Apoptosis
細胞生物學導論 Apoptosis To be or not to be, that is the question 2015-05-06 Outline Types of cell death Apoptosis: morphology and biochemical changes Caspase cascade Bcl-2 family Signal to activate apoptosis: Fas and extrinsic pathway Mitochondria and intrinsic pathway Apoptosis and disease Programmed Cell Death a) b) c) d) a healthy cell a necrotic cell an apoptotic cell an autophagic cell Nikoletopoulou et al. Biochimica et Biophysica Acta (BBA) 2013;1833:3448 Autophagy: dynamic recycling system Degradation of cytoplasmic components through the lysosomal machinery Formation of autophagosome Without chromatin condensation Cell Death and Differentiation (2005) Degraded product Glucose, protein, energy Crosstalk DieDderich et al. Biotech Adv .2014 Other types of cell death Anoikis - : apoptois triggered by loss of anchorage to extracellular matrix (leaving home) Exitotoxicity Wallerian degeneration Blue: nuclei Red: integrin Green: apoptotic cells Biological significance of apoptosis Normal development and homeostasis In response to stress (eg. DNA damage) Dysregulation : development and survival of tumors Terminology • Programmed cell death • Developmental cell death • Caspase-independent apoptosis • Cell death induced by ….. Apoptosis: morphology Chromatin aggregation at nuclear membrane Membrane blebbing，but no loss of integrity Shrinkage of cytoplasm and condensation of nucleus Fragmentation of cells into small bodies Vesicles (apoptotic bodies) Mitochondria become leaky Blebbing Apoptosis 2005; 10: 201 Apoptotic bodies The Biology of Cancer (© Garland Science 2007) Biochemical changes of apoptosis Cleavage of nuclear DNA 1. DNA laddering (Internucleosomal DNA fragmentation) TUNEL (TdT-mediated dUTP nick end labeling) 2. Phosphatidylserine and Annexin V Cytochrome c relocation Electrical potential changes in Mitochondria Internucleosomal DNA fragmentation (DNA laddering) endonuclease Mcllroy et al. Genes Dev. 2000; 14: 549 Terminal deoxynucleotidyl transferase –mediated dUTP-biotin nick end-labeling (TUNEL) 3’ 5’ TdT dUTP Biotin V: villi; L: lumen Gavrieli Y, Sherman Y, Ben‐Sasson SA. JCB (1992) Label Externalization of phosphatidylserine on outer leaflet of plasma membrane Annexin V: compete for phosphatidylserine binding sites with prothrombin Red: Annexin V Green: cytochrome c Nature Cell Biology, cover page (2000;3) Cyotchrome C relocation Ella Bossy-Wetzel et al. EMBO 1998 Caspases ( cysteine aspartate protease) A family of cysteine proteases cleave aspartate residues Proteolysis Specific, irreversible process (cell cycle, cell death) Most proteases are synthesized as precursors Proteases can regulate their own activation Where there are proteases, there are inhibitors Adapted from Thornberry NA, Lazebnik Y. Science 1998;281:1312 Apoptosis - carried out by Caspases Pro-apoptotic stimulus Procaspase Initiator caspases Effector caspases Apoptosis Caspase, cysteine aspartase. Caspase cascade Active form: heterotetramer Thornberry NA. Science 1998:281:1312 Caspases in action Effector caspsases , substrate and morphology Caspase-3 cleaved gelsolin : severe actin filaments Gelsolin: actin-binding protein-actin assembly / disassembly •Neutrophils lacking gelsolin had delayed onset of blebbing Wild type Gelsolin knock-out Kothakota S et al. Science 1997;278:294 Effector caspsases , substrate and morphology Cleave the inhibitor Caspase 3 ICAD CAD: inter-nucleosome cleavage CAD: caspase-activated DNase Nagata S. Exp Cell Res 2000;256:12 Signals to activate caspases Adapted from Ashkenazi A. Nat Rev Cancer 2002;2:420 Pro-apoptotic ligand Extrinsic pathway Pro-apoptotic receptor Caspases BAX Mitochondria BCL2 PUMA Apoptosis Stress BCL2, B-cell chronic lymphocytic leukemia/lymphoma 2; PUMA, p53-upregulated modulator of apoptosis. Intrinsic pathway Extrinsic pathway of apoptosis Adapted from Ashkenazi A. Nat Rev Cancer 2002;2:420 Pro-apoptotic ligand DR DR: death receptor •Intracellular death domain FADD Pro- caspases FADD Caspase 8,10 Caspase 3, 6, 7 Apoptosis Fas-associated death domain The Extrinsic Apoptosis Pathway Activation of death receptors by pro-apoptotic ligands (eg,FAS ligand) Ligand-binding: assembly of the death-inducing signaling complex (DISC) Recruitment of initiator caspases (8 and 10): through the adaptor Fas-associated death domain (FADD) Caspases 8 and 10 activate effector caspases 3, 6, and 7, leading to apoptosis TRAIL (TNF-Related Apoptosis Inducing Ligand) Activate apoptosis in cancer cells Anti-tumor immune surveillance by NK cells TRAIL -/- mice: tumor metastasis and formation Function: Kill transformed cells? Cancer cells sensitive to TRAIL-mediated