Free Interferon-stimulated Gene 15 Research Articles

Free ISG15 inhibits Pseudorabies virus infection by positively regulating type I IFN signaling.

Interferon-stimulated gene 15 (ISG15) is strongly upregulated during viral infections and exerts pro-viral or antiviral actions. While many viruses combat host antiviral defenses by limiting ISG expression, PRV infection notably increases expression of ISG15. However, studies on the viral strategies to regulate ISG15-mediated antiviral responses are limited. Here, we demonstrate that PRV-induced free ISG15 and conjugated proteins accumulation require viral gene expression. Conjugation inhibition assays showed that ISG15 imposes its antiviral effects via unconjugated (free) ISG15 and restricts the viral release. Knockout of ISG15 in PK15 cells interferes with IFN-β production by blocking IRF3 activation and promotes PRV replication. Mechanistically, ISG15 facilitates IFNα-mediated antiviral activity against PRV by accelerating the activation and nuclear translocation of STAT1 and STAT2. Furthermore, ISG15 facilitated STAT1/STAT2/IRF9 (ISGF3) formation and ISGF3-induced IFN-stimulated response elements (ISRE) activity for efficient gene transcription by directly interacting with STAT2. Significantly, ISG15 knockout mice displayed enhanced susceptibility to PRV, as evidenced by increased mortality and viral loads, as well as more severe pathology caused by excessive production of the inflammatory cytokines. Our studies establish the importance of free ISG15 in IFNα-induced antiviral immunity and in the control of viral infections.

Interferon-stimulated gene 15 and ISGylation are upregulated in glioblastoma

Interferon stimulated gene 15 (ISG15) encodes a 15-kDa ubiquitin-like protein that acts as a posttranslational modifier of target proteins via ISGylation, a catalytic process similar to ubiquitination. Protein ISGylation is associated with the modulation of protein stability and protein-protein interactions. Furthermore, non-conjugated ISG15 (free ISG15) is secreted to act as a cytokine-like protein in some cellular contexts. The expression of ISG15 in some cancer types is dysregulated, but its expression status in glioblastoma, a malignant brain tumor highly aggressive and invasive, requires more studies. To explore the potential of ISG15 as a biomarker for glioblastoma, we first evaluated the ISG15 levels in glioblastoma cell lines and the effect of IFN-γ treatment on protein levels and localization of ISG15. In addition, we analyzed the ISG15 levels in glioblastoma samples compared to healthy brain tissue. Our results indicate that ISG15 levels are increased in glioblastoma and are upregulated in response to IFN-γ stimulus, suggesting that ISG15 and ISGylation may play a central role in glioblastoma progression. Thus, ISG15/ISGyaltion may be useful as biomarkers of this type of malignant brain tumors.

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.

Molecular Pathways of Interferon-Stimulated Gene 15: Implications in Cancer.

Human interferon-stimulated gene 15 (ISG15) is a 15-kDa ubiquitin-like protein that can be detected as either free ISG15 or covalently associated with its target proteins through a process termed ISGylation. Interestingly, extracellular free ISG15 has been proposed as a cytokinelike protein, whereas ISGylation is a posttranslational modification. ISG15 is a small protein with implications in some biological processes and pathologies that include cancer. This review highlights the findings of both free ISG15 and protein ISGylation involved in several molecular pathways, emerging as central elements in some cancer types.

Regulation and action of interferon-stimulated gene 15 in breast cancer cells.

Interferon-stimulated gene 15 (ISG15) is a critical ubiquitin-like protein that can be conjugated to proteins via the ISGylation system to modify them posttranslationally. Furthermore, ISG15 can be detected as non-conjugated or free, intracellularly and/or extracellularly. Both conjugated and free ISG15 participate in different cancer types, including breast cancer. Here, we highlighted the findings on ISG15 and protein ISGylation, and their implications in the field of breast cancer research. ISG15 emerges as a central element in mammary tumors and may become a crucial protein in the strategies for detection, prognosis, and therapy of breast cancer.

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.

ISG15 pathway knockdown reverses pancreatic cancer cell transformation and decreases murine pancreatic tumor growth via downregulation of PDL-1 expression.

