Apoptosis: Death Comes for the Cell

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1 Apoptosis: Death Comes for the Cell Joe W. Ramos From Ingmar Bergman s The Seventh Seal 1

2 2

3 Mutations in proteins that regulate cell proliferation, survival and death can contribute to oncogenesis The Chessboard 3

4 Ways to Die From Okada and Mak, Nat. Rev. Cancer 4:

5 The Morphology of Death 5

6 Apoptosis: Programmed Cell Death A term used to describe the morphological changes associated with programmed cell death. The term was originally used by Wyllie and his colleagues and is from the Greek meaning dropping away as the leaves from a tree. 6

7 Apoptosis l Active cell death l Requires energy and RNA and protein synthesis l Characteristic morphological features l DNA cleaved, chromatin condenses l Cells shrink l Formation of apoptotic body l Cleared by phagocytosis l No inflammation=no tissue damage 7

8 Necrosis l Passive cell death l Cells swell up l Membrane breaks down and cellular contents leak out l Nucleus disintegrates l Cell ghosts l Inflammatory=tissue damage 8

9 The function of Cell death l Multicellular development l involved in deletion of entire structures, l sculpting of tissues, l and regulates the neuron number l The immune response l The body s defense against cancer 9

10 Death and the mouse s paw Dark Green fluorescence indicates apoptotic cells. Fig

11 Apoptosis regulates nerve cell targeting Fig

12 Apoptosis in Lymphocyte development 12

13 How do we recognize Programmed Cell Death? 13

14 The Face of Cell Death: Apoptosis 14

15 Detection of apoptotic cells l l l Microscopy l l Cells have classic features (eg. small darkly stained nuclei) Detection of free 3 ends of DNA by TUNEL assay (terminal deoxytransferase-mediated dutp-biotin nick end labeling) Gel electrophoresis l Detect DNA ladder of 180 bp intervals caused by internucleosomal DNA cleavage Flow cytometry l l Measure externalization of phosphatidylserine (PS) with fluorescently labeled Annexin-V Measure DNA fragmentation with propidium iodide 15 fluorescence

16 Analysis of DNA content with a flow cytometer Recall the fluorescence intensity of the DNA dye (amount of DNA) is measured for each cell. Dead Cells 16

17 Determination of Cell Death by Flow Cytometery Annexin V binds PS on the surface of cells in the early phase of apoptosis 17

18 18

19 Triggers of apoptosis l Programmed cell death in which many more cells are produced than survive (e.g. development of lymphocytes) l Toxic stimuli (viruses, chemicals, ionizing radiation) l Extracellular signals (Fas, p75 NGF-R, TNF) l DNA damage (p53) 19

20 C. elegans has played a key role in our understanding of Apoptosis 1090 total cells 131 die Ced-3=no death Ced-4=no death ced-1 mutant (No engulfment) Ced-9=all die ced-1/ced-3 (No cells die) 20 H.R Horvitz and colleagues responsible for much of this work, 2002 Nobel Prize in Medicine with Sulston and Brenner.

21 C. elegans apoptosis CED-9=Blocks apoptosis CED-4=linker molecule forms activating complex with CED-3 CED-3=Protease that executes cell by chewing up proteins EGL-1=Proapoptotic by blocking CED-9 function 21

22 Three classes of proteins function in the apoptotic pathway-conserved in vertebrates Mammalian Bcl-2 can substitute for Ced-9 in c. elegans 22

23 Death s Methods: A protease cascade These proteases are called caspases Fig

24 Caspases l l l l l Caspases are Cysteine directed proteases that cleave after ASPartate residues Ced-3 is the C. elegans homologue At least 14 family members Synthesized as proenzymes with low levels of caspase activity (~1-2 % of active form) Activated upon after aggregation or cleavage to mature form l l l l Caspases 8 and 9 are initiator caspases Caspases 3 is the effector caspase Caspase activation requires a stimulus They proteolyze cellular proteins to carry out cell death program 24

25 The Caspase Family 25

26 Procaspase activation 26

27 Caspase cascade 27

28 Two Pathways that Initiate Apoptosis l Intrinsic/ Mitochondrial Apoptosis l Regulated by Mitochondria l Cytochrome c release l Extrinsic/ Death Receptor Apoptosis l Activated by ligation of Death Receptors l Fas, TNF alpha l These pathways intersect at the effector caspases 28

29 Activation of the Intrinsic Pathway 29

30 Intrinsic/Mitochondrial Pathway CARD domain 30

31 Mitochondrial permeability PT=Permeability transition, bursts outer membrane 31

32 Intrinsic Pathway: Apaf-1 Induced Apoptosis CARD domains 32

33 Smac/Diablo and IAPs Smac=Second mitochondrial activator of caspases IAP=Inhibitor of Apoptosis Proteins 33

Bcl-2 family members l A very large family with 19 members identified l Bcl-2 (homologous to ced-9) is prototype l All have the BH3 domain (Bcl-2 Homology) l BH-3 is the pro-apoptotic domain exposed

