sensitive HL60 AML cells, but not in the ATRA‐resistant NB4R and HL60R cells. Furthermore, short hairpin (sh)RNA‐mediated UBE2L6 depletion in NB4 cells impeded ATRA‐mediated differentiation, suggesting a functional role for UBE2L6 in leukemic cell differentiation. In addition, ATRA induced ISG15 gene expression in NB4 APL cells, leading to increased levels of both free ISG15 protein and ISG15 conjugates. UBE2L6 depletion attenuated ATRA‐induced ISG15 conjugation. Knockdown of ISG15 in NB4 APL cells inhibited ISGylation and also attenuated ATRA‐induced differentiation. In summary, we demonstrate the functional importance of UBE2L6 in ATRA‐induced neutrophil differentiation of APL cells and propose that this may be mediated by its catalytic role in ISGylation.

Loss of TRIM29 suppresses cancer stem cell-like characteristics of PDACs via accelerating ISG15 degradation.

TRIM family proteins are defined as E3 ubiquitin ligases because of their RING-finger domains. The ubiquitin-like protein interferon-stimulated gene 15 (ISG15) encodes a 15-kDa protein, that is implicated in the posttranslational modification of diverse proteins. Both TRIM29 and ISG15 play both pro-tumoral and anti-tumoral functions in cancer cells derived from different histology. In the current study, we demonstrated that correlation expression of TRIM29 and ISG15 in pancreatic ductal adenocarcinomas (PDACs). The current study demonstrated that TRIM29 knockdown destabilized ISG15 protein via promoting its processing by calpain 3 (CAPN3). Importantly, the current study found that TRIM29 knockdown suppressed cancer stem cell-like features of PDACs, which can be rescued by ISG15 independent of its conjugation function. In addition, the current study demonstrated that extracellular free ISG15 played an important role in maintenance of cancer stem cell-like features of PDACs. Thereby, the current study displayed a novel mechanism by which TRIM29 modulates ISG15 stability via CAPN3-mediated processing, and subsequently extracellular ISG15 maintains the cancer stem cell-like features of PDAC via autocrine mode of action.

In silico characterization of interferon-stimulated gene (ISG15) as a biomarker for early pregnancy diagnosis in Bubalus bubalis

Reproductive management of livestock is the foremost requirement for increasing production from any dairy animal. High reproductive efficiency can be achieved by implementation of one of the crucial management practices which involves early differentiation of pregnant and non-pregnant animals. The early and precise pregnancy diagnosis is the major problem in Bubalus bubalis (buffalo) due to absence of estrous signs instead of normal cyclicity of reproductive organs. Therefore, in the present study, one of the conceptus derived protein interferon stimulated protein (15 kDa) released during 18 to 21 days after insemination in response to implantation of embryo was in silico characterized. The protein expressed by interferon-stimulated gene-15 (ISG15) was analyzed by isolation and sequencing of coding region of mRNA. The primary and secondary structures were predicted from the protein sequence to decipher the interaction of ISG15 protein with other molecules. The functional characterization identifies various motifs present in ISG15 protein which are responsible for its interaction with other proteins. Physiochemical properties predicted the ISG15 protein nature during in vitro conditions which are required for any of the assays development. In addition, immunogenic studies revealed ISG15 protein is strongly antigenic in nature and can be used for antibody production. In conclusion, ISG15 protein expression from buffalo is a good indicator of conceptus implantation and has suitable properties for being used as target to develop early pregnancy diagnostic kits.

Interplay between interferon-stimulated gene 15/ISGylation and interferon gamma signaling in breast cancer cells

Interferon-stimulated gene 15 (ISG15) is a ubiquitin-like protein that conjugates to its target proteins to modify them through ISGylation, but the relevance of ISG15 expression and its effects have been not completely defined. Herein, we examined the interplay between ISG15/ISGylation and the interferon-gamma (IFN-γ) signaling pathway in mammary tumors and compared it with that in normal mammary tissues. Our results indicated that mammary tumors had higher levels of ISG15 mRNA and ISG15 protein than the adjacent normal mammary tissue. Furthermore, the expression of IFN-γ signaling components was altered in breast cancer. Interestingly, IFN-γ treatment induced morphological changes in MCF-7 and MDA-MB-231 breast cancer cell lines due to cytoskeletal reorganization. This cellular process seems to be related to the increase in ISGylation of cytoplasmic IQ Motif Containing GTPase Activating Protein 1 (IQGAP1). Interactome analysis also indicated that IFN-γ signaling and the ISGylation system are associated with several proteins implicated in cytoskeletal remodeling, including IQGAP1. Thus, ISG15 may present a potential biomarker for breast cancer, and IFN-γ signaling and protein ISGylation may participate in the regulation of the cytoskeleton in breast cancer cells.

