Valld’Hebron Research InstituteNov 15 2012Regulation of Apoptosis by Bcl-2 Family Membersand XIAP                         ...
Necrosis              vs. Apoptosis passive, „accident“,       active, energy-dependent, alwayspathological         physio...
PROGRAMMED CELL DEATHApoptosis                              Necroptosis                                 (RIPK1/3) Autophag...
TumourCellsOverexpressAnti-Apoptotic GenesFollicular Lymphoma (spleen): t(14;18) IgH(14q32) BCL2(18q21)                   ...
Death Receptor (Extrinsic Pathway)                                                              Intrinsic Pathway         ...
The Bcl-2 Protein Family Regulates the Integrity of the                      Mitochondrial Outer Membrane                 ...
Only XIAP can directly block caspases-3 and -9                                      Riedl and Shi, Nat Rev MCB 2004       ...
Selective Interactions Between BH3-Only and Bcl-2-Like Proteins  Bim, Bid, Puma                           ABT-737   (Olter...
The Bcl-2 Family – Still Many Open Questions                                                 BH3-only                     ...
BOK: A BAX/BAK-Like Protein?BOK: BCL-2 related ovarian killer (Hsu et al. PNAS 1997) BOK is widely expressed       Ke F. e...
BOK is Deleted in Human Cancers with high FrequencyBeroukhim et al., Nature, 2010                                         ...
BOK IS NOT A FUNCTIONAL BAX/BAK HOMOLOGUE• BOK induces intrinsic apoptosis upstream of BAX/BAK• BOK localises predominantl...
Death Receptor-Induced Killing and Crosstalk to  Intrinsic (mitochondrial) Apoptotic Pathway                              ...
APOPTOSIS IS ONLY ONE OF SEVERAL POSSIBLE OUTCOMES IN DR SIGNALING                                              FADD/C8 (?...
TNF-R1: Not Meant to Kill                                       TNFa                                            TNF-R1    ...
LPS plus GalactosamineInjectionModelof TNF-R1-MediatedFulminant Hepatitis• Bacterial LPS -> TNFa(Macrophages, Neutrophils...
Hepatocytesare Type IICellsTYPE I                                                   TYPE II                               ...
The BH3-Only Protein BIM IsRapidlyPhosphorylated in LPS/GalN-Induced Hepatitis                                            ...
Both BID and BIM areInvolved in LPS/GalN-InducedHepatitisKaufmann et al. Immunity (2009)                                  ...
BIM isActivatedby JNK MediatedPhosphorylationA                                     B                                      ...
Both BID and BIM canMediate a Crosstalk fromDeathReceptors to Mitochondria                                       JNK      ...
Fas/CD95/Apo-1• FasL mainly expressed on activated T cells and natural killer cells.• Critical role in the control of the ...
Type I or Type II Fas-Induced Apoptotic Pathway         FasL                                     FasLTYPE I               ...
ManyCancerCells Display a Mandatory Crosstalk                                      Bcl-2           ?   X                  ...
Aim: Uncoupling of DeathReceptorPathway                                  Bcl-2              drugX                         ...
CombinationTherapy             ABT-737     Bcl-2               ABT-737    drug X                     ABT-737              ...
Importance of Apoptosis in Liver                    PathologyAbnormal apoptosis in hepatocytes is cause or contributing fa...
MurineHepatocytesare Type II-LikeCellsJo2 anti-Fas – ALT (200 min)                                   Jo2aFas              ...
Lack of BID Blocks Fas-InducedApoptosis in Type II but not in Type I Cells                                                ...
XIAP isStabilised in Livers of FasL-Treated WT Mice                                       + FasL (0.25 mg/kg)             ...
Absence of XIAP Re-SensitisesBid-DeficientMice To FasL-Induced Hepatitis                                                  ...
Absence of XIAP ConvertsHepatocytesInto Type I Cells                                          DEVD-AMC Assay              ...
Effects of IAP Antagonistic Drug (BV6) Phenocopies Genetic Loss of XIAP                                                   ...
