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Methods for Apoptotic Assay

Methods for Apoptotic Assay. Apoptosis  A physiological mechanism to eliminate excess, damaged or dangerous cells from an organism without damaging surrounding cells and tissues  Necessary for normal embryogenesis  Maintenance of tissue homeostasis. Apoptotic morphology

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Methods for Apoptotic Assay

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  1. Methods for Apoptotic Assay

  2. Apoptosis  A physiological mechanism to eliminate excess, damaged or dangerous cells from an organism without damaging surrounding cells and tissues  Necessary for normal embryogenesis  Maintenance of tissue homeostasis

  3. Apoptotic morphology •  Membrane blebbing •  Aggregation of chromatin at the nuclear membrane • Ends with fragmentation of cell into small bodies • Begin with shrinking of cytoplasm and condensation of nucleus • Formation of apoptotic bodies • Mitochondria become leaky due to pore formation • involving proteins of the bcl-2 family

  4. Apoptotic Pathway • Death receptors • Membrane alteration • Protease cascade • Mitochondrial changes • DNA fragmentation

  5. Death receptors Death inducing signaling complex INTRINSIC PATHWAY Extrinsic pathway cytochromeC Apoptosome APAF-1 Adaptor proteins procaspase9 caspase8 caspase9 Procaspase 8 XIAP Procaspse3 procaspase7 Caspase3 caspase7 Apoptosis

  6. The extrinsic death receptor pathway TNF FASL Apo2/Trial Death receptors Death domain adaptor protein Death effector domain

  7. The intrinsic apoptotic pathway Apoptotic insult AIF nuclease chromatin Caspase activation nuclease chromatin apoptosome Caspase dependent cell death DNA fragmentation

  8. Protease cascade *Conserved domains for caspase interaction: Death Domain( DD) QACRG Death Effector Domain( DED)– hydrophobic interaction Caspase recruitment Domain( CARD)—electrostatic interaction * Amplify a suicide signal in the cell whose death pathways have only weakly initiated

  9. Caspase family Death Effector domain Caspase recruitment Domain

  10. EGL1 CED-9 CED-4 CED-3 apoptosis Nematodes

  11. Pro CED-3 βNAC CED-9 CED-4 Active CED-3 Icd-1( inhibitor of cell death -1)) Early embryo arrest Accumulation of extra die cells Extra die cells in nematode show normal survival Cell death mitochondria

  12. BID, BIM BCL-2 SMAC/DIABLO APAF-1 caspase9 IAP Caspase3,7 mammals Apoptotic stimuli AIF HTR2/OMI EndoG Cytochrome C/dATP Apoptosis

  13. Reaper Hid Grim Sickle Apaf1 DIap1 Dronc Drice Cellular target Apoptosis Fruitfly

  14. Eat me signal PS ( phosphotidylserine) displayed on the plasma membrane on the dying cells Engulfment receptors in mammals: CD91, CD14, CD36, αvβintegrin, phosphotidyl serine receptor( PSR)

  15. ced-2( CRKII) ced5 ( DOCJ-180) ced10( small GTP-ase Rac-1) Cytoskeletal remodeling Cell Engulfment ced1: engulfment receptor ced-6: homologue to the mammalian PTB domain bearing adaptor GULP

  16. nematodes mammalians Phagocytic cells ELMO RAC1 Cytoskeleton remodeling CED-5 Dock180 Phosphotidylserine CED-2 Membrane blebbing CRKII PSR Phosphotidylserine CD19 CD91 DNA degradation CD14 αvβintegrin, Scavenger receptor αvβintegrin, CED-1 CED-6 CED-7 Engulfment of apoptotic cells Apoptotic cells

  17. Decision to die, Apoptotic induction, caspase activation Execution of death, caspase activation Engulfment signal, LPC…. ps ps Anexin I Eat me signal,CD31/ PCAM-1 degradation Anexin I

  18. 180 b.p.

