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OF VET PATHOLOGYinterferon's: interferon's Isaacs and Lindenmann 1957 Did an experiment using chicken cell cultures Found a substance that interfered with viral replication and named interferon Nagano and Kojima also independently discovered this soluble antiviral protein Cont...: Cont... Naturally occurring proteins and glycoprotein's Structurally, helical cytokine family characterized by an amino acid chain 145-166 amino acids long Two types . Type I IFNs viral IFNs include IFN- α (leukocyte), IFN- β (fibroblast) Type II IFN immune IFN (IFN- γ ) synthesized by T cells: ( Clin . Microbiol. Rev. 2001)PowerPoint Presentation: Viral IFN genes in human include 13 IFN- α genes, 1 IFN -β gene Lack introns and are clustered on the short arm of chromosome 9 in human and chromosome 4 in the mouse Single IFN- γ gene three introns and maps to the long arm of chromosome 12 in human and chromosome 10 in mouse ( Clin . Microbiol. Rev. 2001) Cytokine receptor families : Cytokine receptor families _ Roitt's Essential Immunology 10th ed interferon receptor family: interferon receptor family Type 1 IFNs two subunits, IFNAR-1 and IFNAR-2 IFNAR-1, ligand-binding and signalling, IFNAR-2 signalling Type 11 IFNs distinct subunits ,ligand-binding IFNGR-1 and accessory IFNGR-2 Extracellular domain four positionally conserved cysteine residues (CCCC) and lack WSXWS motif as in class I cytokine receptors Protein Tyrosine Kinases : Protein Tyrosine Kinases Subclass of protein kinases Enzyme transfers γ phosphate group from ATP to tyrosine residues of proteins P hosphorylation of tyrosine residues creates binding sites for downstream signalling protein (Hubbard and Till, 2000) 2 classes Transmembrane PTKs Nonreceptor PTKs Insulin receptor Faks,Src Growth factor receptorTarget-selective intervention of RTK signalling : Target-selective intervention of RTK signalling Source: Zwick et al. (2002 )A novel type of tyrosine kinase participates in early events of type 1 IFN signalling : A novel type of tyrosine kinase participates in early events of type 1 IFN signalling Differential effects of tyrosine kinase inhibitors on transcriptional activation of IFN inducible genes suggested their role (Decker et al;1991) Staurosporine and k-252a prevent activation of signalling pathway ( kessler and levy;1991) Janus family of tyrosine kinases, Tyk-2 and Jak-1, constitutively associated with IFNAR1 and IFNAR2 Much larger lack consequences of myristylation ( cobb et al;1991)Genetic approach to identify genes involved in Type 1 IFN signalling: Genetic approach to identify genes involved in Type 1 IFN signalling In 2fTGH expression of gpt of E.coli controlled by IFN inducible gene 6-16 ( pellegrini et al ;1989) Selection for or against gpt expression means to isolate mutants (mulligan and berg;1981) Mutant 11,1 unresponsive to IFN α, IFN β partial sensitivity ( pellegrini et al ;1989) Serial genomic transfection rescued cosmid complementing defectSelection of mutant 11,1 and IFN responsive transfects : Selection of mutant 11,1 and IFN responsive transfects (J Immunol 2011; 187:5479-5488;) cont…: cont… Northern blots of total RNA of parental, mutant and complemented cells to confirm responsiveness to IFN using probe specific for IFN inducible 6-16 transcript Isolation of cosmids from D3-1 transfect: Isolation of cosmids from D3-1 transfect (J Immunol 2011; 187:5479-5488;) Cosmid 3D reverses 11,1 phenotype,contains tyk2 gene : Cosmid 3D reverses 11,1 phenotype,contains tyk2 gene Computer search Gene bank data base revealed complete identity to tyk2 ( crolewski et al;1990) C-terminal domains (272 aa tyk2 and 273 aa jak-1) 59% homology ( wilks et al;1991) N-terminal domains (278 aa tyk2 and 263 aa jak-1) 54% homology ( wilks et al;1991) Similar multidomain structure, high sequences conservation of tyk 2 and