Kaufmann, Strasser&Jost, CDD 2011                                    34
mast cell            35
Neutrophils: - most frequent leukocyte in human blood             - major role in innate immunity             - end-differ...
Primary Neutrophils Die By Classical Apoptosis When Cultured In Vitro                                   C57BL/6 WT        ...
Neutrophils die by Apoptosis in Response to High Doses of TNFa             100             80                             ...
FasLInduced Cell Death is Delayed In Bid-/-Neutrophils                                        Geeringet al.Blood 2011     ...
FasL Trigger Both Apoptosis and Necroptosis in Neutrophils               100                                              ...
Loss of BID Aggravates Dextran Sodium Sulfate-Induced ColitisDSS colitis:                               -> dependent of ne...
Proinflammatory role for BID?                                42
Ex Vivo Generation of Neutrophils Using Conditional Hoxb8Protocol based on Wang et al. Nature Methods 2006                ...
Roles of XIAP in Neutrophils                          SCF-condHoxb neutrophils                                      SCF-co...
Non-apoptotic roles of XIAP  MDP NOD2         XIAP NFkBcytokines                     Damgaard et al. Mol Cell 2012        ...
IAPs Limit Inflammasome Activation In Macrophages                           Vince J. et al. Immunity (2012)               ...
SUMMARY• Several poorly characterised BCL-2 family members• Besides BID, BIM can mediate a crosstalk from DR to  mitochond...
ACKNOWLEDGMENTSUniversity of Bern (CH)     WEHI, Melbourne (AU)   •   Philipp Jost(Munich DE)• NohemyEcheverry           •...
LUBAC: linear ubiquitin chain assembly complex                                                 49
Andrea L 2009                50
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Regulation of Apoptosis by BCL-2 Family Members (Prof. Thomas Kaufmann)

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Seminar led by Prof. Thomas Kaufmann. Institute of Pharmacology, University of Bern, Switzerland, at VHIR (15 November 2012).

Content: We are interested to investigate the molecular mechanisms by which pro- and anti-apoptotic members of the BCL-2 family regulate the intrinsic (mitochondrial) apoptotic pathway. The pathway is initiated by members of the BH3-only protein subgroup, which act as sensors in response to a variety of intracellular stress stimuli. Some BH3-only proteins, including Bid and Bim, can be activated downstream of death receptors (e.g. Fas/CD95, TNF-R1) and thus mediate a crosstalk from the extrinsic to the mitochondrial apoptotic pathway. We investigate these processes in mouse liver (and more recently also in mouse granulocytes), as hepatocytes strongly rely on this crosstalk for death receptor-induced apoptosis to be effective. We are further interested in the role of 'X-linked inhibitor of apoptosis protein' (XIAP) in these same cell death pathways.



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Regulation of Apoptosis by BCL-2 Family Members (Prof. Thomas Kaufmann)

