Gamma-activated Factor Research Articles

A novel kindred with inherited STAT2 deficiency and severe viral illness

A novel kindred with inherited STAT2 deficiency and severe viral illness

Gain-of-Phosphorylation Mutations in Coiled-Coil and DNA-Binding Domain of STAT1 Identified in Japanese Patients with Chronic Mucocutaneous Candidiasis

Abstract 4831Chronic mucocutaneous candidiasis (CMCD) is a rare congenital disorder characterized by persistent or recurrent skin, nails and mucosal membranes infections caused by Candida albicans. Several studies suggest that impairment of development in Th17 lineage and/or IL-17 signaling could be responsible for development of CMCD and seven responsible genes, CARD9, STAT3, IL12B, IL12RB1, IL17RA, IL17F, and AIRE have been identified. Recently, heterozygous mutations in coiled-coil domain (CCD) and DNA-binding domain (DBD) of STAT1 are identified in approximately 40% of patients with CMCD. Signal transducer and activation of transcription 1 (STAT1) is a DNA-binding factor which regulates specific gene transcription. IFN-γ stimulation results in phosphorylation of STAT1 at Tyr701 (pSTAT1) to induce the homodimerization, a gamma-activated factor (GAF), through the conformational change and the nuclear import. The GAF binds to the gamma-activated sequence (GAS) to induce the transcriptional activities. STAT1 mutations identified in patients with CMCD are gain-of-phosphorylation, gain-of-GAF DNA binding and gain-of-GAS transcription activity in response to IFN-γ, IFN-α and IL-27. Based on the results of transient gene experiments, impairment in dephosphorylation of STAT1 has been considered to be a molecular pathogenesis underlying the increased pSTAT1.Here we report six heterozygous missense mutations in CCD and DBD of STAT1, including two novel heterozygous STAT1 mutation, 1060C>A (L354M) and 986C>T (P329L), in two sporadic and four multiplex cases with CMCD in Japan. We investigated functional significance of these mutations by transient gene expression experiments using U3C STAT1 null fibrosarcoma cells. Similar to the previous studies, all mutant proteins showed increased pSTAT1 in response to IFN-α and IFN-γ. Increased GAF-DNA binding and GAS transcription activity were observed in mutant expressed cells. Thus, these mutations are gain-of-function mutations against GAF mediated transcription activity.We then studied dephosphorylation of STAT1 using peripheral blood mononuclear cells (PBMCs) from the patients. The PBMCs were incubated with Staurosporine, tyrosine kinase inhibitor, followed by IFN-γ stimulation and analyzed by flowcytometry and immunoblot. In both experiments, PBMCs from the patients showed increased pSTAT1 after IFN-γ stimulation. Furthermore, we observed persistent pSTAT1 after Staurosporine treatment. These findings suggest that excess pSTAT1 is caused by an impairment of dephosphorylation. The flowcytometry analysis showed no overlap in mean flow intensity of pSTAT1 between 6 patients and 11 healthy controls after Staurosporine treatment. Therefore, this method can be useful for rapid test of STAT1 function in patients with CMCD. Disclosures:No relevant conflicts of interest to declare.

IκB kinase ε (IKKε) regulates the balance between type I and type II interferon responses

Virus infection induces the production of type I and type II interferons (IFN-I and IFN-II), cytokines that mediate the antiviral response. IFN-I (IFN-α and IFN-β) induces the assembly of IFN-stimulated gene factor 3 (ISGF3), a multimeric transcriptional activation complex composed of STAT1, STAT2, and IFN regulatory factor 9. IFN-II (IFN-γ) induces the homodimerization of STAT1 to form the gamma-activated factor (GAF) complex. ISGF3 and GAF bind specifically to unique regulatory DNA sequences located upstream of IFN-I- and IFN-II-inducible genes, respectively, and activate the expression of distinct sets of antiviral genes. The balance between type I and type II IFN pathways plays a critical role in orchestrating the innate and adaptive immune systems. Here, we show that the phosphorylation of STAT1 by IκB kinase epsilon (IKKε) inhibits STAT1 homodimerization, and thus assembly of GAF, but does not disrupt ISGF3 formation. Therefore, virus and/or IFN-I activation of IKKε suppresses GAF-dependent transcription and promotes ISGF3-dependent transcription. In the absence of IKKε, GAF-dependent transcription is enhanced at the expense of ISGF3-mediated transcription, rendering cells less resistant to infection. We conclude that IKKε plays a critical role in regulating the balance between the IFN-I and IFN-II signaling pathways.