jak-1 suggesting involved in related Phosphorylation cascades ( zeimiec and wilks ) Signalling pathways activated by interferon's: Signalling pathways activated by interferon's Jak -Stat pathway Insulin receptor CBL and the Crk substrate (IRS) pathway pathwayJak-Stat pathway in Type I interferon signalling : Jak -Stat pathway in Type I interferon signalling Major pathway for generation of interferon signals by tyrosine kinases activation of Janus family Spontaneous association of JAK and receptor subunit Presence of cytokine, must for JAKs protein tyrosine kinase activity IFN β not IFN α induces association of IFNAR1 and IFNAR2c chains cont…: cont… Joining of SH2 domain on STAT with docking site created by JAK-mediated Phosphorylation of particular tyrosine on receptor subunits Stat1 and Stat2 associates p48 form mature ISGF3 complex which translocates to nucleus initiate gene transcription by binding to interferon stimulated response elements (ISREs)PowerPoint Presentation: ( Kuby immunology 5 th edition) Jak -Stat pathwayPowerPoint Presentation: Signalling pathways activated during engagement of the Type I interferon receptor. (Experimental Hematology 27 (1999) 1583–1592)Insulin receptor substrate (IRS ) pathway in Type I interferon signalling: Insulin receptor substrate (IRS ) pathway in Type I interferon signalling Proteins with multiple tyrosine Phosphorylation sites for the SH2domains of Grb-2, p85 , the SHP-2 phosphatase, Crk Best characterized member of the IRS family, IRS1phosphorylated on tyrosine on IFN stimulation Wortmannin, demonstrated function of PI`3-kinase not required for ISRE-dependent gene transcription Studies demonstrated IRS-1 associates Grb-2 in response to insulin not IFN α conti…: conti… Mouse 32D cells lack expression of IRS-1 and IRS-2 resistant to antiproliferative effects of mouse IFN alpha and INF beta (J Biol Chem ;1996) Signalling elements involved in Type I and II interferon-activated cellular pathways (Experimental Haematology 27 (1999) 1583–1592) Conclusion and future prospects : Conclusion and future prospects Identity between complementary DNAs that reverted mutant 11,1 to IFN responsiveness and tyk-2 DNA are strong evidences pointing physiological role of protein tyk-2 in INF alpha /beta signalling . Receptor tyrosine kinases and the activated signalling cascades represent promising areas for the development of target-selective anticancer drugs . e.g. Gastrointestinal stromal tumours (GIST)….. Imatinib Lung and pancreatic cancer …..Gefitinib Could another kinase perform the role of IFN- tyk for IFN beta …??? References: References Petska S, Langer JA, Zoon KC, Samuel CE (1987) Interferons andtheir actions. Annu Rev Biochem 56:727 A protein tyrosine kinase in the interferon alpha and beta signaling J Immunol 2011; 187:5479-5488; Stark GR, Kerr IM, Williams BRG, Silverman RH, Screiber RD (1998) How cells respond to interferons . Annu Rev Biochem 67:227 Klimpel GR, Fleischmann WR Jr , Klimpel KD (1982) Gamma interferon(IFN gamma) and IFN alpha/beta suppress murine myeloid colonyformation (CFU-C)N: magnitude of suppression is dependentupon level of colony-stimulating factor (CSF). J Immunol 129:76 Novick D, Cohen B, Rubinstein M (1994) The human interferon alpha and beta receptor: characterization and molecular cloning. Cell 77:391 Hanks SK, Quinn AM, Hunter T (July 1988). "The protein kinase family conserved features and deduced phylogeny of the catalytic domains". Science 241 (4861): 42–52 Weinberg, Robert A. (2007). The Biology Of Cancer. New York: Garland Science, Taylor & Francis Group, LLC. pp. 757–759 Roitt's Essential Immunology 10th edition Kuby immonology 5 th edition Ghoreschi K, Laurence A, O’Shea JJ. 2009. Janus kinases in immune cell signaling . Immunol Rev 228: 273–287.PowerPoint Presentation: Thank you You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.