  1. 1. Valld’Hebron Research InstituteNov 15 2012Regulation of Apoptosis by Bcl-2 Family Membersand XIAP Thomas Kaufmann Institute of Pharmacology, University of Bern , Switzerland thomas.kaufmann@pki.unibe.ch
  2. 2. Necrosis vs. Apoptosis passive, „accident“, active, energy-dependent, alwayspathological physiological + pathophys. cellsstayintact, lysis of cells clearedbyphagocytosiswhole (parts of) tissue/ oftendeath of individual organaffected cells no inflammation,inducesinflammation inducestolerance 2
  3. 3. PROGRAMMED CELL DEATHApoptosis Necroptosis (RIPK1/3) Autophagic Cell Death NECROTIC Pyroptosis CELL DEATHAnoikis “accidents”: (casp-1) - lack of energy - physical damage Cornification Pyronecrosis - chemical damage (“keratinization”) 3
  4. 4. TumourCellsOverexpressAnti-Apoptotic GenesFollicular Lymphoma (spleen): t(14;18) IgH(14q32) BCL2(18q21) BCL-2, follicular BCL-2,control lymphoma germinal centre mantlezone 4
  5. 5. Death Receptor (Extrinsic Pathway) Intrinsic Pathway cytokine deprivation, DNA- damage, hypoxia, viral infections, ER stress, DISC anoikis, … procaspase-8 (-10) BH3-only proteins (Bim, Puma, Bad,...) cFLIP caspase-8 Bid cIAP1/2 (Bcl-2, Mcl-1, tBid Bcl-2-like Bcl-xL, ...) Bax/Bak Effector Caspases cytochrome.cXIAP (-3, -6 + -7) Caspase-9/Apaf-1 Apoptosome XIAP 5
  6. 6. The Bcl-2 Protein Family Regulates the Integrity of the Mitochondrial Outer Membrane BH3-only Bcl-2-like Bcl-2 Family Bax-like MOMP: mitochondrial outer membrane permeabilisation MOMP Smac/Diabl cIAP1,2 Cyt.c + adaptor (APAF-1) o XIAPVaux &Silke Nat Rev MCB 2005 Caspase-3, -7 (, -6) Caspase-9 6
  7. 7. Only XIAP can directly block caspases-3 and -9 Riedl and Shi, Nat Rev MCB 2004 7
  8. 8. Selective Interactions Between BH3-Only and Bcl-2-Like Proteins Bim, Bid, Puma ABT-737 (Oltersdorf et al Nature 2005) Obatoclax (Nguyen et al PNAS 2007) A1 Chen et al Mol Cell 2005 (modified) 8
  9. 9. The Bcl-2 Family – Still Many Open Questions BH3-only Bcl-2-like Bax/Bak Strasser, Nat Rev Imm 2005 9
  10. 10. BOK: A BAX/BAK-Like Protein?BOK: BCL-2 related ovarian killer (Hsu et al. PNAS 1997) BOK is widely expressed Ke F. et al., CDD 2012 10
  11. 11. BOK is Deleted in Human Cancers with high FrequencyBeroukhim et al., Nature, 2010 11
  12. 12. BOK IS NOT A FUNCTIONAL BAX/BAK HOMOLOGUE• BOK induces intrinsic apoptosis upstream of BAX/BAK• BOK localises predominantly to non-mitochondrial sites: – Golgi, ER/nuclear outer membrane – nuclear compartment• Bok-/- cells present with aberrant ER stress response (part. BFA) +4-OHT (h): Echeverry et al., in revision 12
  13. 13. Death Receptor-Induced Killing and Crosstalk to Intrinsic (mitochondrial) Apoptotic Pathway 13
  14. 14. APOPTOSIS IS ONLY ONE OF SEVERAL POSSIBLE OUTCOMES IN DR SIGNALING FADD/C8 (?) Proliferation cIAP1/2 Apoptosis Necroptosis NFkB TNFa cell survival 14
  15. 15. TNF-R1: Not Meant to Kill TNFa TNF-R1 RIP1Apoptosis cIAP1/2 Necroptosis NFkB anti-apoptotic genes cytokines => cell survival 15
  16. 16. LPS plus GalactosamineInjectionModelof TNF-R1-MediatedFulminant Hepatitis• Bacterial LPS -> TNFa(Macrophages, Neutrophils, NK T)• Response via soluble, circulating TNFa TNF-R1(Pfeffer et al 1993, Rothe et al 1993, Grivennikov et al 2005)• Sensitisation by D-(+)-galactosamine (GalN) Maeda S et al. Immunity 2003 Kaufmann et al. (2009) 16