34 Bcl-2 family members l A very large family with 19 members identified l Bcl-2 (homologous to ced-9) is prototype l All have the BH3 domain (Bcl-2 Homology) l BH-3 is the pro-apoptotic domain exposed on activation l Act as dimers=either hetero or homodimers l Pro-apoptotic dimers (Bax) increase mitochondrial permeability l Anti-apoptotic members (Bcl-2, Bcl-XL) form dimers with pro-apoptotic members to inactivate them 34

35 The Bcl-2 Family BH domains=protein-protein interaction domains 35

36 Intrinsic Apoptosis 36

37 Some trophic factors prevent apoptosis by inducing inactivation of a pro-apoptotic regulator 37 Figure 23-50

38 Two mechanisms for p53 activation of apoptosis 38

39 Extrinsic/Death Receptor Pathway 39

40 Death Receptors and Ligands CD95=Fas 40

41 TNF receptor family Cysteine-Rich Domains (CRD) Death Domains (DD) Bind DDs of other proteins (e.g. FADD) Recruiting them to the plasma membrane. 41

42 Fas-FasL Apoptosis l In response to antigenic stimulation, peripheral T cells expand l The antigen specific T cells generated must be eliminated (except for the memory cells) l Upon repeated antigenic stimulation via the T Cell receptor: T cells upregulate Fas and FasL l Eliminate neighboring T Cells expressing Fas 42

43 Activation of Apoptosis by Fas Ligand 43

44 Fas Induced Apoptosis The Formation of the Death Initiating Signal Complex (DISC) 44

45 Adaptor Proteins contain conserved protein interaction domains = inhibits apoptosis -CARD domain of Apaf-1 binds CARD domain of procaspase-9. -DED domain of FADD binds DED domain of procaspase-8. -DED domains of FLIP can bind to the DED domain of FADD and block procaspase-8 recruitment. 45

46 Fas and the intrinsic pathway:bid /Bax 46

47 Proteolytic targets of effector caspases l l l l Cytoskeletal regulatory proteins l Actin Nuclear Lamins Poly(ADP-ribose) polymerase (PARP) l PARP activity depletes ATP, thus cleavage of PARP may maintain store of ATP to drive apoptosis DNA-fragmentation factor (DFF) 47

48 Removal of apoptotic cell by phagocytosis 48

49 Removal of cell corpses 49

50 Phagocytosis: Necrosis vs. Apoptosis Production of antiinflammatory cytokines and chemokines No up-regulation of costimulatory molecules Production of proinflammatory cytokines and chemokines Up-regulation of costimulatory molecules 50

51 Phagocytosis tags and receptors 51

52 Two roads to activate apoptosis Extrinsic Intrinsic Cell-extrinsic pathway Pro-apoptotic ligand DR5 Chemotherapy Radiotherapy DR4 DNA damage FLIP Procaspase 8, 10 FADD p53 PUMA, NOXA BID BAX, BAK Mitochondria BCL2, BCLX L, MCL1 Cell-intrinsic pathway Caspase 8, 10 Cytochrome c Caspase 9 APAF1 Caspase 3, 6, 7 IAP Apoptosis SMAC/DIABLO p53 DNA damage 52

53 TNFα Can also activate Necroptosis 53

54 Necroptosis: RIP3 Dependent programed necrosis 54

55 Autophagy Autophagy, literally means self-eating. Two major functions: o to eliminate damaged or superfluous proteins and organelles, commonly referred to as the protein and organelle quality control function of autophagy. o to degrade and recycle intracellular components to sustain sustain metabolism and homeostasis in the absence of external nutrients, commonly referred to as the catabolic function of autophagy. 55

56 The Autophagic Process 56

57 Autophagy under the microscope Autophagic Vesicles LC3-II punctae 57

Signals Controlling Autophagy Genotoxic and oncogenic stresses result in nuclear p53 stabilization and activation, which stimulates autophagy through activation of AMPK AMPK phosphorylates and

58 Signals Controlling Autophagy Genotoxic and oncogenic stresses result in nuclear p53 stabilization and activation, which stimulates autophagy through activation of AMPK AMPK phosphorylates and activates the GAP complex TSC1/TSC2, leading to inactivation of mtorc1 and autophagy induction. Rheb is a GTPase AMPK->mTORC1 PI3K-> AKT activates autophagy blocks autophagy Current Opinion in Cell Biology 2010, 22:

59 Autophagy in Cancer Autophagy Can promote tumor formation or block it depending on context. Clin Cancer Res; 21(3):

60 Questions? 60

Apoptosis: Death comes for the Cell. Apoptosis: Programmed Cell Death. Apoptosis. Necrosis

Apoptosis: Death comes for the Cell. Apoptosis: Programmed Cell Death. Apoptosis. Necrosis poptosis: eath comes for the Cell Mutations in proteins that regulate cell proliferation, survival and death can contribute to oncogenesis Joe W. Ramos jramos@crch.hawaii.edu From Ingmar Bergman s The