Apoptosis - Cell Biology PDF

Cell BIOLOGY Presented by Dr Ghada KHAWAJA Apoptosis Introduction Cell death plays an important part in animal and plant development and it usually continues into adulthood In a healthy adult human, bil...

Cell BIOLOGY Presented by Dr Ghada KHAWAJA Apoptosis Introduction Cell death plays an important part in animal and plant development and it usually continues into adulthood In a healthy adult human, billions of cells die every hour in the bone marrow and intestine Tissues don’t shrink because cell division exactly balances cell death These normal cell deaths are suicides Cells activate an intracellular death program and kill themselves in a controlled way: process called programmed cell death  Definition Apoptosis is the most common form of programmed cell death It comes form the Greek word meaning “falling off” The cell surface of apoptotic cells become chemically altered so that a macrophage rapidly engulfs them before spilling their content Avoiding a damaging inflammatory response. Morphology Apoptotic cells show characteristic morphological changes: – Shrink and condense – Cytoskeleton collapse – Nuclear envelope disassembles – Nuclear chromatin condenses and breaks up into fragments – Cell surface often blelbs and, if the cell is large, often breaks up into membrane-enclosed fragments called apoptotic bodies – Surface of the cell or apoptotic bodies becomes chemically altered Classical examples of apoptosis – In animal development, apoptosis helps in sculpting hands and feet regulating cell numbers in developing nervous system quality control process in developing adaptive immune system – Metamorphosis: elimination of unwanted cells – In adult tissues: Elimination of abnormal, nonfunctional, or potentially dangerous to the animal Elimination of no longer needed cells (T and B lymphocytes after the resolution of an infection) Maintaining a ready supply of short-lived neutrophils in the Bone marrow Biochemistry Biochemical changes can be used to identify apoptotic cells: Cells undergoing apoptosis not only have a characteristic morphology but also display characteristic biochemical changes, which can be used to identify apoptotic cells. During apoptosis, for example, an endonuclease cleaves the chromosomal DNA into fragments of distinctive sizes; because the cleavages occur in the linker regions between nucleosomes, the fragments separate into a characteristic ladder pattern when analyzed by gel electrophoresis  Biochemistry 2. Phosphatiylserine flips to the outer leaflet of the lipid bilayer: – The negatively charged phospholipid phosphatidylserine is normally exclusively located in the inner leaflet of the lipid bilayer of the plasma membrane, but it flips to the outer leaflet in apoptotic cells, where it can serve as a marker of these cells. – Signals neighboring cells and macrophages to phagocytose the dying cell – Blocks the inflammation associated with phagocytosis by inhibiting the production of cytokines by the phagocytotic cell Biochemistry 3. Cell loose the electric potential that exists across the inner membrane of their mitochondria – Cytochrome C is released form the intermembrane space  caspases APOPTOSIS MACHINERY Apoptosis: The beginning Apoptosis Machinery depends on a family of proteases: caspases – Have a cysteine at the active site – Cleave at specific aspartic acid residues Caspases are produced as inactive precursor; procaspases which are typically activated by proteolytic cleavage  Apoptosis: The beginning Healthy cells continuously make the procaspases Thus apoptosis machinery is always in place, all is needed is a trigger to activate it How the first procaspase in the cascade is activated? Initiator procaspases have a long prodomain, which contains a caspase recruitment domain (CARD) CARD enables the assembly with adaptor proteins into activation complexes when the cell receives a signal to undergo apoptosis Apoptosis: The beginning Once incorporated into such a complex: – Initiator procaspases are brought into close proximity – This is sufficient to activate them – They then cleave each other to make the process irreversible  Procaspase activation Procaspases are split into: – Large subunit – Small subunit Together, they form a heterodimer 2 such dimers assemble to form the active tetramer Procaspase activation The first procaspases activated are the initiator procaspases These cleave and activate many executioner procaspases producing an amplifying chain reaction The executioner caspases then cleave a variety of key proteins in the cell, including specific cytosolic proteins and Nuclear lamina, leading to the controlled death of the cell Not all caspases mediate apoptosis Caspases involved in inflammation Caspases involved in apoptosis Caspase 1 (ICE) Interleukin-1- Initiator caspases: Executioner caspases: converting enzyme Caspase 4 Caspase 2 Caspase 3 Caspase 5 Caspase 8 Caspase 6 Caspase 9 Caspase 7 Caspase 10 Target proteins Among the target proteins cleaved by executioner caspases: – Nuclear lamina – Proteins that hold endonucleases in the inactive form – Cytoskeleton – Cell-cell adhesion proteins  Extrinsic pathway Intrinsic pathway SIGNALING PATHWAYS The two best understood signaling pathways that can activate a caspase cascade leading to apoptosis in