Interferon-stimulated gene 15 (ISG15) is a 15kDa protein induced by type I interferons (IFN-α and IFN-β) and is a member of the ubiquitin-like superfamily of proteins. The ISG15 pathway is highly expressed in various malignancies, including pancreatic ductal adenocarcinoma (PDAC), suggesting a potential role of the ISG15 pathway (free ISG15 and ISG15 conjugates) in pancreatic carcinogenesis. However, very little is known about how the ISG15 pathway may contribute to pancreatic tumorigenesis. In the current study, we demonstrate that ISG15 pathway knockdown reverses the KRAS-associated phenotypes of PDAC cells such as increased proliferation and colony formation. Furthermore, clustered regularly interspaced short palindromic repeats(CRISPR)-mediated ISG15 knockdown decreased tumorprogrammed death ligand-1 (PDL-1) expression leading to increased number of CD8+ tumor-infiltrating lymphocytes and decreased pancreatic tumor growth. In addition, the syngeneic subcutaneous mouse model revealed that knocking down the ISG15 pathway significantly decreased the rate of tumor incidence and increased the survival rate. Interestingly, the ISG15 knockdown-mediated PDL-1 downregulation in pancreatic tumors increased the efficacy of anti-programmed cell death protein-1 (PD-1) treatment. ISG15 knockdown in combination with anti-PD-1 treatment synergistically increased the number of CD8+ tumor-infiltrating lymphocytes. Additionally, ISG15 knockdown alone significantly decreased the number of tumor-infiltrating regulatory T cells (Tregs) compared to wild type tumors treated with anti-PD-1 antibody. Overall, these findings suggest that strategies to target the ISG15 pathway by itself or in combination with immunotherapy may lead to improved survival for patients diagnosed with PDAC.

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.

Non-muscle myosin IIA is post-translationally modified by interferon-stimulated gene 15 in breast cancer cells.

ISG15 (interferon-stimulated gene 15) exists as free ISG15 or conjugated ISG15 modifying its target proteins via ISGylation. Few proteins have been identified and studied as ISGylation targets, and their relevance is not completely clear. Here, we isolated ISG15 from MDA-MB-231 breast cancer cells using immunoprecipitation and identified non-muscle myosin IIA (NMIIA) using mass spectrometry as endogenously associated with ISG15. The identification of NMIIA as an ISG15-interacting protein was important, because levels of NMIIA mRNA were not deregulated in all breast cancers, and because our in silico analysis indicated that NMIIA was the target of different posttranslational modifications and had an interactome associated with cytoskeletal remodeling. Furthermore, our experimental assays of co-immunoprecipitation and immunofluorescence confirmed that ISG15 was covalently associated with NMIIA in the cytoplasm of breast cancer cells and that interferon γ (IFN-γ) increased this association without alterations in the NMIIA levels. Thus, NMIIA ISGylation is regulated by IFN-γ, and this modification may modulate its interactions with proteins that remodel the cytoskeleton, participating in the growth and progression of mammary tumors.

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.

Free ISG15 triggers an antitumor immune response against breast cancer: a new perspective.

Interferon-Stimulated Gene 15 (ISG15), an antagonist of the canonical ubiquitin pathway, is frequently overexpressed in various cancers. In cancer cells, ISG15 is detected as free (intracellular) and conjugated to cellular proteins (ISGylation). Free ISG15 is also secreted into the extracellular milieu. ISGylation has protumor functions and extracellular free ISG15 has immunomodulatory properties in vitro. Therefore, whether ISG15 is a tumor suppressor or tumor promoter in vivo remains controversial. The current study aimed to clarify the role of free ISG15 in tumorigenesis. Breast cancer cells stably expressing control, ISG15, and UbcH8 (ISG15-specific E2 ligase) shRNAs were used to assess the immunoregulatory and antitumor function of free ISG15 in cell culture (in vitro) and in nude mice (in vivo). We show that extracellular free ISG15 suppresses breast tumor growth and increases NK cell infiltration into xenografted breast tumors in nude mice, and intracellular free ISG15 enhances major histocompatibility complex (MHC) class I surface expression in breast cancer cells. We conclude that free ISG15 may have antitumor and immunoregulatory function in vivo. These findings provides the basis for developing strategies to increase systemic levels of free ISG15 to treat cancer patients overexpressing the ISG15 pathway.

ISGylation governs the oncogenic function of Ki-Ras in breast cancer

Aberrant expression of the oncogenic Kirsten-Ras (Ki-Ras) and interferon-stimulated gene 15 (ISG15) pathways is common in breast and other cancers. However, whether these dysregulated pathways cooperate to promote malignancy is not known. This study links Ki-Ras and ISG15 in a previously unidentified regulatory loop that may underlie malignant transformation of mammary cells. We show that oncogenic Ki-Ras regulates the expression of the ISG15 pathway (free ISG15 and ISG15 conjugates), and ISG15, in turn, stabilizes Ki-Ras protein by inhibiting its targeted degradation via lysosomes in breast cancer cells. Disruption of this loop by silencing either Ki-Ras or the ISG15 pathway restored the disrupted cellular architecture, a hallmark feature of most cancer cells. We also demonstrate that ISG15 and UbcH8 (ISG15-specific conjugating enzyme) shRNAs reversed Ki-Ras mutation-associated phenotypes of cancer cells, such as increased cell proliferation, colony formation, anchorage-independent growth in soft agar, cell migration, and epithelial-mesenchymal transition. As UbcH8-silenced breast cancer cells are devoid of ISG15 conjugates but have free ISG15, our results using UbcH8-silenced cells suggest that ISG15 conjugates, and not free ISG15, contributes to oncogenic Ki-Ras transformation. We have thus identified the conjugated form of ISG15 as a critical downstream mediator of oncogenic Ki-Ras, providing a potential mechanistic link between ISG15 and Ki-Ras-mediated breast tumorigenesis. Our findings, which show that inhibition of the ISGylation reverses the malignant phenotypes of breast cancer cells expressing oncogenic Ki-Ras, support the development of ISG15 conjugation inhibitors for treating breast and also other cancers expressing oncogenic Ki-Ras.