  17. 17. Hepatocytesare Type IICellsTYPE I TYPE II Bid Caspase-8 tBidBid Caspase-8 Bcl-2-like ? X Bax/Bak Effector Caspases Effector Cyt.c Caspases Apaf1/ Caspase-9 17
  18. 18. The BH3-Only Protein BIM IsRapidlyPhosphorylated in LPS/GalN-Induced Hepatitis * Kaufmann et al. Immunity (2009) 18
  19. 19. Both BID and BIM areInvolved in LPS/GalN-InducedHepatitisKaufmann et al. Immunity (2009) 19
  20. 20. BIM isActivatedby JNK MediatedPhosphorylationA B +/- D-JNKI1 (30 mg/kg, i.p.) * Kaufmann et al. Immunity (2009) 20
  21. 21. Both BID and BIM canMediate a Crosstalk fromDeathReceptors to Mitochondria JNK Corazza et al. JCI (2006) Bim JNK mediated BIM-activation downstream of TRAIL 21
  22. 22. Fas/CD95/Apo-1• FasL mainly expressed on activated T cells and natural killer cells.• Critical role in the control of the immune system• Fas or FasL-mutant mice develop lymphadenopathy and SLE (systemic lupus erythematosus)-like disease and are predisposed to lymphoma development• Many ALPS patients have heterozygous inherited mutations in the Fasgene. (Fisher et al. Cell 1995, Rieux-Laucat et al. Science 1995)• Only the membrane bound form of FasL is inducing cell death (O’Reilly et al. Nature 2009) 22
  23. 23. Type I or Type II Fas-Induced Apoptotic Pathway FasL FasLTYPE I TYPE II Fas Fas ? Bid Caspase-8 tBidBid Caspase-8 Bcl-2-like ? X Bax/Bak Effector Caspases Effector Cyt.c Caspases Apaf1/ Caspase-9 23
  24. 24. ManyCancerCells Display a Mandatory Crosstalk Bcl-2 ? X Mitochondrium X MOMP X X 24
  25. 25. Aim: Uncoupling of DeathReceptorPathway Bcl-2 drugX X MOMP 25
  26. 26. CombinationTherapy ABT-737 Bcl-2 ABT-737 drug X ABT-737 MOMP 26
  27. 27. Importance of Apoptosis in Liver PathologyAbnormal apoptosis in hepatocytes is cause or contributing factor in:- Viral hepatitis -> -> Hepatocellular carcinoma- Alcoholic liver disease (HCC)- Autoimmune hepatitis- Graft-vs-host disease (GvHD)- Endotoxin-induced liver failure- Ischemia/reperfusion-induced liver damage death ligands (activated leukocytes) death receptors (hepatocytes) 27
  28. 28. MurineHepatocytesare Type II-LikeCellsJo2 anti-Fas – ALT (200 min) Jo2aFas wtwt bid-/- lpr wt bid-/- lpr PBS Jo2 FasL (crosslinked) bid-/- +FasL (0.25 mg/kg) scale bar: 50 mm Kaufmann et al. Cell 2007 Jost et al. Nature 2009 (modified) 28
  29. 29. Lack of BID Blocks Fas-InducedApoptosis in Type II but not in Type I Cells Jost PJ et al., 2009 (modified) 29
  30. 30. XIAP isStabilised in Livers of FasL-Treated WT Mice + FasL (0.25 mg/kg) p<0.05 Jost PJ et al. (2009), modified 30
  31. 31. Absence of XIAP Re-SensitisesBid-DeficientMice To FasL-Induced Hepatitis Jost PJ et al., 2009 31
  32. 32. Absence of XIAP ConvertsHepatocytesInto Type I Cells DEVD-AMC Assay Jost PJ et al., 2009 32
  33. 33. Effects of IAP Antagonistic Drug (BV6) Phenocopies Genetic Loss of XIAP 33
  34. 34. Kaufmann, Strasser&Jost, CDD 2011 34
  35. 35. mast cell 35