Roles of miR-138 and ISG15 in oral squamous cell carcinoma.

The aim of the present study was to investigate the roles of microRNA (miR)-138 and interferon-stimulated gene 15 (ISG15) in patients with oral squamous cell carcinoma (OSCC). miR-138 and ISG15 expression in cancer tissues was detected, and the influence on proliferation, migration and invasion of OSCC cell lines was assessed. Reverse transcription-quantitative polymerase chain reaction was performed to analyze the expression of miR-138 and ISG15 in resected cancer tissues and pericancerous tissues harvested from patients with OSCC. The protein level of ISG15 was determined via western blot analysis. The constructed pGCMV/EGFP/miR-138 plasmid was transfected into CAL27 and SCC-15 OSCC cell lines via a liposome method to upregulate miR-138 expression. The transfection efficiency was determined based on miR-138 expression levels, and changes in proliferation, migration and invasion were subsequently compared with those in untransfected cells. The expression of ISG15 mRNA and protein was also detected in OSCC cells. miR-138 was significantly downregulated (P<0.05) in cancer tissues compared with adjacent normal tissues in patients with OSCC, whereas ISG15 mRNA expression levels were significantly higher in pericancerous tissues (P<0.05). ISG15 protein levels were also significantly higher in pericancerous tissues (P<0.05). ISG15 protein and mRNA levels were significantly decreased in the transfected cells compared with the untransfected cells, which indicated that miR-138 overexpression inhibited ISG15 expression. Additionally, the invasion, migration and proliferation abilities of successfully transfected CAL27 and SCC-15 cells were significantly decreased compared with the untransfected cells (P<0.05). The results of the present study suggest that miR-138 functions as a tumor-suppressive miR and serves an important role in OSCC via regulating ISG15 expression. These findings suggest that miR-138 is able to inhibit the proliferation, migration and invasion of OSCC cell lines.

Evidence for the Deregulation of Protein Turnover Pathways in Atm-Deficient Mouse Cerebellum: An Organotypic Study.

Interferon-stimulated gene 15 (ISG15), an antagonist of the ubiquitin pathway, is elevated in cells and brain tissues obtained from ataxia telangiectasia (A-T) patients. Previous studies reveal that an elevated ISG15 pathway inhibits ubiquitin-dependent protein degradation, leading to activation of basal autophagy as a compensatory mechanism for protein turnover in A-T cells. Also, genotoxic stress (ultraviolet [UV] radiation) deregulates autophagy and induces aberrant degradation of ubiquitylated proteins in A-T cells. In the current study, we show that, as in A-T cells, ISG15 protein expression is elevated in cerebellums and various other tissues obtained from Atm-compromised mice in an Atm-allele-dependent manner (Atm+/+ < Atm+/- < Atm-/-). Notably, in cerebellums, the brain part primarily affected in A-T, levels of ISG15 were significantly greater (3-fold higher) than cerebrums obtained from the same set of mice. Moreover, as in A-T cell culture, UV induces aberrant degradation of ubiquitylated proteins and autophagy in Atm-deficient, but not in Atm-proficient, cerebellar brain slices grown in culture. Thus, the ex vivo organotypic A-T mouse brain culture model mimics that of an A-T human cell culture model and could be useful for studying the role of ISG15-dependent proteinopathy in cerebellar neurodegeneration, a hallmark of A-T in humans.

Restriction of Human Cytomegalovirus Replication by ISG15, a Host Effector Regulated by cGAS-STING Double-Stranded-DNA Sensing.