Measles virus C protein suppresses gamma-activated factor formation and virus-induced cell growth arrest

Measles virus (MeV) produces two accessory proteins, V and C, from the P gene. These accessory proteins have been reported to contribute to efficient virus proliferation through the modulation of host cell events. Our previous paper described that Vero cell-adapted strains of MeV led host cells to growth arrest through the upregulation of interferon regulatory factor 1 (IRF-1), and wild strains did not. In the present study, we found that C protein expression levels varied among MeV strains in infected SiHa cells. C protein levels were inversely correlated with IRF-1 expression levels and with cell growth arrest. Forced expression of C protein released cells from growth arrest. C-deficient recombinant virus efficiently upregulated IRF-1 and caused growth arrest more efficiently than the wild-type virus. C protein preferentially bound to phosphorylated STAT1 and suppressed STAT1 dimer formation. We conclude that MeV C protein suppresses IFN-γ signaling pathway via inhibition of phosphorylated STAT1 dimerization.

A Novel Mutation K673R In STAT1 Impaired the STAT1 Signal Transduction In a dominant– Negative Manner Identified In a Japanese Boy with MSMD

Abstract Abstract 1734 Mendelian susceptibility to mycobacterial disease (MSMD) is a rare congenital disorder characterized by susceptibility to infection by poorly virulent intracellular pathogens such as BCG, non-tuberculosis mycobacterium and Salmonellae. MSMD is associated with genetic defects in the IL-12/IFN-γ pathway, and six responsible genes, IFNGR1, IFNGR2, IL12B, IL12RB1, STAT1, and NEMO have been identified so far. Singnal transducer and activation of transcription 1 (STAT1) is a DNA-binding factor which regulates specific gene transcription. IFN-γ stimulation results in phosphorylation of STAT1 at Tyr701 (Y701) to induce the homodimerization, so called a gamma-activated factor (GAF), through the conformational change and the nuclear import. The GAF binds to the gamma-activated sequence (GAS) to induce the transcriptional activities. Patients with STAT1 deficiency were known in both recessive and dominant forms and three mutations in STAT1, L706S, E320Q and Q463H, have previously been reported from three family with an autosomal-dominant form of STAT1 partial deficiency. L706S mutation in the tail segment domain of STAT1 is shown to impair the phosphorylation at Tyr701. Although the phosphorylation is normal in other two mutations, the E320Q or Q463H mutant, in which the DNA-binding ability was abolished. Here we identified a novel heterozygous STAT1 mutation, 2018A>G (K673R), in a Japanese patient with MSMD. This was thought to be first report whose mutation was identified in the SH2 domain of autosomal-dominant form of STAT1. He had a past history of BCGitis after vacctination and multifocal osteomyelitis at the age of 5. Bone biopsy revealed granulomatous change without detecting Mycobacterium. The same mutation was also identified in his father and older sister. Although his sister had a history of BCGitis, his father had no clinical manifestations. A peripheral blood mononuclear cells from the patient could not sufficiently produce TNF-α in response to IFN-γ. EB-transformed B cells from the patient showed that induction of STAT1 phosphorylation by IFN-γ was partially impaired. We generated Flag-tagged STAT1 expression constructs of wild-type (WT) and three mutants including our mutant and analyzed molecular functions along with STAT1 signaling. K673R STAT1 showed partial impairment in the phosphorylation in response to both IFN-α and IFN-γ. Further, the electrophoretic mobility shift assay revealed that the K673R mutant abolished the DNA-binding ability. The nuclear translocation was normally detected. Curiously, dose-dependent negative effect on WT STAT1 was observed in K673R mutant by the reporter assay. K673R mutant interacted with WT to abrogate the DNA-binding activity, thus, exerted a dominant-negative effect on the transcription activity of WT STAT1. Based on these results, a novel heterozygous mutation, K673R, in SH2 domain of the STAT1 gene impaired the STAT1 signal transduction in a dominant-negative manner. K673R mutation in STAT1 may play an important role in molecular pathogenesis of MSMD. Disclosures: No relevant conflicts of interest to declare.

