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

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poptosis: eath comes for the Cell Mutations in proteins that regulate cell proliferation, survival and death can

edu From Ingmar Bergman s The Seventh Seal poptosis: Programmed Cell eath term used to describe the

Cancer 4:592-603 The term was originally used by Wyllie and his colleagues and is from the Greek meaning

poptosis ctive cell death Requires energy and RN and protein synthesis Characteristic morphological features N

1 poptosis: eath comes for the Cell Mutations in proteins that regulate cell proliferation, survival and death can contribute to oncogenesis Joe W. Ramos From Ingmar Bergman s The Seventh Seal poptosis: Programmed Cell eath term used to describe the morphological changes associated with programmed cell death. From Okada and Mak, Nat. Rev. Cancer 4: The term was originally used by Wyllie and his colleagues and is from the Greek meaning dropping away as the leaves from a tree. poptosis ctive cell death Requires energy and RN and protein synthesis Characteristic morphological features N cleaved, chromatin condenses Cells shrink Formation of apoptotic body Cleared by phagocytosis No inflammation=no tissue damage Necrosis Passive cell death Cells swell up Membrane breaks down and cellular contents leak out Nucleus disintegrates Cell ghosts Inflammatory=tissue damage 1

2 The function of Cell death eath and the mouse s paw Multicellular development involved in deletion of entire structures, sculpting of tissues, and regulates the neuron number The immune response The body s defense against cancer ark Green fluorescence indicates apoptotic cells. Fig poptosis regulates nerve cell targeting poptosis in Lymphocyte development Fig How do we recognize Programmed Cell eath? The Face of Cell eath: poptosis 2

etection of apoptotic cells Microscopy Cells have classic features (eg.

labeling) Gel electrophoresis etect N ladder of 180 bp intervals caused by internucleosomal N cleavage nalysis of N content with a

Flow cytometry Measure externalization of phosphatidylserine (PS) with fluorescently labeled nnexin-v Measure N fragmentation with

development of lymphocytes) Toxic stimuli (viruses, chemicals, ionizing radiation) Extracellular signals (Fas, p75 NGF-R, TNF) N

3 etection of apoptotic cells Microscopy Cells have classic features (eg. small darkly stained nuclei) etection of free 3 ends of N by TUNEL assay (terminal deoxytransferase-mediated dutp-biotin nick end labeling) Gel electrophoresis etect N ladder of 180 bp intervals caused by internucleosomal N cleavage nalysis of N content with a flow cytometer Recall the fluorescence intensity of the N dye (amount of N) is measured for each cell. Flow cytometry Measure externalization of phosphatidylserine (PS) with fluorescently labeled nnexin-v Measure N fragmentation with propidium iodide fluorescence Triggers of apoptosis Programmed cell death in which many more cells are produced than survive (e.g. development of lymphocytes) Toxic stimuli (viruses, chemicals, ionizing radiation) Extracellular signals (Fas, p75 NGF-R, TNF) N damage (p53) C. elegans has played a key role in our understanding of poptosis C. elegans apoptosis 1090 total cells 131 die Ced-3=no death Ced-4=no death Ced-9=all die ced-1 mutant (No engulfment) ced-1/ced-3 (No cells die) CE-9=Blocks apoptosis CE-4=linker molecule forms activating complex with CE-3 CE-3=Protease that executes cell by chewing up proteins EGL-1=Proapoptotic by blocking CE-9 function H.R Horvitz and colleagues responsible for much of this work, 2002 Nobel Prize in Medicine with Sulston and Brenner. 3

Three classes of proteins function in the apoptotic pathway-conserved in vertebrates These

cleave after SPartate residues Ced-3 is the C.

caspase activity (~1-2 % of active form) ctivated upon after aggregation or cleavage to

4 Three classes of proteins function in the apoptotic pathway-conserved in vertebrates These proteases are called caspases eath s Methods: protease cascade Mammalian Bcl-2 can substitute for Ced-9 in c. elegans Fig Caspases The Caspase Family Caspases are Cysteine directed proteases that cleave after SPartate residues Ced-3 is the C. elegans homologue t least 14 family members Synthesized as proenzymes with low levels of caspase activity (~1-2 % of active form) ctivated upon after aggregation or cleavage to mature form 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 Procaspase activation Caspase cascade 4