mammalian cells: the extrinsic pathway & intrinsic pathway. Each uses its own initiator procaspases and activation Complex. Extrinsic pathway Cell-surface Fas death receptors activate the extrinsic pathway They belong to the tumor necrosis factor (TNF) receptor family They are homotrimers transmembrane proteins containing: – Extra cellular ligand-binding domain – Single transmembrane domain – Intacellular death domain required to activate the apoptotic program  Extrinsic pathway; Fas activation The extrinsic pathway of apoptosis activated through Fas death receptors: Fas ligand on the surface of a killer lymphocyte activates Fas death receptors on the surface of the target cell. The cytosolic tail of Fas then recruits the adaptor protein FADD via the death domain on each protein (FADD stands for Fas-associated death domain). Each FADD protein then recruits an initiator procaspase (8 or 10 or both) via a death effector domain present on both FADD and the procaspase, forming a death-inducing signaling complex (DISC). Within the DISC, the initiator procaspase molecules are brought into close proximity, which activates them; the activated procaspases then cleave one another to stabilize the activated protease, which is now a caspase. Activated caspase-8 and caspase-10 then cleave and activate executioner procaspases, producing a caspase cascade which leads to apoptosis. Extrinsic pathway; inhibition Many cells produce inhibitory proteins that act either extracellularly or intracellularly to restrain the extrinsic pathway. some produce cell surface decoy receptors, which have a ligand-binding domain but not a death domain:because they can bind a death ligand but cannot activate apoptosis, the decoys competitively inhibit the death receptors Cells can also produce intracellular blocking proteins such as FLIP which resembles an initiator procaspase but lacks the proteolytic domain; it competes with procaspase-8 and procaspase-10 for binding sites in the DISC and thereby inhibits the activation of these initiator procaspases. Such inhibitory mechanisms help prevent the inappropriate activation of the extrinsic pathway of apoptosis.  Intrinsic pathway In response to injury, DNA damage, lack of oxygen/nutrients….. Release of cytochrome c, component of the electron transport chain, from the inter-membrane space of mitochondria Release of cytochrome c from mitochondria during apoptosis. Fluorescence micrographs of human cancer cells in culture (A) The control cells were transfected with a gene encoding a fusion protein consisting of cytochrome c linked to green fluorescent protein( cytochrome-c-GFP); they were also treated with a positively charged red dye that accumulates in mitochondria. The over lapping distribution of the green and red indicate that the cytochrome-c-GFP is located in mitochondria. (B ) Cells expressing cytochrome-c-GFP were irradiated with ultraviolet light to induce apoptosis, and after 5 hours they were stained with antibodies (in red) against cytochrome c; the cytochrome-c-GFP is also shown( in green). The six cells in the bottom half of the micrographs in B have released their cytochrome c from mitochondria into the cytosol, whereas the cells in the upper half of the micrograph have not yet done so; Intrinsic pathway When released into the cytosol, Cytochrome c binds to Apaf1 (apoptotic protease activating factor-l) causing it to oligomerize into a wheel like heptamer; apoptosome Apaf1 in the apoptosome then recruit initiator procaspase 9 Activated caspase 9 then activates down stream executioner procaspases  Regulation Intrinsic pathway Bcl2 proteins regulate the intrinsic pathway of apoptosis They control the release of cytochrome c pro-apoptotic anti-apoptotic BH123 (Bax, Bak) Bcl2 (Bcl2, Bcl-XL) BH3-only (Bad, Bim..) Regulation Intrinsic pathway Role of Bcl2 proteins: BH123 When activated by an apoptotic stimulus the BH123 proteins aggregate on the outer mitochondrial membrane and release cytochrome c and other proteins from the intermembrane space into the cytosol by an unknown mechanism  Regulation Intrinsic pathway Role of Bcl2 proteins: Bcl2 In the absence of an apoptotic stimulus, anti-apoptotic Bcl2 proteins bind to and inhibit the BH123 proteins on the mitochondrial outer membrane Regulation Intrinsic pathway Role of Bcl2 proteins: BH3-only In the presence of an apoptotic stimulus, BH3-only proteins are activated and bind to the anti-apoptotic Bcl2 proteins so they can no longer inhibit the BH123 proteins, which now become activated and aggregate in the mitochondrial outer membrane and promote the release of mitochondrial proteins in the cytosol.  Intrinsic pathway regulation Bcl2 proteins are not the only intracellular regulators of apoptosis The IAPs (inhibitors of apoptosis) proteins also play an important part in suppressing apoptosis Intrinsic pathway regulation; inhibition: IAPs IAPs are inhibitors of apoptosis First discovered in viruses Now it is known that it is produced in many animal cells Intrinsic pathway regulation;over coming IAPs: anti-IAPs Extracellular Survival Factors Inhibit Apoptosis in various ways Summary Either Excessive or insufficient apoptosis can contribute to disease  Refrence Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 5th edition. New York: Garland Science; 2008