Receptor complex and signalling pathway of the two type II IFNs, IFN-γ and IFN-γrel in mandarin fish or the so-called Chinese perch Siniperca chuatsi.

IFN-γ, as the sole member of mammalian type II IFN, is a multifunctional cytokine which exerts its effects through two distinct IFN-γ receptors, IFNGR1 and IFNGR2. However, in teleost fish, another IFN-γ homologous gene, namely IFN-γ related gene (IFN-γrel), has been identified. Although IFN-γ and IFN-γrel genes have been described in some fish species, many important aspects remain poorly understood in relation with their signalling and function. In the present study, IFN-γ and IFN-γrel, as well as their receptors, cytokine receptor family B (CRFB) 17, CRFB13, two of which are homologous to IFNGR1 in mammals, and CRFB6, homolomous to IFNGR2, have been characterized in mandarin fish, Siniperca chuatsi. It was revealed that the two type IFN members exhibit antiviral activity, and IFN-γ transduces downstream signalling through CRFB13 and CRFB6, while IFN-γrel interacts with CRFB17 to activate downstream signalling. Moreover, IFN-γ and IFN-γrel have been shown to exert antiviral biological activity in a STAT1-dependent manner. Intracellular domain analysis of CRFB17 and CRFB13 demonstrated that the Y386 tyrosine residue of CRFB13 is required for the activation of the IFN-γ-mediated biologic response, and the Y324 and Y370 residues in CRFB17 are required to activate IFN-γrel signalling.

Interferons: Success in anti-viral immunotherapy.

The interferons (IFNs) are glycoproteins with strong antiviral activities that represent one of the first lines of host defense against invading pathogens. These proteins are classified into three groups, Type I, II and III IFNs, based on the structure of their receptors on the cell surface. Due to their ability to modulate immune responses, they have become attractive therapeutic options to control chronic virus infections. In combination with other drugs, Type I IFNs are considered as "standard of care" in suppressing Hepatitis C (HCV) and Hepatitis B (HBV) infections, while Type III IFN has generated encouraging results as a treatment for HCV infection in phase III clinical trials. However, though effective, using IFNs as a treatment is not without the need for caution. IFNs are such powerful cytokines that affect a wide array of cell types; as a result, patients usually experience unpleasant symptoms, with a percentage of patients suffering system wide effects. Thus, constant monitoring is required for patients treated with IFN in order to reach the treatment goals of suppressing virus infection and maintaining quality of life.

The role of interferons in early pregnancy.

The interferons (IFNs) form part of the large family of glycoproteins known as cytokines. They are secreted by host cells as a line of defence against pathogens and certain tumours. IFNs affect cell proliferation and differentiation and also play a very important role in the functioning of the immune system. Miscarriage in both humans has been associated with higher levels of IFN, particularly IFN-γ. However, this cytokine is evidently vital in successful murine pregnancies since it is involved in maintaining the decidual layer in addition to remodelling of the vasculature in the uterus. The effects of IFN on human pregnancies are more difficult to study. Hence, there is still a lot more to be discovered in the hope of reaching a definite conclusion regarding the impact of IFN.

IFN-epsilon mediates TNF-alpha-induced STAT1 phosphorylation and induction of retinoic acid-inducible gene-I in human cervical cancer cells.

Retinoic acid inducible gene-I (RIG-I) plays important roles during innate immune responses to viral infections and as a transducer of cytokine signaling. The mechanisms of RIG-I up-regulation after cytokine stimulation are incompletely characterized. It was previously reported that IFN-gamma induces the expression of RIG-I in endothelial cells. In this study, we characterized the mechanism of type I IFN-mediated up-regulation of RIG-I in HeLa cells and found that, in addition to type I IFN, TNF-alpha, a cytokine that regulates innate immune responses, induced expression of RIG-I. To investigate whether TNF-alpha- and type I IFN-mediated up-regulations of RIG-I were causally related, we studied the kinetics of these responses. Our results were consistent with a model in which TNF-alpha functioned upstream of type I IFNs. The ability of TNF-alpha to up-regulate RIG-I required protein synthesis, expression of functional type I IFNRs, and STAT1 signaling. We also found that IFN-epsilon was the only IFN isoform expressed constitutively in HeLa cells and that its expression was up-regulated in response to stimulation with TNF-alpha. The mechanism of up-regulation involved stabilization of IFN-epsilon mRNA in the absence of transcriptional activation. Silencing the expression of IFN-epsilon attenuated STAT1 expression and phosphorylation and inhibited RIG-I expression, providing additional support for the participation of IFN-epsilon upstream of STAT1. Our findings support a sequential mechanism whereby TNF-alpha leads to stabilization of IFN-epsilon mRNA, increased IFN-epsilon synthesis, engagement of type I IFNRs, increased STAT1 expression and phosphorylation, and up-regulation of RIG-I expression. These findings have implications for our understanding of the immune responses that follow cytokine stimulation.