Two Pathways that Initiate poptosis ctivation of the Intrinsic

Mitochondria Cytochrome c release Extrinsic/ eath Receptor

These pathways intersect at the effector caspases Two mechanisms

domain Intrinsic Pathway: paf-1 Induced poptosis Smac/iablo and

5 Two Pathways that Initiate poptosis ctivation of the Intrinsic Pathway Intrinsic/ Mitochondrial poptosis Regulated by Mitochondria Cytochrome c release Extrinsic/ eath Receptor poptosis ctivated by ligation of eath Receptors Fas, TNF alpha These pathways intersect at the effector caspases Two mechanisms for p53 activation of apoptosis Intrinsic/Mitochondrial Pathway CR domain Intrinsic Pathway: paf-1 Induced poptosis Smac/iablo and IPs CR domains Smac=Second mitochondrial activator of caspases IP=Inhibitor of poptosis Proteins 5

Bcl-2 family members very large family with 19 members identified Bcl-2 (homologous to ced-9) is prototype ll have the BH3 domain (Bcl-2 Homology) BH-3 is the

members (Bcl-2, Bcl-XL) form dimers with pro-apoptotic members to inactivate them The Bcl-2 Family BH domains=protein-protein interaction domains Some trophic factors

and Bad have distinct functions to activate apoptosis Cell, Vol 111, 331-342, 1 November 2002 Bid, Bax, and Lipids Cooperate to Form Supramolecular Openings in the

1, Mason R. Mackey 2, Guy Perkins 2, Mark H. Ellisman 2, Martin Latterich 3, Roger Schneiter 4, ouglas R. Green 1, and onald.

6 Bcl-2 family members very large family with 19 members identified Bcl-2 (homologous to ced-9) is prototype ll have the BH3 domain (Bcl-2 Homology) BH-3 is the pro-apoptotic domain exposed on activation ct as dimers=either hetero or homodimers Pro-apoptotic dimers (Bax) increase mitochondrial permeability nti-apoptotic members (Bcl-2, Bcl-XL) form dimers with pro-apoptotic members to inactivate them The Bcl-2 Family BH domains=protein-protein interaction domains Some trophic factors prevent apoptosis by inducing inactivation of a pro-apoptotic regulator Mitochondrial permeability Figure PT=Permeability transition, bursts outer membrane Bid and Bad have distinct functions to activate apoptosis Cell, Vol 111, , 1 November 2002 Bid, Bax, and Lipids Cooperate to Form Supramolecular Openings in the Outer Mitochondrial Membrane Bax+ BH3 Peptide (irect ctivation) Bax + N/C-Bid + Bcl-xL+ BH3 Peptide (e-repression) Liposome ssay (cardiolipin+bax+bid) Tomomi Kuwana 1, Mason R. Mackey 2, Guy Perkins 2, Mark H. Ellisman 2, Martin Latterich 3, Roger Schneiter 4, ouglas R. Green 1, and onald. Newmeyer 1 * * * BH3 Peptide tbid irectly activates Bax pore formation Bad indirectly activates Bax pore formation (Binds Bcl-xL releasing Bax) N/C-Bid=recombinant activated Bid Kuwana et al., Molecular Cell, 17, ,

Extrinsic/eath Receptor Pathway eath Receptors and

poptosis Cysteine-Rich omains (CR) eath omains ()

F) Recruiting them to the plasma membrane.

cells expand The antigen specific T cells generated

Upon repeated antigenic stimulation via the T Cell

neighboring T Cells expressing Fas ctivation of

7 Extrinsic/eath Receptor Pathway eath Receptors and Ligands C95=Fas TNF receptor family Fas-FasL poptosis Cysteine-Rich omains (CR) eath omains () Bind s of other proteins (e.g. F) Recruiting them to the plasma membrane. In response to antigenic stimulation, peripheral T cells expand The antigen specific T cells generated must be eliminated (except for the memory cells) Upon repeated antigenic stimulation via the T Cell receptor: T cells upregulate Fas and FasL Eliminate neighboring T Cells expressing Fas ctivation of poptosis by Fas Ligand Fas Induced poptosis The Formation of the eath Initiating Signal Complex (ISC) 7

daptor Proteins contain conserved protein interaction domains Fas and the

procaspase-9. -E domain of F binds E domain of procaspase-8.

