Trends Biochem. Sci.


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Trends Biochem. Sci.

  1. 1. Immunological Synapse Formation between T and APC Science 2002 295:1539
  2. 2. Immunological Synapse
  3. 3. Signal Transduction Determines Cell Response ?? ? TCR IL-2 APC
  4. 4. <ul><li>Role of cytokines in immune system and hematopoiesis </li></ul><ul><li>Basic concepts of signal transduction </li></ul><ul><li>JAK-STAT pathway in cytokine signaling </li></ul><ul><li>Regulation of cytokine response </li></ul><ul><li>Negative regulators for cytokine signal transduction, SOCS and PIAS families </li></ul>OUTLINE
  5. 5. General Properties of Cytokines <ul><li>Polypeptides </li></ul><ul><li>Produced in response to microbes and antigens </li></ul><ul><li>Mediate and regulate immune and inflammatory responses </li></ul><ul><li>Pleiotropic and redundant </li></ul><ul><li>Regulate the synthesis and actions of other cytokines </li></ul><ul><li>Actions are local or systemic (autocrine, paracrine, or endocrine) </li></ul>
  6. 6. 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
  7. 7. 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
  8. 8. 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 +
  9. 9. 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
  10. 10. Roles of Cytokines in Hematopoiesis Cell. Mol. Immunol. 2003 Abbas & Lichtman 5 th ed.
  11. 11. What Happens When Ligands Bind to Receptors <ul><li>Conformational change of the receptors: </li></ul><ul><li>Opens ion channel </li></ul><ul><li>Facilitates binding of intracellular signaling proteins such as chemokine receptor </li></ul><ul><li>Dimerization (Oligomerization) of the receptors: </li></ul><ul><li>Bring signaling molecules into juxtaposition </li></ul><ul><li>-induces post-translational modification such as phosphorylation in the receptors or signal meidators </li></ul><ul><li>-activates downstream mediators </li></ul>
  12. 12. Phosphorylation of Proteins as a Controling Mechanism for Signal Transduction <ul><li>Advantages </li></ul><ul><li>Rapid: does not require new protein synthesis or protein degradation </li></ul><ul><li>Reversible : easily reversed by action of protein phosphatases </li></ul><ul><li>Easy to relay signals: phosphorylation on Tyr, Thr, or Ser creating binding sites for other proteins </li></ul>
  13. 13. 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
  14. 14. 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.
  15. 15. Major Events in Signal Transduction Mediated by Receptor Tyrosine Kinase (RTK) or non-RTK <ul><li>Ligand-induced receptor dimerization </li></ul><ul><li>Activation of kinases </li></ul><ul><li>Activation of signal mediators </li></ul><ul><li>Activation of transcription factors </li></ul><ul><li>Translocation of transcription factors into nucleus and transactivation </li></ul>
  16. 16. Cytokine Receptor-mediated Signaling Pathways
  17. 17. 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
  18. 18. 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
  19. 19. 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
  20. 20. Mol. Cell. Biol. 5 th ed. 2004 Lodish et al. Genetic Approach for Elucidating IFN Signaling Pathway ICR191 (frame shift mutagen)
  21. 21. 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
  22. 22. Animation for JAK-STAT signaling
  23. 23. Structural and Functional Domains of JAK Family JAK family : JAK1, JAK2, JAK3 and TYK2 Nat. Rev. Mol. Cell Biol. 2002 3:651
  24. 24. Nat. Rev. Mol. Cell Biol. 2002 3:651 Structural and Functional Domains of STATs STAT family: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6
  25. 25. 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
  26. 26. Cytokine Receptor Families by Their Structures (wsxws motif for  -helical cytokine) (serpentine)
  27. 27. Molecular Structure of Class I Receptors Cytokine Growth Factor Rev 2001 12:19 One chain for Ligand Binding One chain for Signal transducing
  28. 28. Nat. Rev. Immunol.2001 1:200 Critical Roles of  C Family in Lymphocyte Development and Function
  29. 29. N. Eng. J. Med. 2000 343:1313 SCID Resulting from Defects in IL-7R  , JAK3 or  c Chain
  30. 30. Nat. Rev. Mol. Cell Biol. 2002 3:651 Phenotypes of JAK-Knockout Mice
