Type I Interferonopathy due to a Homozygous Loss-of-Inhibitory Function Mutation in STAT2.

PURPOSE: STAT2 is both an effector and negative regulator of type I interferon (IFN-I) signalling. We describe the characterization of a novel homozygous missense STAT2 substitution in a patient with a type I interferonopathy. METHODS: Whole-genome sequencing (WGS) was used to identify the genetic basis of disease in a patient with features of enhanced IFN-I signalling. After stable lentiviral reconstitution of STAT2-null human fibrosarcoma U6A cells with STAT2 wild type or p.(A219V), we performed quantitative polymerase chain reaction, western blotting, immunofluorescence, and co-immunoprecipitation to functionally characterize the p.(A219V) variant. RESULTS: WGS identified a rare homozygous single nucleotide transition in STAT2 (c.656C > T), resulting in a p.(A219V) substitution, in a patient displaying developmental delay, intracranial calcification, and up-regulation of interferon-stimulated gene (ISG) expression in blood. In vitro studies revealed that the STAT2 p.(A219V) variant retained the ability to transduce an IFN-I stimulus. Notably, STAT2 p.(A219V) failed to support receptor desensitization, resulting in sustained STAT2 phosphorylation and ISG up-regulation. Mechanistically, STAT2 p.(A219V) showed defective binding to ubiquitin specific protease 18 (USP18), providing a possible explanation for the chronic IFN-I pathway activation seen in the patient. CONCLUSION: Our data indicate an impaired negative regulatory role of STAT2 p.(A219V) in IFN-I signalling and that mutations in STAT2 resulting in a type I interferonopathy state are not limited to the previously reported R148 residue. Indeed, structural modelling highlights at least 3 further residues critical to mediating a STAT2-USP18 interaction, in which mutations might be expected to result in defective negative feedback regulation of IFN-I signalling.

FBXW7-mediated stability regulation of signal transducer and activator of transcription 2 in melanoma formation.

In this study, we provide critical evidence that STAT2 stability regulation plays an essential role in melanoma cell proliferation and colony growth. We found that the interaction of FBXW7 and STAT2 induced STAT2 destabilization via a ubiquitination-mediated proteasomal degradation pathway. Notably, GSK3ß-mediated STAT2 phosphorylation facilitated STAT2-FBXW7 interactions via the DNA binding domain of STAT2 and domains 1, 2, 6, and 7 of FBXW7 WD40. Importantly, the inverse correlation between protein levels of STAT2 and FBXW7 were observed not only in human melanoma cells but also in a human skin cancer tissue array. The relationship between protein levels of STAT2 and FBXW7, cell proliferation, and colony growth were similarly observed in the melanoma cell lines SK-MEL-2, -5, and -28. Moreover, STAT2 knockdown in melanoma cells suppressed melanoma cell proliferation and colony formation. These data demonstrated that FBXW7-mediated STAT2 stability regulation plays an essential role in melanoma cell proliferation and cancer growth.

Anti-proliferative effect of 8α-tigloyloxyhirsutinolide-13-O-acetate (8αTGH) isolated from Vernonia cinerea on oral squamous cell carcinoma through inhibition of STAT3 and STAT2 phosphorylation.

BACKGROUND: The high mortality rate of oral cancers has stimulated the search for effective herbal medicines and their pharmacological targets. Vernonia cinerea, a perennial tropical herb, is wildly used as a traditional folk medicine for treatment of intestinal diseases and various skin diseases in addition to possessing anti-cancer activity. However, the effect of 8α-tigloyloxyhirsutinolide-13-O-acetate (8αTGH) as a major sesquiterpene lactone compound found in V. cinerea and the underlying mechanism of its action on oral cancer cells remains unknown. PURPOSE: To investigate the anti-cancer activity of 8αTGH extracted from V. cinerea and the underlying mechanism of its action in oral cancer cells. METHODS: The anti-proliferative effect of 8αTGH on oral squamous cell carcinoma (HSC4) and lung carcinoma (A549) was determined using the SRB colorimetric method. The molecular mechanism of 8αTGH was explored using kinase inhibitors, followed by Western blotting or RT-qPCR. Flow cytometry and Western blotting were used to assess cell cycle arrest. RESULTS: 8αTGH inhibited cancer cell growth more effectively on HSC4 than A549 and was much less effective on tested normal oral cells. 8αTGH inhibited STAT3 phosphorylation on both cancer cells. Notably, 8αTGH was able to suppress the constantly activated STAT2 found only in HSC4. The STAT2 inhibition by 8αTGH consequently caused down-regulation of ISG15 and ISG15 conjugates. As a result, decreased expression of CDK1/2 and Cyclin B1 was detected leading to G2/M cell cycle arrest. CONCLUSION: 8αTGH isolated from V. cinerea preferentially inhibits the proliferation of oral cancer cells by causing G2/M cell cycle arrest via inhibition of both STAT3 and STAT2 phosphorylation. The results provide molecular bases for developing 8αTGH as a drug candidate or a complementary treatment of oral cancer.