  36. 36. Neutrophils: - most frequent leukocyte in human blood - major role in innate immunity - end-differentiated, short-lived - apoptotic clearance of activated neutrophils essential 36
  37. 37. Primary Neutrophils Die By Classical Apoptosis When Cultured In Vitro C57BL/6 WT 100 80 Survival (%) 60 untreated GM-CSF 1ng/ml G-CSF 10ng/ml 40 Q-VD oph 20uM 20 0 0 24 48 72 96 Time (h) 37
  38. 38. Neutrophils die by Apoptosis in Response to High Doses of TNFa 100 80 wt untreated bid-/- untreatedSurvival % 60 wt TNFa 40 bid-/- TNFa 20 0 0 24 48 72 96 Time (h) 100 80 60 wt TNFa TNFα 50 ng/ml bid-/- TNFa Q-VD oph 20 μM 40 wt TNFa + Q-VD-oph 20 bid-/- TNFa + Q-VD-oph 0 0 24 48 72 96 38
  39. 39. FasLInduced Cell Death is Delayed In Bid-/-Neutrophils Geeringet al.Blood 2011 39
  40. 40. FasL Trigger Both Apoptosis and Necroptosis in Neutrophils 100 100 wt bid-/- 80 80Survival % Survival % FasL FasL 60 60 FasL + Q-VD-oph FasL + Q-VD-oph 40 40 20 20 0 0 0 24 48 72 96 h 0 24 48 72 96 h 100 wt 100 bid-/- 80 FasL + Q-VD-oph 80 FasL + Q-VD-oph Survival % 60 FasL + Q-VD-oph + Nec Survival % 60 FasL + Q-VD-oph + Nec 40 40 20 20 0 0 0 24 48 72 96 h n>3 0 24 48 72 96 h FasL 100 ng/ml, Q-VD oph 20 μM, Necrostatin-1 20uM 40
  41. 41. Loss of BID Aggravates Dextran Sodium Sulfate-Induced ColitisDSS colitis: -> dependent of neutrophils + macrophages -> independent of adaptive immune system DSS in drinking water water d0 d1 d2 d3 d4 d5 d6 d7 d8 105 animal weight (%) 100 * ** *** *** 95 *** wt n.s. bid-/- 90 n.s. * 85 0 1 2 3 4 5 6 7 8 days => BID is anti-inflammatory 41
  42. 42. Proinflammatory role for BID? 42
  43. 43. Ex Vivo Generation of Neutrophils Using Conditional Hoxb8Protocol based on Wang et al. Nature Methods 2006 d5 d0 43
  44. 44. Roles of XIAP in Neutrophils SCF-condHoxb neutrophils SCF-condHoxb8 neutrophils 100 80 wt TNF-a TNFa (pg/ml) XIAP-/- TNF-aViability [%] 60 40 20 0 0 24 48 72 hours 44
  45. 45. Non-apoptotic roles of XIAP MDP NOD2 XIAP NFkBcytokines Damgaard et al. Mol Cell 2012 45
  46. 46. IAPs Limit Inflammasome Activation In Macrophages Vince J. et al. Immunity (2012) 46
  47. 47. SUMMARY• Several poorly characterised BCL-2 family members• Besides BID, BIM can mediate a crosstalk from DR to mitochondria• XIAP is a crucial discriminator between type I and type II Fas-induced apoptosis• FasL triggers mix of apoptosis and necroptosis in neutrophils• Non-apoptotic roles of IAPs and DRs become increasingly evident (important) 47
  48. 48. ACKNOWLEDGMENTSUniversity of Bern (CH) WEHI, Melbourne (AU) • Philipp Jost(Munich DE)• NohemyEcheverry •Andreas Strasser • MadsGyrd-Hansen (Kopenhagen, DK)• UrsinaGurzeler •Francine Ke • ChristophBorner (Freiburg i.Brsg., DE)• Tatiana Rabachini •Paul Ekert• Daniel Bachmann • Georg Häcker(Freiburg i. Brsg., DE) •John Silke• Simone Wicki • Thomas Brunner (Konstanz, DE) •David Huang• Nicole Tochtermann • Frank Essmann(Tübingen, DE) •UeliNachbur• LaetitiaRoh • Julia Fernandez-Rodriguez (Gothenburg, SE)• Hans-Uwe Simon• Mario Tschan • Kurt Ballmer (Villigen, CH)• ShidaYousefi• Clemens Dahinden 48
  49. 49. LUBAC: linear ubiquitin chain assembly complex 49
  50. 50. Andrea L 2009 50

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