Inhibition of STAT 1 Phosphorylation by Human Parainfluenza Virus Type 3 C Protein

The P mRNA of the viruses belonging to the subfamily Paramyxovirinae possesses a unique property of giving rise to several accessory proteins by a process that involves the utilization of overlapping open reading frames (the C proteins) and by an "RNA-editing" mechanism (the V proteins). Although these proteins are considered accessory, numerous studies have highlighted the importance of these proteins in virus transcription and interferon signaling, including our previous observation on the role of human parainfluenza virus type 3 (HPIV 3) C protein in the transcription of viral genome (Malur et al., Virus Res. 99:199-204, 2004). In this report, we have addressed its role in interferon signaling by generating a stable cell line, L-C6, by using the lentiviral expression system which expresses HPIV 3 C protein. The L-C6 cells were efficient in abrogating both alpha and gamma interferon-induced antiviral states and demonstrated a drastic reduction in the formation of gamma-activated factor complexes in the cell extracts. Western blot analysis subsequently revealed a defect in the phosphorylation of STAT 1 in these cells. Taken together, our results indicate that HPIV 3 C protein is capable of counteracting the interferon signaling pathway by specifically inhibiting the activation of STAT 1.

Inhibition of the gamma interferon response by a Sendai virus C protein mutant with no STAT1-binding ability

Sendai virus C protein interacts with the signal transducer and activator of transcription (STAT) 1. This interaction is believed to be essential for the Sendai virus inhibition of the interferon (IFN) response. We here analyzed C F170S (a C protein mutant with the F170S mutation) with no STAT1-binding ability. C F170S lacked the ability to inhibit the IFN-α response, but retained the ability to inhibit the IFN-γ response. IFN-γ stimulation caused STAT1 phosphorylation, formation of the gamma-activated factor capable of binding to a gamma-activated sequence DNA probe, and STAT1 nuclear translocation, even in the presence of C F170S. These results suggest that C protein has the STAT1-binding-independent anti-IFN-γ mechanism, which targets processes after the STAT1 nuclear translocation event.

The C-terminal half-fragment of the Sendai virus C protein prevents the gamma-activated factor from binding to a gamma-activated sequence site

Sendai virus C protein associates with the signal transducer and activator of transcription (STAT) 1 and inhibits the interferon (IFN) response. We report a molecular basis for the anti-IFN-γ mechanism of Sendai virus. The C-terminal half-fragment of the C protein (D1) retains both the STAT1-binding and the anti-IFN-γ abilities comparable to those of the full-size C. IFN-γ stimulation generates phosphory lated-STAT1 even in the presence of the C or the D1. The phosphorylated-STAT1 generated in the D1-expressing cells forms an aberrant complex, which does not bind to a γ-activated sequence (GAS) probe. Purified D1, indeed, inhibits in vitro the binding of the phosphory lated-STAT1 dimer to the GAS probe. The D1, however, binds to the STAT1 N-terminal domain, but not the DNA binding domain. These results suggest the possibility that the C protein prevents the γ-activated factor from binding to GAS elements through its interaction with the STAT1 N-terminal domain.

Resistance to interferons in melanoma cells does not correlate with the expression or activation of signal transducer and activator of transcription 1 (Stat1).