Phosphotyrosine proteomic study of interferon alpha signaling pathway using a combination of immunoprecipitation and immobilized metal affinity chromatography.

Tyrosine phosphorylation is a type of post-translational modification that plays a crucial role in signal transduction. Thus, the study of this modification at the proteomic level has great biological significance. However, because of the low abundance of tyrosine-phosphorylated proteins in total cell lysate, it is difficult to evaluate the dynamics of tyrosine phosphorylation at a global level. In this work, proteins carrying phosphotyrosine (pTyr) were first purified from whole cell lysate by immunoprecipitation using anti-pTyr monoclonal antibodies. After tryptic digestion, phosphopeptides were further enriched by IMAC and analyzed by LC-MS. Quantitative changes of tyrosine phosphorylation at the global level were evaluated using isotopic labeling (introduced at the methyl esterification step prior to IMAC). Using this double enrichment approach, we characterized interferon alpha (IFNalpha)-induced pTyr proteomic changes in Jurkat cells. We observed induced phosphorylation on several well documented as well as novel tyrosine phosphorylation sites on proteins involved in IFNalpha signal transduction, such as Tyk2, JAK1, and IFNAR subunits. A specific site on alpha-tubulin (Tyr-271) was observed to be phosphorylated upon treatment as well. Furthermore, our results suggest that LOC257106, a CDC42 GAP-like protein, is potentially involved in this pathway.

The human interferon system: characterization and classification after discovery of novel members.

The human interferon (IFN) system is the best characterized of all animal IFN systems. Until recently it is thought that all IFNs and IFN-related genes and proteins have been discovered. However, in the last three years, the discovery and characterization of IFNs including IFN-epsilon (IFN-epsilon), IFN-kappa (IFN-kappa) and a novel IFN-lambda (IFN-lambda) family, in particular, substantially changed this opinion. In this article, we attempt to review recent developments in the field of interferon discovery and present an overview of current classification of the human IFN system. Characterization of the constituent parts of the human IFN system including ligands, receptors and players involved in the signal transduction pathway are discussed.

The type-I interferon receptor. The long and short of it.

The type-I interferon receptor is a multisubunit receptor of the cytokine receptor superfamily. The production of specific monoclonal antibodies against the receptor and the cloning of different receptor subunits have contributed to understanding the type-I interferon receptor structure and function. The present article analyzes these new advances and the role of the different receptor subunits in type-I interferon signaling.

Differential tyrosine phosphorylation of the IFNAR chain of the type I interferon receptor and of an associated surface protein in response to IFN-alpha and IFN-beta.

The human interferon alpha-receptor (IFNAR gene product) is a transmembranal protein of 557 amino acids with an intracytoplasmic domain of 100 amino acids containing four tyrosines. Antibodies to a C-terminal peptide (residues 521-536) were developed which efficiently immunoprecipitate the 105 kDa IFNAR protein from detergent extracts of human cells. We show that the IFNAR protein becomes tyrosine phosphorylated within 5 min after treatment of human myeloma U266 cells with IFN-alpha 2, IFN-alpha 8 or IFN-beta. The IFNAR chain interacts with both IFN-alpha 2 and IFN-beta, as demonstrated by cross-linking. Among elements involved in signal transduction by type I IFNs, the tyrosine kinase Tyk2 but not Jak1, and the ISGF3 transcription factor subunit Stat2 (p113) but not Stat1 (p91), are found associated with the IFNAR protein. After IFN-beta treatment for 5 min, a tyrosine-phosphorylated protein of approximately 95 kDa (beta-PTyr) is found bound to IFNAR, but can be dissociated by denaturation. The beta-PTyr protein is present on the cell surface, like IFNAR, as shown by extracellular biotin tagging. The ratio of beta-PTyr to IFNAR tyrosine phosphorylation is much higher with IFN-beta than with IFN-alpha 2 or 8. Both are IFN dependent and abrogated by a monoclonal antibody which blocks IFNAR action. The beta-PTyr component may represent an important difference in the action of IFN-beta as compared with IFN-alpha in their shared receptor system.