STAT2 is an essential adaptor in USP18-mediated suppression of type I interferon signaling.

Type I interferons (IFNs) are multifunctional cytokines that regulate immune responses and cellular functions but also can have detrimental effects on human health. A tight regulatory network therefore controls IFN signaling, which in turn may interfere with medical interventions. The JAK-STAT signaling pathway transmits the IFN extracellular signal to the nucleus, thus resulting in alterations in gene expression. STAT2 is a well-known essential and specific positive effector of type I IFN signaling. Here, we report that STAT2 is also a previously unrecognized, crucial component of the USP18-mediated negative-feedback control in both human and mouse cells. We found that STAT2 recruits USP18 to the type I IFN receptor subunit IFNAR2 via its constitutive membrane-distal STAT2-binding site. This mechanistic coupling of effector and negative-feedback functions of STAT2 may provide novel strategies for treatment of IFN-signaling-related human diseases.

Hepatitis B virus hijacks CTHRC1 to evade host immunity and maintain replication.

Hepatitis B virus (HBV) infection causes acute and chronic liver diseases, but is not directly cytopathic. Liver injury results from repeated attempts of the cellular immune response system to control the viral infection. Here, we investigate the roles of cellular factors and signaling pathways involved in the regulation of HBV replication to reveal the mechanism underlying HBV infection and pathogenesis. We show that collagen triple helix repeat containing 1 (CTHRC1) expression is elevated in HBV-infected patients and in HBV-transfected cells through epigenetic modification and transcriptional regulation. CTHRC1 facilitates HBV replication in cultured cells and BALB/c mice by activating the PKCα/ERK/JNK/c-Jun cascade to repress the IFN/JAK/STAT pathway. HBV-activated CTHRC1 downregulates the activity of type I interferon (IFN), the production of IFN-stimulated genes (ISGs), and the phosphorylation of signal transducer and activator of transcription 1/2 (STAT1/2), whereas it upregulates the phosphorylation and ubiquitination of type I IFN receptors (IFNARα/ß). Thus, our results show that HBV uses a novel mechanism to hijack cellular factors and signal cascades in order to evade host antiviral immunity and maintain persistent infection. We also demonstrate that CTHRC1 has a novel role in viral infection.

The role of signal transducer and activator of transcription-2 in the interferon response.

The signal transducer and activator of transcription-2 (STAT2) was discovered as a cellular component of the DNA binding complex known as interferon (IFN) stimulated gene factor-3. Numerous studies have confirmed that STAT2 operates as a positive regulator in the transcriptional activation response elicited by IFNs. In this article, we highlight the progress made in elucidating the pivotal role of STAT2 in driving the expression of IFN-induced genes, innate antiviral immunity, apoptosis, and cancer. A better understanding of the functional role of STAT2 in the IFN response and how STAT2 is regulated will uncover new clues to its role in diseases.

Physical requirements and functional consequences of complex formation between the cytomegalovirus IE1 protein and human STAT2.

Our previous work has shown that efficient evasion from type I interferon responses by human cytomegalovirus (hCMV) requires expression of the 72-kDa immediate-early 1 (IE1) protein. It has been suggested that IE1 inhibits interferon signaling through intranuclear sequestration of the signal transducer and activator of transcription 2 (STAT2) protein. Here we show that physical association and subnuclear colocalization of IE1 and STAT2 depend on short acidic and serine/proline-rich low-complexity motifs in the carboxy-terminal region of the 491-amino-acid viral polypeptide. These motifs compose an essential core (amino acids 373 to 420) and an adjacent ancillary site (amino acids 421 to 445) for STAT2 interaction that are predicted to form part of a natively unstructured domain. The presence of presumably "disordered" carboxy-terminal domains enriched in low-complexity motifs is evolutionarily highly conserved across all examined mammalian IE1 orthologs, and the murine cytomegalovirus IE1 protein appears to interact with STAT2 just like the human counterpart. A recombinant hCMV specifically mutated in the IE1 core STAT2 binding site displays hypersensitivity to alpha interferon, delayed early viral protein accumulation, and attenuated growth in fibroblasts. However, replication of this mutant virus is specifically restored by knockdown of STAT2 expression. Interestingly, complex formation with STAT2 proved to be entirely separable from disruption of nuclear domain 10 (ND10), another key activity of IE1. Finally, our results demonstrate that IE1 counteracts the antiviral interferon response and promotes viral replication by at least two distinct mechanisms, one depending on sequestration of STAT2 and the other one likely involving ND10 interaction.