phagocytosis Cytoskeletal regulatory proteins ctin Nuclear Lamins Poly(P-ribose)

8 daptor Proteins contain conserved protein interaction domains Fas and the intrinsic pathway:bid = inhibits apoptosis -CR domain of paf-1 binds CR domain of procaspase-9. -E domain of F binds E domain of procaspase-8. -E domains of FLIP can bind to the E domain of F and block procaspase-8 recruitment. /Bax Proteolytic targets of effector caspases Removal of apoptotic cell by phagocytosis Cytoskeletal regulatory proteins ctin Nuclear Lamins Poly(P-ribose) polymerase (PRP) PRP activity depletes TP, thus cleavage of PRP may maintain store of TP to drive apoptosis N-fragmentation factor (FF) Removal of cell corpses Phagocytosis tags and receptors 8

Two roads to activate apoptosis TNFα receptors also signal to NFkB Extrinsic Intrinsic

example) IKKK=IkB Kinase kinase NFkB activates transcription of several anti-apoptotic

PE-15 Structure and Binding Partners Characterization of phospho-epitope antibodies PKC

15-ka protein containing 130 amino acids N-terminus consists of a eath Effector omain and

9 Two roads to activate apoptosis TNFα receptors also signal to NFkB Extrinsic Intrinsic Ubiquitylation is a common signal transduction mechanism (see regulation of cyclins for example) IKKK=IkB Kinase kinase NFkB activates transcription of several anti-apoptotic proteins including IPs and Bcl-2. PE-15 Structure and Binding Partners Characterization of phospho-epitope antibodies PKC CamK II NES N- E -C p104 p116 s s PKC CamK II F Omi PL1 ERK1/2 Rsk2 KT NES N- E -C ka protein containing 130 amino acids N-terminus consists of a eath Effector omain and NES Regulated at Ser104 and Ser116 by phosphorylation Effect of PE-15 phosphorylation on its binding to ERK ps116 PE-15 binds F ps104 ps116 GST-PE15 pulldown 9

10 GROWTH SIGNLS PE-15 is nti-apoptotic FS RECEPTOR GROWTH SIGNLS PE-15 is nti-apoptotic FS RECEPTOR KT PKC P E P E P- P- F C S P S E F C S P S E X CSPSE CSPSE KT PKC P E P- P- F F PE-15 blocks Fas and TNFα apoptosis in Hela, MCF7, NIH3T3 CaMKII CaMKII PE-15 blocks TRIL apoptosis in glioma lines poptosis SURVIVL PE-15 null astrocytes more sensitive to TNFα GROWTH SIGNLS RS RF MEK KT CaMKII PKC POPTOTIC SIGNLING Example Question Stathmin ERK MNK RSK2 RSK1 PROLONGE ERK PE-15 regulates both ERK and apoptosis SSOCITION WITH RSK2 pathways ERK PE RSK2 SURVIVL p- PROLIFERTION p- p- p- RSK2 RSK2 PE ERK IRECTE TO RSK2 TRNSCRIPTION SURVIVL PROLIFERTION PE -p -p INCRESE PHOSPHORYLTION N/OR LTERE CONFORMTION ERK p- PE p- RSK2 PHOSPHORYLTE PE-15 IS RECRUITE TO ISC Compare the formation of the eath Initiation Signaling Complex (ISC) of the extrinsic pathway to the formation of the apoptosome of the intrinsic pathway. rawings could help. What signal initiates the formation of each (an aggregation step)? Where are the complexes formed in the cell? What adaptor proteins mediate the formation of each complex? What are the initiator and effector caspases for each? How are the caspases activated? What do they do? 10

Apoptosis: Death Comes for the Cell

Apoptosis: Death Comes for the Cell Joe W. Ramos joeramos@hawaii.edu From Ingmar Bergman s The Seventh Seal 1 2 Mutations in proteins that regulate cell proliferation, survival and death can contribute