Defects in expression or activation signal transducer and activator of transcription-1 (Stat1) in response to interferon-alpha2 (IFN-alpha2) have been implicated as a mechanism for IFN resistance in melanoma cells. To further determine the significance of this observation, 17 melanoma cell lines sensitive or resistant to the antiproliferative effects of IFN-alpha2 and IFN-beta, as well as 30 melanoma patient samples, were analyzed for Stat1 levels by either Western blot analysis or immunohistochemistry. Although the expression level varied between samples, all the cell lines except one and all melanoma biopsy specimens expressed Stat1. IFN-stimulated levels of Stat1 and Stat2, which constitute the transcriptional activation complexes, such as, gamma activated factor (GAF) and IFN-stimulated gene factor 3 (ISGF3), for IFN-stimulated gene (ISG) induction were assessed in melanoma cell lines. Both IFN-alpha2 and INF-beta induced equivalent amounts of Stat1 and Stat2 proteins in cell lines, although compared with IFN-alpha2, IFN-beta had greater antiproliferative effects. No significant differences were observed in tyrosine or serine phosphorylation of Stat1 or the formation of GAF or ISGF3 complexes following IFN-alpha2 or IFN-beta treatment of IFN-resistant or IFN-sensitive cell lines. Comparable induction of two ISGs, ISG54 and IFN regulatory factor-1 (IRF-1), was observed in both sensitive WM9 and resistant A375 cells. Therefore, we report that defects in expression or activation of Stat1 or Stat2 were infrequent in melanoma cell lines and tumor samples and did not correlate with IFN resistance. Cellular resistance to IFNs likely results from defective quantitative or qualitative expression of specific ISGs.

Interferon regulatory factor-two restricts expression of interferon-stimulated genes to the endometrial stroma and glandular epithelium of the ovine uterus.

Interferon tau (IFNtau) is the signal for maternal recognition of pregnancy in ruminants. The positive effects of IFNtau on IFN-stimulated gene (ISG) expression are mediated by ISG factor 3 (ISGF3), which is composed of signal transducer and activator of transcription (Stat) 1, Stat 2, and IFN regulatory factor-9 (IRF-9), and by gamma-activated factor (GAF), which is a Stat 1 homodimer. Induction of ISGs, such as ISG17 and 2',5'-oligoadenylate synthetase, by IFNtau during pregnancy is limited to the endometrial stroma (S) and glandular epithelium (GE) of the ovine uterus. The IRF-2, a potent transcriptional repressor of ISG expression, is expressed in the luminal epithelium (LE). This study determined effects of the estrous cycle, pregnancy, and IFNtau on expression of Stat 1, Stat 2, IRF-9, IRF-1, and IRF-2 genes in the ovine endometrium. In cyclic ewes, Stat 1, Stat 2, IRF-1, and IRF-9 mRNA and protein were detected at low levels in the S and GE. During pregnancy, expression of these genes increased only in the S and GE. Expression of IRF-2 was detected only in the LE and superficial GE (sGE) of both cyclic and pregnant ewes. In cyclic ewes, intrauterine administration of IFNtau stimulated Stat 1, Stat 2, IRF-9, and IRF-1 expression in the endometrium. Ovine IRF-2 repressed transcriptional activity driven by IFN-stimulated response elements that bind ISGF3, but not by gamma-activation sequences that bind GAF. These results suggest that IRF-2 in the LE and sGE restricts IFNtau induction of ISGs to the S and GE. In the S and GE, IFNtau hyperactivation of ISG expression likely involves formation and actions of the transcription factors ISGF3 and, perhaps, IRF-1.

The V protein of simian virus 5 inhibits interferon signalling by targeting STAT1 for proteasome-mediated degradation.

To replicate in vivo, viruses must circumvent cellular antiviral defense mechanisms, including those induced by the interferons (IFNs). Here we demonstrate that simian virus 5 (SV5) blocks IFN signalling in human cells by inhibiting the formation of the IFN-stimulated gene factor 3 and gamma-activated factor transcription complexes that are involved in activating IFN-alpha/beta- and IFN-gamma-responsive genes, respectively. SV5 inhibits the formation of these complexes by specifically targeting STAT1, a component common to both transcription complexes, for proteasome-mediated degradation. Expression of the SV5 structural protein V, in the absence of other virus proteins, also inhibited IFN signalling and induced the degradation of STAT1. Following infection with SV5, STAT1 was degraded in the absence of virus protein synthesis and remained undetectable for up to 4 days postinfection. Furthermore, STAT1 was also degraded in IFN-pretreated cells, even though the cells were in an antiviral state. Since pretreatment of cells with IFN delayed but did not prevent virus replication and protein synthesis, these observations suggest that following infection of IFN-pretreated cells, SV5 remains viable within the cells until they eventually go out of the antiviral state.