Nonstructural Protein 2 of Porcine Reproductive and Respiratory Syndrome Virus Inhibits the Antiviral Function of Interferon-Stimulated Gene 15

Type I interferon (alpha/beta interferon [IFN-α/β]) stimulates the expression of interferon-stimulated gene 15 (ISG15), which encodes a ubiquitin-like protein, ISG15. Free ISG15 and ISG15 conjugates function in diverse cellular pathways, particularly regulation of antiviral innate immune responses. In this study, we demonstrate that ISG15 overexpression inhibits porcine reproductive and respiratory syndrome virus (PRRSV) replication in cell culture and that the antiviral activity of interferon is reduced by inhibition of ISG15 conjugation. PRRSV nonstructural protein 2 (nsp2) was previously identified as a potential antagonist of ISG15 production and conjugation. The protein contains a papain-like protease domain (PLP2) that plays a crucial role in the proteolytic cleavage of the PRRSV replicase polyproteins. PLP2 was also proposed to belong to the ovarian tumor domain-containing superfamily of deubiquitinating enzymes (DUBs), which is capable of inhibiting ISG15 production and counteracting ISG15 conjugation to cellular proteins. To determine whether this immune antagonist function could be selectively inactivated, we engineered a panel of mutants with deletions and/or mutations at the N-terminal border of the nsp2 PLP2-DUB domain. A 23-amino-acid deletion (amino acids 402 to 424 of the ORF1a-encoded protein) largely abolished the inhibitory effect of nsp2 on ISG15 production and conjugation, but no viable recombinant virus was recovered. A 19-amino-acid deletion (amino acids 402 to 420), in combination with a downstream point mutation (S465A), partially relieved the ISG15 antagonist function and yielded a viable recombinant virus. Taken together, our data demonstrate that ISG15 and ISGylation play an important role in the response to PRRSV infection and that nsp2 is a key factor in counteracting the antiviral function of ISG15.

Crystal Structure of Human ISG15 Protein in Complex with Influenza B Virus NS1B

ISG15 (interferon-stimulated gene 15), the first ubiquitin-like protein (UBL) identified, has emerged as an important cellular antiviral factor. It consists of two UBL domains with a short linker between them. The covalent attachment of ISG15 to host and viral proteins to modify their functions, similar to ubiquitylation, is named ISGylation. Influenza B virus NS1B protein antagonizes human but not mouse ISGylation because NS1B exhibits species specificity; it only binds human and non-human primate ISG15. Previous studies have demonstrated that the N-terminal UBL domain and linker of ISG15 are required for the binding by NS1B and that the linker plays a large role in the species specificity, but the structural basis for them has not been elucidated. Here we report the crystal structure of human ISG15 in complex with NS1B at a resolution of 2.0 Å. A loop in the ISG15 N-terminal UBL domain inserts into a pocket in the NS1B dimer, forming a high affinity binding site. The nonspecific van der Waals contacts around the ISG15 linker form a low affinity site for NS1B binding. However, sequence alignment reveals that residues in the high affinity site are highly conserved in primate and non-primate ISG15. We propose that the low affinity binding around the ISG15 linker is important for the initial contact with NS1B and that the stable complex formation is largely contributed by the following high affinity interactions between ISG15 N-terminal UBL domain and NS1B. This provides a structural basis for the species-specific binding of ISG15 by the NS1B protein.

Interferon-Stimulated Gene 15 and the Protein ISGylation System

Interferon-stimulated gene 15 (ISG15) is one of the most upregulated genes upon Type I interferon treatment or pathogen infection. Its 17 kDa protein product, ISG15, was the first ubiquitin-like modifier identified, and is similar to a ubiquitin linear dimer. As ISG15 modifies proteins in a similar manner to ubiquitylation, protein conjugation by ISG15 is termed ISGylation. Some of the primary enzymes that promote ISGylation are also involved in ubiquitin conjugation. The process to remove ISG15 from its conjugated proteins, termed de-ISGylation, is performed by a cellular ISG15-specific protease, ubiquitin-specific proteases with molecular mass 43 kDa (UBP43)/ubiquitin-specific proteases 18. Relative to ubiquitin, the biological function of ISG15 is still poorly understood, but ISG15 appears to play important roles in various biological and cellular functions. Therefore, there is growing interest in ISG15, as the study of free ISG15 and functional consequences of ISGylation/de-ISGylation may identify useful therapeutic targets. This review highlights recent discoveries and remaining questions important to understanding the biological functions of ISG15.