2.Apoptosis, Programmed cell death Biological functions of apoptosis Morphological and biochemical characteristics of apoptosis  Contrast ofApoptosis and necrosis 3.Molecular mechanisms of apoptosis Early researches (MIT: Robert Horrid , 1986)  Apoptosis is carried out by  a proteolytic system —caspase How to activate caspases? The target proteins of caspase are the following:  Molecular pathways of apoptosis *  * Chapter 13 Cell apoptosis Extracellular cotrol of cell division,  cell growth, and apoptosis Mitogens stimulate G1-Cdk and G1/S-Cdk activities PRIZED Horvitz, and Sulston share Physiology or Medicine Nobel  （2002 ）“for their discoveries concerning genetic regulation of organ development and programmed cell death”1090 –131= 959(cells) 机体结构细胞增殖细胞分化细胞凋亡细胞信号转导染色体（DNA 与蛋白质的相互作用）衰老A simplified model of one way that mitogens stimulate cell division Human cells have a built-in limitation on the number of times they can divide The cell division is cotrolled not only by extracellular mitogens but also by intracellular mechanisms that can limit cell proliferation. Cell-cycle arrest or apoptosis induced by excessive stimulation of mitogenic pathways. Extracellular growth factors stimulate cell growth Activation of cell-surface receptors Activation of PI 3-kinase  Activation of elF4E (translation initiation factor) and S6 kinase(phosphorylates ribosomal protein S6)  Increasing protein synthesis and cell growth  In development, homeostasis, tumor surveillance, and the function of the immune system.  细胞凋亡是多细胞生物在发育过程中，一种由基因控制的主动的细胞生理性自杀行为。Morphologi changes: Early : Chromosome condensation, cell body shrink  Later : Blebbing and Nucleus and cytoplasm fragment—Apoptotic bodies At last: Phagocytosed A、Normal cell B、Apoptosis: Apoptotic bodies  Biochemical characteristics of apoptosis : Apoptosis induced  by Cyto C Lane 1．0 h 2．1 h 3．2 h 4．3 h 5．4 h 6．Control  7．Marker 2.0 kbp 1.0  0.5 0.2 180~200 bp DNA ladder, Accumulation of tTG, PS flip-flop Apoptosis  Necrosis  Death by apoptosis is a neat, orderly process C. elegans:1090 cells,  The Finding of CED3 mutant Without losing any of their cells to apoptosis. CED3 gene play a crucial role in the process of apoptosis. C elegans: a millimeter long, transparent body only a few cell types, from zygote to mature adult only in 3.5days.  131 cells death. 线虫体细胞凋亡研究：PCD 相关基因15个，分为四组：1、与PCD 有关的基因，负责PCD 控制：ced-3、ced-4 和ced-9。2、与PCD 过程吞噬作用有关：ced-1、ced-2、ced-5-8 和ced-10。3、核酸酶基因（nuc-1）, 控制DNA 降解，但非PCD 所必须。4、影响特异细胞类型PCD 的基因：ces-1、ces-2、cgl-1、和her-1. 保守性高，在哺乳动物中有相应同源物：Ced-3=ICE, Ced-4=Apaf-1, Ced-9=Bcl-2. Mammalian: CED3 is related to mammalian interleukin-  1 –converting enzyme (ICE or caspase-1) (1) Why called caspase? Active site: Cysteine Cleavage site: Asparatic acid Cysteine Asparatic acid specific protease Aps-Xxx 天冬氨酸特异性的半光氨酸蛋白水解酶Apoptosis can be divided into two phases：Activation phase：The cell responds to “death signals”that commit it to undergoing self-destruction. Execution phase：The death sentence is carried out. Apoptosis cells are recognized by phagocytes because they carry exposed markers, called “eat me”signals. The best studied “eat me”signal is the presence of phosphatidylserine molecules in the outer leaflet of PM of apoptotic cells (by flop-flipase). All caspases expressed as proenzymes—Procaspases Procaspase  NH2-terminal prodomain: Highly variable Large subunit (20kD) Small subunit (10kD) How are procaspases activated to initiate the caspase cascade? The activation is triggered by adaptor proteins that bring multiple copies of specific procaspases. 3 groups of caspase: 1、apoptotic initiators: caspase-2, caspase-8, caspase-9 and caspase-10  2、apoptotic executioners: caspase-3, caspase-6 ，caspase-7 and 14 (morphology change)  3、inflammatory mediateors: caspase-1,  and caspase-11 Procaspases are activated by binding to adaptor proteins The caspase cascade involved in apoptosis Procaspase activation by proteolytic cleavage. Caspase cascade More than a dozen protein kinase, including FAK, PKC, and Raf1. FAK–disrupt cell adhesion for the apoptotic cell. Lamins. Cleavage of lamins leads to the disassembly of the nuclear lamina and shrinkage of the nucleus. Proteins required for cell structure. Such as IF, actin, and gelsilin. Cleavage and inactivation of these proteins lead to changes in cell shape. Induce cell display signals marked it for phagocytosis . The inhibitor of CAD (Caspase-activated Dnase, an endonuclease). Cleavage of CAD inhibitor lead to activation of CAD, once activated, CAD translocates from the cytosol to the nucleus severing DNA into fragments. Enzymes involved in DNA repair. Which are inactivated by caspase cleavage. DNA repair is a homeostatic activity that is inappropriate in an apoptotic cell. Two principle pathways Extrinsic pathway Intrinsic pathway