  31. 31. Prevention of Organ Allograft Rejection by a Specific Janus Kinase 3 Inhibitor Science 2003 302:875 NHP: non-human primates Low dose high dose
  32. 32. Nat. Rev. Mol. Cell Biol. 2002 3:651 Phenotypes of STAT Knockout Mice
  33. 33. Nature Genet. 2003 33:388 (U. Paris) Impaired Response to IFN  /  and Lethal Viral Disease in Human STAT1 Deficiency
  34. 34. <ul><li>Receptor-mediated endocytosis and degradation </li></ul><ul><li>Dephosphorylation by tyrosine phosphatases </li></ul><ul><li>Naturally occurring dominant negative STATs such as STAT1  and STAT3  that don’t have transactivating domain </li></ul><ul><li>S uppressor o f c ytokine s ignaling (SOCS) family </li></ul><ul><li>P rotein i nhibitor of a ctivated S tats (PIAS) </li></ul>Negative Regulation of the JAK-STAT pathway
  35. 35. Cell 2002 109:S121-S131
  36. 36. Negative Regulations of Cytokine Signaling Nat. Rev. Immunity 2003 3:900
  37. 37. Structural and Functional Domains of SOCS Family Nat. Rev. Immunol. 2002 2:410 K: Kinase inhibitory region
  38. 38. Inhibitory Mechanisms of SOCS Molecules Trend. Immunol. 2003 24:659
  39. 39. SOCS Family Members Target Signaling Proteins for Degradation by Proteasome Trends Biochem. Sci. 2002 27:235
  40. 40.                                                                                                                    SOCS Targeting Key Signaling Proteins for Degradation by the Proteasome Sci. STKE, Vol. 2003 169:pe6
  41. 41. Liver Degeneration and Lymphoid Deficiencies in Mice Lacking SOCS1 Proc. Natl. Acad. Sci. 1998 95: 14395 SOCS1-/- SOCS1+/+
  42. 42. SOCS1 Is a Critical Inhibitor of IFN   Signaling and Prevents Fatal Neonatal Actions of this Cytokine Cell 1999 98:597 JBC 2006 281:11135
  43. 43. Nat. Rev. Mol. Cell Biol. 2002 3:651 Phenotypes of SOCS Knockout Mice
  44. 44. Hypersensitivity of SOCS-1 KO Mice to LPS In Vivo Immunity 2002 Vol. 17:583
  45. 45. SOCS1 Negatively Regulates TLR Signaling by Mal/ TIRAP Degradation Nat. Immunol. 2006 7:148 Forward
  46. 46. TLR Signaling Pathways Cell Death Diff. 2006 13:816 back
  47. 47. SOCS1 Is a Suppressor of Liver Fibrosis and Hepatitis-induced Carcinogenesis JEM 2004 199:1701
  48. 48. 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
  49. 49. Nat. Rev. Immunol. 2005 5:593 PIAS-protein Regions Involved in Protein–protein Interactions
  50. 50. Nat. Rev. Immunity 2003 3:900 Proposed Mechanisms for Inhibiting the JAK–STAT Pathway by PIAS Proteins
  51. 51. Negative Regulation of PIAS on Gene Transactivation Nat. Rev. Immunol. 2005 5:593
  52. 52. Positive Regulation of PIAS on Gene Transactivation Nat. Rev. Immunol. 2005 5:593
  53. 53. Some but not All IFN  -inducible Genes Are Enhanced in Pias1 -/- MEF Nat. Immunol. 2004 5:891
  54. 54. Enhanced Antiviral Response and Sensitivity to LPS-induced Toxic Shock in Pias1 -/- Mice Nat. Immunol. 2004 5:891
  55. 55. Enhanced Expression of NF-  B-Regulated Genes in Response to TNF-  and LPS in Pias1 null Cells Mol. Cell. Biol. 2005, 25:1113
  56. 56. PIAS1 Affects the Binding of p65 to the Endogenous Promoters Revealed by ChIP Assays Mol. Cell. Biol. 2005, 25:1113
  57. 57. Nat. Rev. Immunol. 2005 5:593 Specificity of PIAS1-mediated Transcriptional Repression in STAT1 and NF-  B Signaling
  58. 58. Proc. Natl. Acad. Sci. USA 2004 101:99 PIAS1/PIAS3 Activates Smad Transcriptional Activity
  59. 59. <ul><li> Suggested Readings </li></ul><ul><li>JAK-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins Science 1994 264:1415 </li></ul><ul><li>2. STAT: transcriptional control and biologic impact. </li></ul><ul><li>Nat. Rev. Mol. Cell Biol. 2002 3:651 </li></ul><ul><li>3. Regulation of gene-activation pathways by PIAS proteins in the immune system </li></ul><ul><li>Nat. Rev. Immunol. 2005 5:593 </li></ul><ul><li>Textbook: </li></ul><ul><li>Immunobiology Janeway 6 th ed. 2005, Chapter 6 </li></ul><ul><li>Cellular and Molecular Immunology 5th Ed. 2003, by Abbas and Lichtman, Chapter 11 </li></ul>
  60. 60. back R-SMAD: Receptor-regulated SMAD ex. SAMD2 and SMAD3 Co-SMAD: Common-mediated SMAD ex. SMAD4
  61. 61. 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 back
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