Lecture 9 Apoptosis 2023 PDF

Cell Cycle III: Apoptosis 1 Apoptosis • “Programmed cell death”. • Number of cells is, in part, controlled by regulation of cell death. • Differs from necrosis: trauma or cytotoxicity leading to ¯ATP and ¯Na+/K+-ATPase activity, followed by lysis. • Why does apoptosis occur? Apoptosis Necrosis...

Cell Cycle III: Apoptosis 1 Apoptosis • “Programmed cell death”. • Number of cells is, in part, controlled by regulation of cell death. • Differs from necrosis: trauma or cytotoxicity leading to ¯ATP and ¯Na+/K+-ATPase activity, followed by lysis. • Why does apoptosis occur? Apoptosis Necrosis 2 Apoptosis • Apoptosis in development. • Balance of cell division with death in adult tissues. Mouse paw Cells undergoing apoptosis Loss of cells between digits 3 General Characteristics • In order for apoptosis to occur, a cell must undergo significant biochemical and morphological change. • Initiation by intracellular or extracellular signal. • Activation of a series of proteins involved in promoting apoptosis (or inhibition of those that prevent it). • Important intracellular proteins necessary for survival are cleaved during apoptosis. • Orderly disposal of dead cell. 4 Structural Changes During Apoptosis 1 2 3 Normal cell Normal Loss of adhesion “Blebbing” 5 Phagocytosis • Asymmetric distribution of plasma membrane is lost. • Negatively charged phosphatidylserine then becomes exposed on the outside of cell. • The cell is then marked for phagocytosis by a macrophage. 6 Cell-death Mutants Normal • Caenorhabditis elegans is a nematode. • 959 somatic cells. • 131 apoptose every time. • Discovery of ced-3 gene that encodes proteins similar to mammalian protease. • “caspases”. • ced-4 encodes Apaf-1; ced-9 encodes Bcl-2. ced-3 mutant 7 ced-3 mutation prevents apoptosis in all 131 cells Ellis and Horvitz 1986. Cell 44:817-829. Caspases • Identification of ced-3 gene led to the discovery of homologous family of proteins, called “caspases”, in mammals. • They are proteases that cleave essential proteins. • Involved in most changes observed during cell death. • This enzyme has a cysteine residue at its catalytic site and cleaves other protiens at an aspartate site. • Caspases may also cleave each other, leading to their activation. 8 Caspases Cleave Essential Proteins (+) = cleavage = activation Caspases (+) Protein Kinases e.g. FAK, disrupt cell adhesion Lamins Disassembly of nuclear lamina Cytoskeleton Changes in cell shape CAD DNA fragmentation From Karp 2008. Cell and Molecular Biology. CAD = caspase-activated DNAse; FAK = focal adhesion kinase 9 Procaspase Activation • Initiator (or “pro”) caspases are inactive. • An apoptotic signal triggers assembly of adaptor proteins that activate caspases. • These caspases go on to activate executioner caspases by proteolytic interactions (cleavage). 10 11 The Caspase Cascade • • • • An active caspase activates another caspase… Irreversible, “all-or-none”. Amplification. Result: intracellular proteins are cleaved. 12 Figure 18-5b Molecular Biology of the Cell (© Garland Science 2008) 2 Major Apoptotic Pathways Extrinsic pathway • Procaspase activation triggered from outside the cell. • “Cell death” receptors. Intrinsic pathway • Procaspase activation triggered from inside the cell. • e.g. intracellular damage. • e.g. via Bcl-2 family of proteins (note: this may involve an extracellular survival factor). 13 Extrinsic Pathway • Extracellular signal (Fas ligand) activates death receptor (Fas protein). • Recruitment of adaptor proteins and procaspase activation. • Caspase cascade. 14 FADD = Fas-associated death domain; DISC = death-inducing signalling complex Intrinsic Pathway • An intracellular death signal initiates caspase cascade and cell death. • Initiated by e.g. DNA damage or loss of survival factor. • Bcl-2 protein family Cytochrome c in mitochondria DNA in nucleus – Bcl-2 inhibits apoptosis – Bax and Bak act on mitochondria and release cytochrome c. • Balance of these determines fate (i.e. life or death) of cell. Cytochrome c released DNA fragmented (Karp, 2008) 15 Intrinsic Pathway • Apoptotic signal activates Bcl2 family proteins (e.g. Bax) to form aggregates in outer mitochondrial membrane. • Induces cytochrome c release from intermembrane space. 16 Intrinsic Pathway • Formation of “apoptosome”. • Caspase cascade. 17 Apaf-1 = apoptotic protease activating factor-1; CARD = caspase recruitment domain Human Apoptosome at 12.8 A Resolution 18 Yu et al. 2005 Structure 13:1725-1735 Some Mechanisms of the Intrinsic Pathway Y survival factor DNA damage (-) Cytochrome c release from mitochondria (synthesis) ­Bax (-) (irreversible) Caspase cascade Cleavage of intracellular proteins 19 Apoptosis in the Developing Nervous System • • • • Recall NGF. A growth factor, but also a survival factor. Released by “target” cells. Bax is an important mediator*. 20 *Lindsten et al. 2005. Neuroscientist 11:10-15 Apoptosis in the Developing Nervous System • Mutations in genes encoding important proteins in the apoptotic pathway have drastic consequences. • In developing mouse embryo, forebrain protrusion and excessive mitosis and differentiation result. Nijhawan et al. 2000. Annu Rev Neurosci 23:73-87 21 A Summary Apoptosis in vitro 22 Things to Consider... How do extrinsic and intrinsic pathways of apoptosis differ? For example, think about how the caspase cascade is initiated in each case. 23