Molecular dynamics simulations of the conformational changes in signal transducers and activators of transcription, Stat1 and Stat3.

All signal transducers and activators of transcription (STAT) factors are a family of cytoplasmic transcription factors that mediate the signal response to cytokines, growth factors, and hormonal factors. The phosphorylation and subsequent activation of Stat3, a member of the STAT family, has been found to be elevated in a large number of diverse human cancers. Understanding of the dynamics of the Stat3 dimer interface is pertinent to designing small molecule inhibitors to activated Stat3 dimer. To this end, we have performed molecular dynamics simulations in explicit water of the activated Stat3 homodimer, and also its closely related member of the STAT family, activated Stat1 homodimer. We observed a large-scale domain motion in the Stat3 dimer while the structure of the monomer remains intact. The driving force for this conformational change is enhanced binding of the Stat3 dimer to the DNA, thereby regulating gene expression. Our model shows that the carboxy terminus of one monomer wraps around the core of the SH2 domain of the other monomer, and this region that makes up the dimer interface remains intact during the dynamics. Water diffuses into a cavity under this dimer interface, thus expanding a pre-existing cavity that gets gated and closed by the loops in the SH2 domain. This cavity could serve as a potential binding pocket for inhibitor design.

Isolation and expression profile of a gene encoding for the Signal Transducer and Activator of Transcription STAT2 in Atlantic salmon (Salmo salar).

Signal Transducer and Activator of Transcription (STAT)-2 is a molecule involved in the type I interferon (IFN) signalling pathway. The full length cDNA sequence of Atlantic salmon (Salmo salar) ssSTAT2 was determined and phylogenetic analysis of the amino acid sequence grouped this novel salmon gene to the STAT2 clade. This represents the first fish STAT2 report. The gene encodes for a 802 aa polypeptide that has 38% identity to the human or murine STAT2. The expression was monitored by qPCR in the kidney of animals over the time of infection with the Infectious Salmon Anaemia Virus (ISAV) and in TO cells infected with Infectious Pancreatic Necrosis Virus (IPNV) or with the Salmon Alphavirus (SAV). SAV and ISAV induced an approximate 10-fold increase in the level of expression of ssSTAT2 gene whilst IPNV only induced a 1.5-fold increase.

A Mutation in the SH2 domain of STAT2 prolongs tyrosine phosphorylation of STAT1 and promotes type I IFN-induced apoptosis.

Type I interferons (IFN-alpha/beta) induce apoptosis in certain tumor cell lines but not others. Here we describe a mutation in STAT2 that confers an apoptotic effect in tumor cells in response to type I IFNs. This mutation was introduced in a conserved motif, PYTK, located in the STAT SH2 domain, which is shared by STAT1, STAT2, and STAT3. To test whether the tyrosine in this motif might be phosphorylated and affect signaling, Y631 of STAT2 was mutated to phenylalanine (Y631F). Although it was determined that Y631 was not phosphorylated, the Y631F mutation conferred sustained signaling and induction of IFN-stimulated genes. This prolonged IFN response was associated with sustained tyrosine phosphorylation of STAT1 and STAT2 and their mutual association as heterodimers, which resulted from resistance to dephosphorylation by the nuclear tyrosine phosphatase TcPTP. Finally, cells bearing the Y631F mutation in STAT2 underwent apoptosis after IFN-alpha stimulation compared with wild-type STAT2. Therefore, this mutation reveals that a prolonged response to IFN-alpha could account for one difference between tumor cell lines that undergo IFN-alpha-induced apoptosis compared with those that display an antiproliferative response but do not die.

Isoform selective inhibition of STAT1 or STAT3 homo-dimerization via peptidomimetic probes: structural recognition of STAT SH2 domains.

The identification of constitutively activated STAT (Signal Transducers and Activators of Transcription) proteins in aberrant cell signaling pathways has led to investigations targeting the selective disruption of specific STAT isoforms directly associated with oncogenisis. We have identified, through the design of a library of peptidomimetic inhibitors, agents that selectively disrupt STAT1 or STAT3 homo-dimerization at low micromolar concentrations. ISS840 has 20-fold higher inhibition of STAT1 homo-dimerization (IC(50) value of 31 microM) relative to STAT3 (IC(50) value of 560 microM).