14 apoptosis - Objectives • To recognize some remarkable...

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Unformatted text preview: Objectives • To recognize some remarkable morphological and biochemical features of apoptosis • To identify some key molecules in apoptosis • To understand some better-characterized cell signal transduction pathways and the ways the key molecules interact during apoptosis 1 Apoptosis • The number of cells in multicellular organism is regulated by controlling both the rate of cell division and the rate of cell rate death (by activating an intracellular cell death program) A type of Programmed Cell Death Programmed From a Greek word meaning “falling off”, as leaves from a tree 2 - • Very important in both embryonic development and embryonic adult homeostasis: adult - The nerve cells that exceed the target cells for innervation (up (up to half or more normally die) - Those T lymphocytes that can attack the body’s own cells - The cells in those structures that are no longer needed (e.g., (e.g., development of hand with individual digits, sex determination, etc.) 3 - In a healthy adult human, billions of cells die in the bone marrow and intestine every hour - Activated T cells that have responded to an infectious agent that has been eliminated - Those cells that have sustained irreparable genetic damage and have the potential to progress into cancer cells (one of the body’s (one main weapons against the development of cancer) 4 • 1. A neat, orderly process: neat orderly Overall shrinkage in shrinkage volume of the cell and its nucleus Loss of adhesion to neighboring cells Formation of blebs at the blebs cell surface Dissection of the chromatin into small fragments Rapid engulfment via phagocytosis of phagocytosis of neighboring cells 2. 3. 4. 5. 5 Accidental / Passive cell death 6 APOPTOSIS Morphologic Criteria Deletion of single cells Membrane blebbing, but no loss of integrity Cells shrink, ultimately forming apoptotic bodies No inflammatory response Phagocytosis by adjacent normal cells, and some macrophages Lysosomes intact Compaction of chromatin into uniformly dense masses Induced by physiological stimuli Tightly regulated process with synthetic and activation steps Requires energy Requires macromolecular synthesis De novo gene transcription Nonrandom oligonucleosomal length fragmentation of DNA NECROSIS Death of cell groups Loss of membrane integrity Cells swell Significant inflammatory response Phagocytosis by macrophages Lysosomal leakage Clumpy, ill-defined aggregation of illchromatin Evoked by non-physiological disturbances nonLoss of regulation of ion homeostasis No requirement of energy No requirements for protein or nucleic acid synthesis No new gene transcription Random digestion of DNA Biochemical Criteria 7 DNA Gel Electrophoresis (topoisomerase inhibitor - an anticancer drug) 8 DNA Fragmentation and Laddering 9 Loss of Phosphatidylserine Asymmetry at the Plasma Membrane 10 Caspases • The enzymes of apoptotic cell death • The molecular basis of apoptosis was first revealed in studies on the nematode worm Caenorhabditis Caenorhabditis elegans - The adult has 959 cells; 131 are destined to die by apoptosis - The worm carrying a mutation on the CED-3 gene proceed CED through development without losing any of their cells to apoptosis - Discovery of a homologous family of proteins in mammals (caspases) 11 • A family of proteolytic enzymes that have a cysteine at their active site and cleave their target proteins at specific aspartic acids • Some of them are activated at an early stage of apoptosis and are responsible for triggering most, if not all, of the changes observed during the execution phase Cysteinyl aspartate-specific proteases (caspases) 12 • Caspases are constitutively expressed as inactive pro-enzymes (zymogens) or procaspases: - Consists of N-terminal pro-domain, a large subunit, and a small subunit • Activation involves proteolytic processing at a specific aspartic acid between the large and small subunits (pro-domain is removed) (pro - The active site is formed by a heterodimer (one large and one small subunit); fully active caspase is a tetramer composed of 2 such heterodimers 13 Pro-caspase 14 15 Extrinsic / Death Receptor Pathway Endoplasmic Reticulum Pathway Intrinsic / Mitochondrial Pathway 16 Extrinsic / Death Receptor Pathway Tumor necrosis factor Tumor TNF receptor 1 Death receptors (e.g., Fas, TRAIL-R, etc.) (e.g., TRAILDeath ligands (e.g., CD95/FasL, TRAIL, etc.) TNF-R1-associated death domain TNF- R1Fas-associated death domain Fas- (Death-inducing Signaling Complex) (Death DISC 17 Bcl-2 Family Proteins • Guardians of cell death; regulate the activation of procaspases • Growing family of homologous genes are divided into at least 3 distinct subfamilies: the Bcl-2 and the subfamilies Bax subfamily, and the BH-3 only proteins: - Although the overall amino acid sequence homology is relatively low, there are highly conserved domains: Bcl-2 homology domains (BH1-4) (for hetero-complex (for formation and cell-death-inducing capacity) 18 Three Subfamilies of the Bcl-2 Family Proteins Bcl-2 Subfamily Bax Subfamily BH-3 Only Subfamily • Activators interact with Bax subfamily proteins, e.g., Bax/Bak, and activate them • Sensitizers bind with Bcl-2 subfamily proteins, .e.g., Bcl-2/Bcl-XL, BclBcl-2/Bclso as to prevent them from interacting with Bax subfamily proteins 19 Intrinsic / Mitochondrial Pathway Intrinsic Bax (monomer in the cytosol) → Conformational switch in the Nterminus and the C-terminus → Translocation to the mitochondria → Oligomerization to multimeric complexes → Formation of new channels and / or activation of permeability pores → Depolarization of the mitochondrial membrane / Effluxes of cytochrome c and other pro-apoptotic factors → other Formation of apoptosome [cytochrome c + Apaf-1 (apoptotic protease-activating factor 1) + pro-caspase 9] → Activation of caspase 9 Bax Apoptosome 20 • Some of the targets of caspases: 1. More than a dozen protein kinases (e.g., focal adhesion (e.g., kinase FAK) → detachment of the apoptotic cell from its neighbors 2. Lamins → disassembly of the nuclear lamina and shrinkage of the nucleus 3. Proteins required for cell structure (e.g., intermediate (e.g., filaments, actin, etc.) → changes in cell shape 4. An endonuclease (caspase-activated DNase CAD) is activated following cleavage if its inhibitory protein (ICAD) → CAD translocates to the nucleus where it attacks DNA, severing it into fragments 5. Proteins involved in DNA repair (e.g., GADD45) (e.g., 21 Crosstalk between the Extrinsic and the Intrinsic Pathways Type I Type II 22 • BH-3 only proteins act as sensors that mediate activation of Bax and Bak - e.g., Bid is proteolytically cleaved by caspase-8 to a pro-apoptotic form (truncated Bid tBid, p15), which appears to interact physically with Bax to mediate a conformational change in the Bax N-terminus 23 • The models of how Bcl-2 inhibits apoptosis are under current debate: 1. Sequestration of BH-3 only proteins by Bcl-2 that serves as a sink 2. Inhibition of Bax activation 3. Interference with mitochondrial events, such as opening of channels and release of pro-apoptotic factors 24 Different Models of How Bcl-2 Inhibits Apoptosis 25 How Bcl-2 Family Proteins Interact and Lead to Apoptosis 26 ...
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