1
Recettore per l’IFNα
Università degli Studi di TorinoScuola Universitaria per le Biotecnologie
Corso di Laurea Specialistica in Biotecnologie Molecolari
Riccardo Pizzo
2
Complesso ifnar1-ifnar2
3
Organizzazione dei geni
Locus del recettore delle citochine di classe II nell’uomo,nel topo, nel pollo
Hardy MP. et al., “Multiple regions within the promoter of murine Ifnar-2 gene confer basal and inducible expression”, Biochem.J. 365:355,2002
4
Organizzazione del gene IFNAR1
Diop G. et al., “Exhaustive genotyping of the interferon alpha receptor 1 (IFNAR1) gene and association of an IFNAR1 protein variant with AIDS progression or susceptibility to HIV-1 infection in a French AIDS cohort”, Biomed Pharmacother. 60:569,2006
5
Organizzazione del gene IFNAR2
Varianti di splicing
huIFNAR-2a = forma solubilehuIFNAR-2b = forma tronca
huIFNAR-2c = forma completa = 515 aminoacidiHardy MP. et al., “Multiple regions within the promoter of murine Ifnar-2 gene confer basal and inducible expression”, Biochem.J. 365:355,2002
6
Formazione del complesso IFNAR1-IFNAR2-IFNα
IFNAR2 EC
Chill J.H.. et al., “The human type I interferon receptor: NMR structure reveals the molecular basis of ligand binding”, Structure 11:791,2003
Istidine nel sito di legame
Meccanismo di dissociazione del complesso ternario
Domini SD perpendicolari
Strisce idrofobiche e idrofiliche parallele
7
IFNAR1
SD1
SD2
SD3
SD4
IFNAR1 IFNAR2
IFNα
SD4 corretta orientazione dei domini SD1-2-3 Alta efficienza di
reclutamento di IFNAR1 nel complesso ternario
SD1-2-3 legame a bassa affinità con IFNα
IFNAR1 aumenta di 20 volte l’affinità di legame di IFNAR2
Cajean-Feroldi C. et al., “Identification of residues of the IFNAR1 chain of type I human interferon receptor critical for ligand binding and biological activity”, Biochemistry 43:12498,2004
8
Lamken P. et al., “Functional cartography of the ectodomain of type I interferon receptor subunit ifnar1”, J.Mol.Biol. 350:476 ,2005
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Trasduzione del segnale
Blocco della proliferazione+apoptosi
Takaoka A. et Yanai H., “Interferon signaling network in innate defence”, Cell Microbiol . 8:907 ,2006
Jak/Stat
Takaoka A. et Taniguchi T., “New aspects of IFNα-β signalling in immunity, oncogenesis and bone metabolism”, Cancer sci. 94:405 ,2003
ERK2/p38
Stat5-CrkL
PKCδ
PI3K
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PI3K sopravvivenzaapoptosi VS
Pokrovskaja K. et al., “Alternative signaling pathways regulating type I interferon-induced apoptosis”, J Interferon Cytokine Res. . 25:799 ,2005
Caraglia M. et al., “Alpha-interferon and its effests on signal trasduction pathways”, J Cell Physiol . 202:323 ,2005
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1)Trasduzione del segnale mediato dal dominio citoplasmatico ICD di IFNAR2
IFNAR2
1°clivaggioformazione del membrane stub =58kD
2°clivaggio formazione dell’ICD =34kD
Proteolisi attivata da IFNα+ PMA+ TNFα+ p38MAPK+ PKCδ+ EGF
Preseniline coinvolte nel clivaggio di IFNAR2
INFNAR2 ICD localizza nel nucleo
Inibizione della trascrizione mediato da HDAC per costrutti contenenti promotore TK
Attivazione della trascrizione mediato da Stat2 per costrutti contenenti promotore α-globina+VHL
Saleh A.ZM. et al., “Regulated proteolysis of the IFNaR2 subunit of the interferon-alpha receptor”, Oncogene. 23:7076,2004
El Fiky A. et al., “Intracellular domain of the IFNaR2 interferon receptor subunit mediates transcription via Stat2”, J Cell Physiol . 204:567 ,2005
Modulazione del segnale
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Ifnar1 proteina2)Modulazione del segnale mediato da IFNAR2 solubile
IFNAR2a vs IFNAR2c
Western blots di siero,liquido peritoneale,urina di topo adulto
Quantificazione dell’espressione dell’mRNA di IFNAR2 in tessuti di topo adulto
Inibizione dell’attività indotta dall’INFα dell’ 2’5’ OASLuc L ad opera di IFNAR-2a
Inibizione dell’effetto antiproliferativo dell’IFNα su timociti primari ad opera di IFNAR-2a ricombinante
IFNAR-2a inibisce l’attività dell’IFNα Hardy MP. et al., “The soluble murine type I interferon receptor Ifnar-2 is present in serum, is independently regulated,and has both agonistic and antagonist properties”, Blood 97:473,2001
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Complementazione dell’attività antiproliferativa indotta dall’INFα in timociti IFNAR-2 -/- ad opera di IFNAR-2a
IFNAR-2a può formare un complesso con IFNAR1 capace di mediare una parziale risposta antiproliferativa
Il segnale indotto da IFNα può essere trasdotto in assenza del dominio intracellulare di IFNAR-2c
IFNAR-2a inibitorio
IFNAR-2a stimolatorio