The role of protein tyrosine phosphatase SHP-1 in the regulation of IFN-gamma signaling in neural cells.

The role for protein tyrosine phosphatase SHP-1 in controlling signal transduction by IFN-gamma in astrocytes was studied. IFN-gamma induced the gamma-activated factor (GAF) within 30 min and GAF subsequently declined by 8 h after treatment. However, treatment with IFN-gamma in the presence of protein tyrosine phosphatase inhibitor vanadate blocked the decrease in GAF activity. The increased stability of GAF in vanadate-treated cultures was similarly observed in astrocytes of motheaten mice, which specifically lack the protein tyrosine phosphatase SHP-1. Prolongation of GAF activity coincided with increased expression of the IFN-inducible transcription factor, IFN-regulatory factor-1 (IRF-1). Increased IRF-1 was coincident with increased expression of MHC class I molecules in astrocytes in accordance with the activity of IRF-1 in the promoter region. These data implicate an important role for protein tyrosine phosphatases, including SHP-1, in the regulation of IFN-gamma-signaling and IFN-gamma-inducible genes in neural cells.

Modulation of major histocompatibility complex class I genes by interferon-gamma and ganglioside GT 1b in astrocytes: involvement of protein tyrosine phosphatases.

We have previously reported that the polysialoganglioside GT1b suppresses the induction of major histocompatibility complex class I molecules by interferon-gamma in astrocytes. Suppression by GT1b depended on the presence of sialic acid moieties because asialo-GM1 was not suppressive. In the present report, GT1b was found to act transcriptionally to suppress class I genes because both the interferon-gamma induction of RNA and the activity of class I promoter constructs were inhibited. Furthermore, GT1b suppressed promoter activity through interferon regulatory factor elements, indicating an effect on the transcription activation factor, interferon regulatory factor 1. Interferon-gamma induced interferon regulatory factor 1 within 8 h, and GT1b suppressed this induction. The suppression of interferon regulatory factor 1 by GT1b correlated with the suppression of gamma-activated factor binding at the promoter of the interferon regulatory factor 1 gene. The suppression of gamma-activated factor by GT1b appeared to involve increased protein tyrosine phosphatase activity because treatment of the cells with pervanadate reversed the effect of GT1b on the gamma-activated factor and, correspondingly, phosphotyrosine content. In sum, GT1b displays specific effects on interferon-gamma signaling and negative feedback regulatory molecules in astrocytes.

Interferon Regulatory Factor Element and Interferon Regulatory Factor 1 in the Induction of Major Histocompatibility Complex Class I Genes in Neural Cells

The role of the MHC-IRF-E and interferon regulatory factor 1 (IRF-1) in the regulation of MHC class I genes in astrocytes was analyzed. Transcriptional activation of MHC class I genes after treatment of astrocytes with various inducers occurred over a period of hours and correlated with cell surface expression. Functional analysis of the MHC class I gene promoter region confirmed that induction was controlled by a restricted region of 88 base pairs containing two well-defined inducible enhancers, the MHC-CRE and the MHC-IRF-E. Further analysis showed that potential MHC-CRE enhancer activity was silent. Therefore, the MHC-IRF-E, rather than the MHC-CRE, appeared responsible for enhancement of the MHC class I gene and was supported by three findings: (1) site-directed mutation of the MHC-IRF-E-abrogated induction, (2) promoter constructs containing IRF-Es as the sole enhancers were highly inducible in astrocytes, and (3) the expression of transcription factor IRF-1, which acts through the MHC-IRF-E to induce MHC class I genes, was induced to high levels in parallel with that of MHC class I induction. The induction of the IRF-1 gene correlated with the prior induction of the gamma-activated factor (GAF) or NF-kappa B, depending on the inducer, indicating that both gamma activation sites (GAS) and kappa B sites in the IRF-1 promoter are important.(ABSTRACT TRUNCATED AT 250 WORDS)

Prolactin induces rapid p95/p70 tyrosine phosphorylation, and protein binding to GAS-like sites in the anx Icp35 and c-fos genes