Accumulation of the interferon-stimulated gene 15 (ISG15) protein product, which is reversibly conjugated to numerous polypeptide targets, impacts the proteome and physiology of uninfected and infected cells. While many viruses, including human cytomegalovirus (HCMV), blunt host antiviral defenses by limiting ISG expression, the overall abundance of ISG15 monomer and protein conjugates rises in HCMV-infected cells. However, the molecular signals underlying ISG15 accumulation and whether the ISG15 polypeptide itself influences HCMV infection biology remain unknown. Here, we establish that the ISG15 gene product itself directly regulates HCMV replication and that its accumulation restricts productive virus growth. Although ISG15 monomer and protein conjugate accumulation was induced in cells infected with UV-inactivated HCMV, it was subsequently reduced, but not eliminated, by an immediate-early (IE) or early (E) virus-encoded function(s). Instead, HCMV-induced ISG15 monomer and protein conjugate accumulation was dependent upon the double-stranded DNA (dsDNA) sensor cyclic GMP-AMP synthase (cGAS), the innate immune adaptor STING, and interferon signaling. Significantly, dsDNA itself was sufficient to induce cGAS-, STING-, and interferon signaling-dependent ISG15 monomer and conjugate protein accumulation in uninfected cells. Accumulation of ISGylated proteins in uninfected cells treated with dsDNA was prevented by expressing the HCMV multifunctional IE1 transactivator. This demonstrates that expression of a single host interferon-stimulated gene, ISG15, restricts HCMV replication, and that IE1 is sufficient to blunt ISGylation in response to dsDNA sensing in uninfected cells. Moreover, it establishes that ISGylation modifies the proteomes of virus-infected and uninfected normal cells in response to cell-intrinsic dsDNA sensing dependent upon cGAS-STING.IMPORTANCE By antagonizing type I interferon production and action, many viruses, including human cytomegalovirus (HCMV), evade host defenses. However, levels of the interferon-induced ISG15 protein, which is covalently conjugated to host and viral proteins, increase in HCMV-infected cells. How ISG15 accumulation is regulated and whether the ISG15 polypeptide influences HCMV replication remain unknown. This study establishes that ISG15 itself restricts HCMV replication and that HCMV-induced ISG15 accumulation is triggered by host defenses that detect cytoplasmic double-stranded DNA (dsDNA). Remarkably, dsDNA triggered ISG15 accumulation even in uninfected cells, and this was reduced by HCMV IE1 expression. This shows that ISG15 itself controls the replication of HCMV, which causes life-threatening disease among the immunocompromised and is a significant source of congenital morbidity and mortality among newborns. Moreover, it demonstrates that ISG15 modifies the uninfected cell proteome in response to dsDNA, potentially impacting responses to DNA vaccines, gene therapy, and autoimmune disease pathogenesis.

Cell type-dependent regulation of free ISG15 levels and ISGylation.

Interferon-stimulated gene 15 (ISG15) is an ubiquitin-like protein, which can either be found as a free protein or covalently-bound to target proteins via ISGylation. The functions of free and conjugated ISG15 are ambiguous in tumorigenesis owing to its roles as an oncogene and a tumour suppressor gene. This dual role for ISG15 could be a result of the cancer cell type and the cellular context. Here, we report that ISG15 expression is upregulated in different cancer cells compared to normal cells. Furthermore, we found higher endogenous, free ISG15 protein levels in MCF7 breast cancer cells than in other cells, suggesting that non-conjugated ISG15 levels are cell type-specific. Additionally, we demonstrated that interferon gamma (IFN-Ɣ) increased both free and conjugated levels of ISG15 in MCF7 cells. Interestingly, endogenous conjugated and free ISG15 levels were differentially regulated by IFN-Ɣ in several cell lines. On characterisation of the subcellular distribution of ISG15 in several cell types, our results indicated that free ISG15 was mainly localised to the cytoplasm of MCF7 cells, whereas ISGylation marks were also found in the cytoplasm, but mainly in the nucleus, with a specific distribution pattern in each cell type. Thus, free and conjugated ISG15 protein levels and their subcellular distribution are cell type-dependent, whereas IFN-Ɣ signalling may differentially control the abundance of both ISG15 forms in transformed and normal cells.

Consecutive Inhibition of ISG15 Expression and ISGylation by Cytomegalovirus Regulators.

Interferon-stimulated gene 15 (ISG15) encodes an ubiquitin-like protein that covalently conjugates protein. Protein modification by ISG15 (ISGylation) is known to inhibit the replication of many viruses. However, studies on the viral targets and viral strategies to regulate ISGylation-mediated antiviral responses are limited. In this study, we show that human cytomegalovirus (HCMV) replication is inhibited by ISGylation, but the virus has evolved multiple countermeasures. HCMV-induced ISG15 expression was mitigated by IE1, a viral inhibitor of interferon signaling, however, ISGylation was still strongly upregulated during virus infection. RNA interference of UBE1L (E1), UbcH8 (E2), Herc5 (E3), and UBP43 (ISG15 protease) revealed that ISGylation inhibits HCMV growth by downregulating viral gene expression and virion release in a manner that is more prominent at low multiplicity of infection. A viral regulator pUL26 was found to interact with ISG15, UBE1L, and Herc5, and be ISGylated. ISGylation of pUL26 regulated its stability and inhibited its activities to suppress NF-κB signaling and complement the growth of UL26-null mutant virus. Moreover, pUL26 reciprocally suppressed virus-induced ISGylation independent of its own ISGylation. Consistently, ISGylation was more pronounced in infections with the UL26-deleted mutant virus, whose growth was more sensitive to IFNβ treatment than that of the wild-type virus. Therefore, pUL26 is a viral ISG15 target that also counteracts ISGylation. Our results demonstrate that ISGylation inhibits HCMV growth at multiple steps and that HCMV has evolved countermeasures to suppress ISG15 transcription and protein ISGylation, highlighting the importance of the interplay between virus and ISGylation in productive viral infection.