apoptosis DR4 Apo2L : apoptosis-inducing ligand 2 TRAIL: TNF - Related Apoptosis-Inducing Ligand DR5 Ashkenazi A. Nat Rev Cancer 2002;2:420 Activated caspase 3, 6, and 7 execute apoptosis Pro-apoptotic ligand DR5 Cell-extrinsic pathway DR4 DNA damage FADD FLIP FLIP, (FADD-like interleukin 1β-converting enzyme) inhibitory protein Procaspase 8, 10 Caspase 8, 10 Caspase 3, 6, 7 Apoptosis Activation of Fas by Fas-ligand on killer lymphocyte DISC: death inducing signal complex Figure 18-6 Molecular Biology of the Cell (© Garland Science 2008) Death receptors Combination therapy of established cancer using a histone deacetylase inhibitor and a TRAIL receptor agonist MD5-1: TRAIL receptor agonist PNAS 2008; 105:11317 To avoid inappropriate activation of death receptor Decoy receptors (without death domain) Intracellular blocking protein: competes for binding on DISC and lacks proteolytic domain (eg,FLIP) FLIP: FADD-like interleukin 1βconverting enzyme inhibitory protein Extrinsic pathway Gewirtz et al. Apoptosis, senescence, and cancer Pathways leading to necroptosis Nature 2015;517:311 Adapted from Ashkenazi A. Nat Rev Cancer 2002;2:420–430 Intrinsic pathway of apoptosis Chemotherapy Radiotherapy Hypoxia DNA damage p53 Apaf 1: apoptotic protease activating factor-1 Mitochondria Cytochrome c Caspase 9 Caspase 3, 6, 7 Apoptosis Apaf 1 Apoptosome Cytochrome C, Apaf1 and apoptosome CARD: caspase recruitment domain Figure 18-8 Molecular Biology of the Cell (© Garland Science 2008) Bcl-2 proteins regulate the intrinsic pathway By controlling the release of cytochrome c into cytosol Science 1985;228:1440 bcl-2 (B-cell leukemia/lymphoma 2) gene Bcl-2 families BH:Bcl2-homology TM: transmembrane domain Shibue T.et al. Int J Cancer 2006;119:2036 Binding selectivies of BH3-only proteins Proapoptotic BH123 BH3-only Anti-apoptotic Shibue T.et al. Int J Cancer 2006;119:2036 Translocation of BAX from cytosol to mitochondria BAK Translocation of BAX : Anti-apoptotic Bcl-2 protein, eg Bcl-xL Figure 18-10 Molecular Biology of the Cell (© Garland Science 2008) The induction of apoptosis via Bcl-2 family proteins starts from the activation of BH3-only proteins Shibue Int. J. Cancer 2006: 119, 2036 Regulation by Bcl-2 families BH123 BH1234BH3-only BH3-only •Neutralize Bcl-2 •Bind to BaK Gewirtz et al. Apoptosis, senescence, and cancer BH3-only protein activation •Pro-apoptotic •Link between stimuli and intrinsic pathway •Link between extrinsic and intrinsic pathway Shibue T.et al. Int J Cancer 2006;119:2036 Interaction between PUMA and Bcl-XL or Bax in the mitochondria of hippocampus after cerebral ischemia Niizuma, K. et al. Stroke 2009;40:618 Inhibitors of apoptosis (IAPs) Regulatory Mechanisms Converging on Caspases Found in insect viruses (Baculoviruses) Prevent the hosts from killing themselves by apoptosis Bind to and inhibit activated caspases Human IAP proteins NIAP:neuronal IAP CIAP: cellular IAP XIAP: X chromosome-linked IAP Anti-IAPs - bind to and neutralize of IAPs Drosophila : Reaper, Grim, Hid Mammals: Smac (DIABLO), Omi Produced in response to various apoptotic stimuli Regulation by IAP proteins (mammals) SMAC: second mitochondria-derived activator of caspase Gewirtz et al. Apoptosis, senescence, and cancer Extracellular survival factors inhibit apoptosis Animal cells require signaling from other cells to avoid apoptosis Find-me and eat-me : signals in apoptotic cell clearance Ravichandran J. Exp. Med. 2010:207:1807 Disease associated with apoptosis Decreased apoptosis Increased apoptosis Cancer Ischemic injury Auto immune disorder Neurodegenerative disorder Virus infection AIDS … Myelodysplastic syndrome … Adapted from Ashkenazi A. Nat Rev Cancer 2002;2:420–430 Two Major Apoptosis Signaling Pathways Pro-apoptotic ligand DR5 Extrinsic pathway Chemotherapy Radiotherapy DR4 DNA damage p53 FADD FLIP PUMA, NOXA BID BAX, BAK Procaspase 8, 10 Intrinsic pathway BCL2, BCLXL, MCL1 Mitochondria Caspase 8, 10 Caspase 9 Caspase 3, 6, 7 Cytochrome c SMAC/DIABLO APAF1 IAP p53 Apoptosis DNA damage APAF1, apoptotic protease activating factor-1; BAK, BCL2 homologous antagonist/killer; BAX, BCL2-associated protein; BCLXL, BCL2-like 1; BID, BH3-interacting domain death agonist; DR, death receptor; FADD, Fas-associated death domain; FLIP, FLICE (FADD-like interleukin 1β-converting enzyme) inhibitory protein; IAP, inhibitor of apoptosis protein; MCL1, myeloid cell leukemia sequence 1 (BCL2-related); PUMA, p53-upregulated modulator of apoptosis; SMAC/DIABLO: second mitochondria-derived activator of caspase/direct IAP binding protein with low pI.
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