Trends Biochem. Sci.
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    Trends Biochem. Sci. Trends Biochem. Sci. Presentation Transcript

    • Immunological Synapse Formation between T and APC Science 2002 295:1539
    • Immunological Synapse
    • Signal Transduction Determines Cell Response ?? ? TCR IL-2 APC
      • Role of cytokines in immune system and hematopoiesis
      • Basic concepts of signal transduction
      • JAK-STAT pathway in cytokine signaling
      • Regulation of cytokine response
      • Negative regulators for cytokine signal transduction, SOCS and PIAS families
      OUTLINE
    • General Properties of Cytokines
      • Polypeptides
      • Produced in response to microbes and antigens
      • Mediate and regulate immune and inflammatory responses
      • Pleiotropic and redundant
      • Regulate the synthesis and actions of other cytokines
      • Actions are local or systemic (autocrine, paracrine, or endocrine)
    • Modified from Cell. Mol. Immunol. Abbas & Lichtman 5 th Ed. 2003 Cytokines/Chemokines Involved in Innate Immunity TNF  , IL-1, Chemokines IFN  IL-12 Microbes NK DC Neutrophils Macrophage Blood Vessel TLR NKR TLR
    • Cytokines/Chemokines Involved in Innate Immunity TNF  IL-1 IL-12 IFN  IFN  Chemokine Cytokine Producers Targets:Effects M Ø, DC, T cells Neutrophils: activation Endothelial cells: activation (inflammation) Hypothalamus: Fever Many cells: apoptosis M Ø, endothelial Ditto Modified from Cell. Mol. Immunol. Abbas & Lichtman 5 th Ed. 2003 M Ø, DC T cells & NK: IFN  synthesis and CTL activity T cells: Th1 differentiation IFN  : M Ø All cells: antiviral state, increase MHC I IFN  : fibroblast NK cells: activation NK, NKT MØ: activation (increased microbicidal) T cells, CD8 B cells: isotype switching to IgG2A Many cells: Increase MHC I & MHC II M Ø, endothelial Leukocytes: chemotaxis, activation, Fibroblast, T cells migration to tissues
    • How Adaptive Immunity Works Modified from Cell. Mol. Immunol. Abbas & Lichtman 5 th Ed. IL-2 CD4 + T Cell IL2, IFN  IL4, IL5 Plasma Cell Ab production B Cell BCR T Cell TCR MHC: peptide DC Macrophage CD8 + CTL Granzyme, Perforin IL2, IFN  CD8 +
    • Cytokines Involved in Adaptive Immunity IL-2 IL-4 IL-5 Lymphotoxin IFN  Cytokine Producers Targets:Effects T cells T cells: proliferation, cytokine production B cells: proliferation, antibody production NK: proliferation and acitvation Th2 B cells: isotype switching to IgE T cells: Th2 differentiation, proliferation Modified from Cell. Mol. Immunol. Abbas & Lichtman 5 th Ed. 2003 Th2 Eosinophils: activation, increase prod B cells: proliferation, IgA production T cells Recruitment and activation of neutrophils Th1, CD8 MØ: activation (increased microbicidal) NK, NKT B cells: isotype switching to IgG2A Many cells: Increase MHC I & MHC II
    • Roles of Cytokines in Hematopoiesis Cell. Mol. Immunol. 2003 Abbas & Lichtman 5 th ed.
    • What Happens When Ligands Bind to Receptors
      • Conformational change of the receptors:
      • Opens ion channel
      • Facilitates binding of intracellular signaling proteins such as chemokine receptor
      • Dimerization (Oligomerization) of the receptors:
      • Bring signaling molecules into juxtaposition
      • -induces post-translational modification such as phosphorylation in the receptors or signal meidators
      • -activates downstream mediators
    • Phosphorylation of Proteins as a Controling Mechanism for Signal Transduction
      • Advantages
      • Rapid: does not require new protein synthesis or protein degradation
      • Reversible : easily reversed by action of protein phosphatases
      • Easy to relay signals: phosphorylation on Tyr, Thr, or Ser creating binding sites for other proteins
    • Tyrosine Phosphorylation Initiates Signaling (In general) PTK : Protein Tyrosine Kinase PTP : Protein Tyrosine Phosphatase Note: Src-family kinase PTP Active Form P PTK Inactive Form Signal mediator
    • Regulation of Activity of Src Family Kinase Src family: B cells: Lyn, Fyn, Blk T cells: Lck, Fyn, Immunobiology 6 th ed. 2005, Janeway et al.