Abstract 24: Loss of ubiquitin-specific peptidase 18 (USP18) causes cold sensitive mice by destabilizing the critical regulator of thermogenesis: uncoupling protein-1 (UCP-1)

Abstract USP18 is the major ISG15 (Interferon-Stimulated Gene 15) deconjugase that removes ISG15 from substrate proteins. We recently reported that USP18 null mice spontaneously develop leiomyosarcomas. Intriguingly, these USP18 null mice are also markedly cold sensitive as compared to their wild-type littermates. When briefly exposed to cold stimuli, USP18 null mice significantly (P &amp;lt; 0.05) decreased their body temperature. In contrast, wild-type mice exposed to the same experimental conditions exhibited transient changes in their body temperature. We sought to elucidate the engaged mechanism. Expression profiles of critical thermogenic regulatory proteins were examined by immunoblot analyses of brown fat tissues of USP18 null mice. Strikingly, UCP-1 expression was substantially reduced in these tissues of USP18 null versus wild-type mice. To independently confirm that reduced UCP-1 expression was caused by loss of USP18, stable knock-down of USP18 was independently achieved in a panel of murine cell lines by use of transfected small hairpin RNAs (shRNAs). The obtained results were compared to that of transfected control vectors. USP18 down-regulation by different shRNAs markedly reduced UCP-1 protein as compared to controls. As expected, engineered gain of USP18 expression in the same murine cell lines stabilized UCP-1 protein. We explored if UCP-1 destabilization was due to its complex formation with the ubiquitin-like protein ISG15. This was the expected experimental outcome since USP18 is the distinct deconjugase that removes ISG15 from complexed proteins. Immunoprecipitation assays were performed to establish this direct association. These studies revealed a complex formed between ISG15 and UCP-1 protein. Thus, engineered loss of USP18 in mice led to cold sensitive mice from repression of UCP-1. As a consequence of its complex with ISG15, the thermogenic regulator UCP-1 was destabilized. Taken together, this study extends prior work by showing a previously unrecognized link between USP18 and regulation of thermogenesis. This defines a novel role for the USP18 protease in metabolism. Citation Format: Xi Liu, Yun Lu, Lin Zheng, David J. Sekula, Sarah J. Freemantle, Ethan Dmitrovsky. Loss of ubiquitin-specific peptidase 18 (USP18) causes cold sensitive mice by destabilizing the critical regulator of thermogenesis: uncoupling protein-1 (UCP-1). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 24.

Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor 1 Interacts with a Member of the Interferon-Stimulated Gene 15 Pathway.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus known to establish lifelong latency in the human host. We and others have previously shown that three KSHV homologs of cellular interferon regulatory factors (IRFs), known as viral IRFs (vIRFs), participate in evasion of the host interferon (IFN) response. We report that vIRF1 interacts with the cellular interferon-stimulated gene 15 (ISG15) E3 ligase, HERC5, in the context of Toll-like receptor 3 (TLR3) activation and IFN induction. The ISG15 protein is covalently conjugated to target proteins upon activation of the interferon response. Interaction between vIRF1 and HERC5 was confirmed by immunoprecipitation, and the region between amino acids 224 and 349 of vIRF1 was required for interaction with HERC5. We further report that expression of vIRF1 in the context of TLR3 activation results in decreased ISG15 conjugation of proteins. Specifically, TLR3-induced ISG15 conjugation and protein levels of cellular IRF3, a known ISG15 target, were decreased in the presence of vIRF1 compared to the control. vIRF1 itself was also identified as a target of ISG15 conjugation. KSHV-infected cells exhibited increased ISG15 conjugation upon reactivation from latency in coordination with increased IFN. Furthermore, knockdown of ISG15 in latently infected cells resulted in a higher level of KSHV reactivation and an increase in infectious virus. These data suggest that the KSHV vIRF1 protein affects ISG15 conjugation and interferon responses and may contribute to effective KSHV replication. The KSHV vIRF1 protein can inhibit interferon activation in response to viral infection. We identified a cellular protein named HERC5, which is the major ligase for ISG15, as a vIRF1 binding partner. vIRF1 association with HERC5 altered ISG15 modification of cellular proteins, and knockdown of ISG15 augmented reactivation of KSHV from latency.