  19. Increased low molecular weight DNA in apoptotic cells

  20. Terminal dideoxynucleotidyl transferase

  21. annexinV: Ca+2 dependent phospholipid binding protein, high affinity to PS

  22. Substrate cleavage Caspase activation

  23. Apoptin誘發細胞凋亡機制之探討 Mechanisms Involved in Apoptosis induced by Apoptin Associated Proteins

  24. Chicken Anemia Virus diameter 23 - 25 nm single-stranded circular DNA (2319 nt) polycistronic mRNA VP1 51.6 kDa VP2 24 kDa VP3 13.6 kDa (Apoptin)

  25. CAV infection cause depletion of erythroblastoid and lymphoid cell in young chicken (Jerissen & Noteborn et al, J Viorogy,1992) Apoptosis is responsible for the thymocyte depletion in young chicken (Jerissen et al, J Virology,1992) VP3 protein of CAV is sufficient to induce apoptosis in chicken mononuclear cells ( Zhung & Noteborn et al, Leukaemia1995)

  26. Apoptin p53 Bcl-2 BAG-1 CrmA ICE Apoptosis Noteborn et al, Cancer Res, Carcinogenesis,1995

  27. Apoptin induces apoptosis in human transformed and malignant cells but not in normal cells Danen-Van Oorschot et al, PNAS 1997

  28. Apoptin activity and localization in human normal vs, transformed/tumorgenic cells Human cells Apoptin Apoptotic activity localization Normal primaryNone cytoplasmic Tumor-derived/transformedyesnuclear Noteborn etal, 1998, Mutation Research

  29. The effect of transforming genes and UV-irradiation on Apoptin induced apoptosis Donors of fibroblast Apoptin induced Apoptosis Apoptin expression plus none SV40 largeTUV Healthy no yes no Cancer prone no yes yes Noteborn etal, 1998, Mutation Research

  30. Apoptin –expressing adenoviral vector specifically kills tumor cells of xenogenic HepG2 tumor cells in Balb/Cnu/nu Pietersen et al 1999 Gene Therapy Apoptin induced apoptosis requires activation of caspases in human tumor cells Oorschot et al 2000 J Virology A tumor specific kinase activity regulate the viral death protein Apoptin Rohn et al 2002 JBC Apoptin induces tumor specific apoptosis as a globular multimer Leviveld et al 2002 JBC

  31. Purpose To study the molecular mechanisms involved in Apoptin induced apoptosis

  32. Identification of Apoptin-Associated Proteins by Yeast Two-Hybrid System

  33. b-gal filter assay Growth on SD/-Trp/-Leu/-His APAP1 APAP2

  34. b-gal filter assay Growth on SD/-Trp/-Leu/-His APAP3 APAP4

  35. APAP1 Identical to DEDAF (Death Effector Domain- Associated Factor) and human RYBP (Ring1 and YY1 Binding Protein) APAP2 An unknown gene with 6000b.p. with ten ankyrin repeats APAP3 Identical to a Interferon induced IFP35 APAP4 Identical to Hippi/Hip-1 protein interactor

  36. APAP1 Identical to DEDAF (Death Effector Domain- Associated Factor) and human RYBP (Ring1 and YY1 Binding Protein) APAP2 An unknown gene with 6000b.p. with ten ankyrin repeats APAP3 Identical to a Interferon induced IFP35 APAP4 Identical to Hippi/Hip-1 protein interactor

  37. PART1 Cloning and characterization of Apoptin associated protein 1 ( APAP1)

  38. APAP1 is identical to DEDAF (Death Effector Domain-Associated Factor) and RYBP (Ring1 and YY1 Binding Protein)

  39. Peptide structure of APAP1 APAP1 226 a.a. NLS Zinc finger KKEKK KKTKPK NLS CSVCTFRNSAEAFKCSIC

  40. Purpose Demonstration of in vivo and in vitro binding of Apoptin and APAP1

  41. GST pull down of Apoptin and APAP1 pGEX/ Apoptin pET32a(+)/ APAP1 E.coli transformation( BL-21) IPTG induction 4oC sonication GST pull down SDS PAGE Western Blot( anti-His detection)

  42. Apoptin associate with APAP1 in vitro

  43. Co-immunoprecipitation of Apoptin and APAP1 GFP pRK5F/ APAP1 pEGFPC2/ Apoptin Flag DNA transfection HeLa cells 24hrs, 37oC cell harvest, IP/ -GFP SDS-PAGE Blot -Flag and -GFP

  44. Apoptin associate specifically with APAP1 in vivo

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