    • Major Events in Signal Transduction Mediated by Receptor Tyrosine Kinase (RTK) or non-RTK
      • Ligand-induced receptor dimerization
      • Activation of kinases
      • Activation of signal mediators
      • Activation of transcription factors
      • Translocation of transcription factors into nucleus and transactivation
    • Cytokine Receptor-mediated Signaling Pathways
    • P STAT2 JAK1 TYK2 IFN  R STAT1 STAT3 Cytosol Nucleus ISRE STAT2 p48 STAT1 STAT3 STAT1 GAS GAS STAT3 STAT1 STAT1 GAS STAT3 Interferon-  Receptor Signaling Pathway P P P P P P P P P P Kinase Signal Transducer Activator of Transcription
    • Chris Schindler Cloning STAT1 Xin-Yuan Fu STAT2 David Levy STAT3 Jim Darnell Rockefeller U Biochemical Approach George Stark Cleveland Clinic Fund Ian Kerr ICRF UK Genetic Approach Sandra Pellegrini
    • Biochemical Approach for Elucidating IFN Signaling Pathway HeLa Microsequencing and cDNA cloning IFN  or IFN  Analysis promoter of IFN-inducible genes Consensus binding sequence GAS (gamma activated site) : TTN 4-6 AA ISRE (IFN-stimulated response element) :AGTTN 3 TTC Purification of ISGF3 or GAF Using GAS or ISRE-column
    • Mol. Cell. Biol. 5 th ed. 2004 Lodish et al. Genetic Approach for Elucidating IFN Signaling Pathway ICR191 (frame shift mutagen)
    • Complementation group Response to Ligands IFN  IFN  Complementing Protein U1 U2 U3 U4 U5 U6  1  2 - + TYK2 IRF9(P48) STAT1 JAK1 IFNAR2 STAT2 JAK2 IFNGR2 - - ± - - - - - + + + + ± - Complementation Groups for IFN  (U1-U6) or IFN  (  and  ) responses ± :indicated that some genes do not respond, whereas others do respond well Q: Why there is no STAT3 mutation in the complementation group ? Modified from Science 1994 264:1415
    • Animation for JAK-STAT signaling
    • Structural and Functional Domains of JAK Family JAK family : JAK1, JAK2, JAK3 and TYK2 Nat. Rev. Mol. Cell Biol. 2002 3:651
    • Nat. Rev. Mol. Cell Biol. 2002 3:651 Structural and Functional Domains of STATs STAT family: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6
    • Activation of JAKs and STATs by Cytokines Modified from Gene 2002 285:1 Ligand Jak kinases STATs IFN family Type I IFN- IFN  or IFN  Tyk2, Jak1 STAT1, STAT2 Type II IFN-IFN  Jak1, Jak2 STAT1  C family IL-2 Jak1, Jak3 STAT5 IL-4 Jak1, Jak3 STAT5 IL-7 Jak1, Jak3 STAT5 gp130 family IL-6 Jak2 STAT3 IL-11 Jak2 STAT3
    • Cytokine Receptor Families by Their Structures (wsxws motif for  -helical cytokine) (serpentine)
    • Molecular Structure of Class I Receptors Cytokine Growth Factor Rev 2001 12:19 One chain for Ligand Binding One chain for Signal transducing
    • Nat. Rev. Immunol.2001 1:200 Critical Roles of  C Family in Lymphocyte Development and Function
    • N. Eng. J. Med. 2000 343:1313 SCID Resulting from Defects in IL-7R  , JAK3 or  c Chain
    • Nat. Rev. Mol. Cell Biol. 2002 3:651 Phenotypes of JAK-Knockout Mice
    • Prevention of Organ Allograft Rejection by a Specific Janus Kinase 3 Inhibitor Science 2003 302:875 NHP: non-human primates Low dose high dose
    • Nat. Rev. Mol. Cell Biol. 2002 3:651 Phenotypes of STAT Knockout Mice
    • Nature Genet. 2003 33:388 (U. Paris) Impaired Response to IFN  /  and Lethal Viral Disease in Human STAT1 Deficiency
      • Receptor-mediated endocytosis and degradation
      • Dephosphorylation by tyrosine phosphatases
      • Naturally occurring dominant negative STATs such as STAT1  and STAT3  that don’t have transactivating domain
      • S uppressor o f c ytokine s ignaling (SOCS) family
      • P rotein i nhibitor of a ctivated S tats (PIAS)
      Negative Regulation of the JAK-STAT pathway
    • Cell 2002 109:S121-S131