Hardy MP. et al., “The soluble murine type I interferon receptor Ifnar-2 is present in serum, is independently regulated,and has both agonistic and antagonist properties”, Blood 97:473,2001
14
3)Modulazione del segnale mediato da cross-competizione
EpoR/IFNAR1 chimera downmodula la risposta a IFNα
IL-12Rβ1 downmodula la risposta a IFNα
EpoR/IFNAR1 chimera+ IL-12Rβ1 interferiscono con il pathway JAK-STAT
EpoR/IFNAR1 chimera+ IL-12Rβ1 riducono i livelli di IFNAR1 in membrana
Riduzione di IFNAR1 dipende da Tyk2
Dondi E. et al., “Down-modulation of type I interferon responses by receptor cross-competition for a shared Jak kinase”, J.Biol.Chem. 276:47004,2001
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Downregolazione del segnale
Fosfatasi= SHP1+SHP2+TcPTP
SOCS
PIAS
Crm1
David M., “Signal transduction by type I interferons”, BioTechniques. 33:S58,2002
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Dowregolazione di IFNAR1
IFNα induce degradazione
Degradazione nei lisosomi
Stimolazione con IFNα attiva Tyk2
Tyk2 induce la fosforilazione di Ser535 di IFNAR1
Marijanovic Z. et al., “TYK2 activity promotes ligand-induced IFNAR1 proteolysis”, Biochem.J. 397:31,2006
Ser535 di IFNAR1 è necessaria per il legame con β-Trcp
Ser535 di IFNAR1 è necessaria per l’ubiquitinazione
Kumar K.G. et al., “Phosphorylation and specific ubiquitin acceptor sites are required for ubiquitination and degradation of tha IFNAR1 subunit of type I interferon receptor”, J.Biol.Chem. 279:46614,2004
17
Hardy MP. et al., “Multiple regions within the promoter of murine Ifnar-2 gene confer basal and inducible expression”, Biochem.J. 365:355,2002
Diop G. et al., “Exhaustive genotyping of the interferon alpha receptor 1 (IFNAR1) gene and association of an IFNAR1 protein variant with AIDS progression or susceptibility to HIV-1 infection in a French AIDS cohort”, Biomed Pharmacother. 60:569,2006
Chill J.H.. et al., “The human type I interferon receptor: NMR structure reveals the molecular basis of ligand binding”, Structure 11:791,2003
Cajean-Feroldi C. et al., “Identification of residues of the IFNAR1 chain of type I human interferon receptor critical for ligand binding and biological activity”, Biochemistry 43:12498,2004
Lamken P. et al., “Functional cartography of the ectodomain of type I interferon receptor subunit ifnar1”, J.Mol.Biol. 350:476 ,2005
Takaoka A. et Taniguchi T., “New aspects of IFNα-β signalling in immunity, oncogenesis and bone metabolism”, Cancer sci. 94:405 ,2003
Takaoka A. et Yanai H., “Interferon signaling network in innate defence”, Cell Microbiol . 8:907 ,2006
Pokrovskaja K. et al., “Alternative signaling pathways regulating type I interferon-induced apoptosis”, J Interferon Cytokine Res. 25:799 ,2005
Caraglia M. et al., “Alpha-interferon and its effests on signal trasduction pathways”, J Cell Physiol . 202:323 ,2005
Saleh A.ZM. et al., “Regulated proteolysis of the IFNaR2 subunit of the interferon-alpha receptor”, Oncogene. 23:7076,2004
El Fiky A. et al., “Intracellular domain of the IFNaR2 interferon receptor subunit mediates transcription via Stat2”, J Cell Physiol . 204:567 ,2005
Hardy MP. et al., “The soluble murine type I interferon receptor Ifnar-2 is present in serum, is independently regulated,and has both agonistic and antagonist properties”, Blood 97:473,2001
Bibliografia
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Kumar K.G. et al., “Phosphorylation and specific ubiquitin acceptor sites are required for ubiquitination and degradation of tha IFNAR1 subunit of type I interferon receptor”, J.Biol.Chem. 279:46614,2004
Dondi E. et al., “Down-modulation of type I interferon responses by receptor cross-competition for a shared Jak kinase”, J.Biol.Chem. 276:47004,2001
Pestka S. et al., “Interferons, interferon-like cytokines, and their receptors”, Immunol. rev. 202:8,2004
Asselin-Paturel E. et al., “Production of type I interferons:plasmocytoid dendritic cells and beyond”, J Exp Med. . 202:461,2005
Lamken P. et al., “Ligand-induced assembling of type I interferon receptor on supported lipid bilayers”, J.Mol.Biol. 341:303,2004
Cali Cutrone E. et al., “Identification of critical residues in bovine IFNAR-1 responsible for interferon binding”, J.Biol.Chem. 276:17140,2001
Bekisz J. et al., “Human interferons alpha, beta and omega”, Growth factors. 22:243,2004
Deonarain R. et al., “Interferon-α-β receptor interactions:a complex story unfolding”, Curr Pharm Des. . 8:2131,2002
David M., “Signal transduction by type I interferons”, BioTechniques. 33:S58,2002
Marijanovic Z. et al., “TYK2 activity promotes ligand-induced IFNAR1 proteolysis”, Biochem.J. 397:31,2006
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