Several growth factors and cytokines have been proposed to act through signaling pathways related by their dependence on tyrosine phosphorylation of latent transcription factors, and their use of similar transcription factor binding sites. Related mechanisms have previously not been reported for prolactin, growth hormone, or any other glandular hormone. We have identified sequences in the annexin Icp35 gene that are related to regulatory sequences in mammary gland milk protein genes, and then used DNA binding assays to show that PRL induces transient expression of factors specific for these sequences. The sequences in question are partially related to the core homology of the interferon (IFN)gamma-activated sequence (GAS). Considering that the prolactin-regulated factors might be related to the p91 component of IFN gamma-activated factor, we used immunoassays to show that prolactin induced tyrosine phosphorylation of a protein that comigrated with immunoreactive relatives of p91, and that antibody to p91 specifically interfered with the prolactin-induced binding activity on the annexin Icp35 gene.

Prolactin induces rapid p95/p70 tyrosine phosphorylation, and protein binding to GAS-like sites in the anx Icp35 and c-fos genes.

Several growth factors and cytokines have been proposed to act through signaling pathways related by their dependence on tyrosine phosphorylation of latent transcription factors, and their use of similar transcription factor binding sites. Related mechanisms have previously not been reported for prolactin, growth hormone, or any other glandular hormone. We have identified sequences in the annexin Icp35 gene that are related to regulatory sequences in mammary gland milk protein genes, and then used DNA binding assays to show that PRL induces transient expression of factors specific for these sequences. The sequences in question are partially related to the core homology of the interferon (IFN)gamma-activated sequence (GAS). Considering that the prolactin-regulated factors might be related to the p91 component of IFN gamma-activated factor, we used immunoassays to show that prolactin induced tyrosine phosphorylation of a protein that comigrated with immunoreactive relatives of p91, and that antibody to p91 specifically interfered with the prolactin-induced binding activity on the annexin Icp35 gene.

Sublocalization of the human interferon-gamma receptor accessory factor gene and characterization of accessory factor activity by yeast artificial chromosomal fragmentation.

A chromosomal fragmentation procedure was employed to produce a deletion set of yeast artificial chromosomes (YACs) from a parental YAC, GART D142H8, known to map to human chromosome 21q and to encode the human interferon-gamma receptor (Hu-IFN-gamma R) accessory factor gene as well as the phosphoribosylglycinamide formyltransferase (GART) gene. When expressed in Chinese hamster ovary cells, these deleted YACs retain accessory factor activity, as judged by major histocompatibility complex class I antigen inducibility, until the deletions from the acentric end exceed 390 kilobases (kb). Therefore, the accessory factor (AF-1) gene can be localized to a 150-kb region at the left (centric) end of the parental 540-kb GART YAC. Cells containing functional YACs are also able to induce the ISGF3 gamma and gamma-activated factor (GAF) transcription factors, but were not protected against encephalomyocarditis virus (EMCV) upon treatment with Hu-IFN-gamma. Therefore, the Hu-IFN-gamma R and the AF-1 are sufficient for some, but not all, of the actions of Hu-IFN-gamma. We postulate that an additional accessory factor (AF-2) required for antiviral activity against EMCV is encoded on chromosome 21q.

Activation of transcription by IFN-gamma: tyrosine phosphorylation of a 91-kD DNA binding protein.

Interferon-gamma (IFN-gamma) induces the transcription of the gene encoding a guanylate binding protein by activating a latent cytoplasmic factor, GAF (gamma-activated factor). GAF is translocated to the nucleus and binds a DNA element, the gamma-activated site. Through cross-linking and the use of specific antibodies GAF was found to be a 91-kilodalton DNA binding protein that was previously identified as one of four proteins in interferon-stimulated gene factor-3 (ISGF-3), a transcription complex activated by IFN-alpha. The IFN-gamma-dependent activation of the 91-kilodalton DNA binding protein required cytoplasmic phosphorylation of the protein on tyrosine. The 113-kilodalton ISGF-3 protein that is phosphorylated in response to IFN-alpha was not phosphorylated nor translocated to the nucleus in response to IFN-gamma. Thus the two different ligands result in tyrosine phosphorylation of different combinations of latent cytoplasmic transcription factors that then act at different DNA binding sites.