    • Negative Regulations of Cytokine Signaling Nat. Rev. Immunity 2003 3:900
    • Structural and Functional Domains of SOCS Family Nat. Rev. Immunol. 2002 2:410 K: Kinase inhibitory region
    • Inhibitory Mechanisms of SOCS Molecules Trend. Immunol. 2003 24:659
    • SOCS Family Members Target Signaling Proteins for Degradation by Proteasome Trends Biochem. Sci. 2002 27:235
    •                                                                                                                    SOCS Targeting Key Signaling Proteins for Degradation by the Proteasome Sci. STKE, Vol. 2003 169:pe6
    • Liver Degeneration and Lymphoid Deficiencies in Mice Lacking SOCS1 Proc. Natl. Acad. Sci. 1998 95: 14395 SOCS1-/- SOCS1+/+
    • SOCS1 Is a Critical Inhibitor of IFN   Signaling and Prevents Fatal Neonatal Actions of this Cytokine Cell 1999 98:597 JBC 2006 281:11135
    • Nat. Rev. Mol. Cell Biol. 2002 3:651 Phenotypes of SOCS Knockout Mice
    • Hypersensitivity of SOCS-1 KO Mice to LPS In Vivo Immunity 2002 Vol. 17:583
    • SOCS1 Negatively Regulates TLR Signaling by Mal/ TIRAP Degradation Nat. Immunol. 2006 7:148 Forward
    • TLR Signaling Pathways Cell Death Diff. 2006 13:816 back
    • SOCS1 Is a Suppressor of Liver Fibrosis and Hepatitis-induced Carcinogenesis JEM 2004 199:1701
    • Nat. Rev. Immunol. 2005 5:593 SAP: S caffold Attachment factor A and B, A cinus, and P IAS RLD: R ING finger- l ike Zinc binding d omain AD: A cidic d omain SIM: S UMO- i nteracting m otif S/T: S erine and T hreonine-rich Structural and Functional Domains of PIAS Family SIM
    • Nat. Rev. Immunol. 2005 5:593 PIAS-protein Regions Involved in Protein–protein Interactions
    • Nat. Rev. Immunity 2003 3:900 Proposed Mechanisms for Inhibiting the JAK–STAT Pathway by PIAS Proteins
    • Negative Regulation of PIAS on Gene Transactivation Nat. Rev. Immunol. 2005 5:593
    • Positive Regulation of PIAS on Gene Transactivation Nat. Rev. Immunol. 2005 5:593
    • Some but not All IFN  -inducible Genes Are Enhanced in Pias1 -/- MEF Nat. Immunol. 2004 5:891
    • Enhanced Antiviral Response and Sensitivity to LPS-induced Toxic Shock in Pias1 -/- Mice Nat. Immunol. 2004 5:891
    • Enhanced Expression of NF-  B-Regulated Genes in Response to TNF-  and LPS in Pias1 null Cells Mol. Cell. Biol. 2005, 25:1113
    • PIAS1 Affects the Binding of p65 to the Endogenous Promoters Revealed by ChIP Assays Mol. Cell. Biol. 2005, 25:1113
    • Nat. Rev. Immunol. 2005 5:593 Specificity of PIAS1-mediated Transcriptional Repression in STAT1 and NF-  B Signaling
    • Proc. Natl. Acad. Sci. USA 2004 101:99 PIAS1/PIAS3 Activates Smad Transcriptional Activity
      • Suggested Readings
      • JAK-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins Science 1994 264:1415
      • 2. STAT: transcriptional control and biologic impact.
      • Nat. Rev. Mol. Cell Biol. 2002 3:651
      • 3. Regulation of gene-activation pathways by PIAS proteins in the immune system
      • Nat. Rev. Immunol. 2005 5:593
      • Textbook:
      • Immunobiology Janeway 6 th ed. 2005, Chapter 6
      • Cellular and Molecular Immunology 5th Ed. 2003, by Abbas and Lichtman, Chapter 11
    • back R-SMAD: Receptor-regulated SMAD ex. SAMD2 and SMAD3 Co-SMAD: Common-mediated SMAD ex. SMAD4
    • Cross-link protein to DNA Break cells and sonicate chromatin Add pre-blocked protein A/G beads Add primary Ab Immunoprecipitate and enrich chromatin Degrade protein and reverse crosslinks Detect specific DNA with PCR Chromatin Immunoprecipitation (ChIP) for Detecting Protein-DNA Interactions in Vivo http://www.activemotif.com/catalog/cell_biology/chipit back