1、,细胞增生和凋亡的分子机制,Fate of cells Undergoing cell cycle proliferation differentiating to specific cell Death,细胞分裂增生的研究20世纪60年代 细胞周期分子机制的研究Hartwell L, Nurse P, Hunt T 2001 Nobel prize for physiology and medicine,细胞增生（proliferation）细胞在严密调控下有序进入细胞周期而分裂繁殖。 细胞增殖的意义细胞增殖为细胞分化提供来源补充因死亡而消失的细胞,细胞凋亡的研究始于上世纪60年代， 上世纪
2、80年代在线虫首次阐明 2002年诺贝尔医学和生理学奖,Nobel Prize for Physiology and Medicine 2002,For “genetic regulation of organ development and programmed cell death” Sydney Brenner (English) H. Robert Horvitz (American) John Sulston (English),Sydney Brenner H. Robert Horvitz John Sulston,细胞凋亡的概念 机体细胞在生理或病理状态下发生的自发性的程序性死
3、亡 细胞凋亡的意义 清除错误细胞 清除多余细胞，使各组织的细胞达到平衡,第一节 生长因子信号转导活化细胞周期是细胞增生的分子机制,一、细胞经历细胞周期而增生,The 4 phases of a typical cell cycle and the events occurring during each phase are outlined,M phase is the period when cells prepare for and then undergo cytokinesis. During mitosis the chromosomes are paired and then di
4、vided prior to cell division. G1phase corresponds to the gap in the cell cycle that occurs following cytokinesis. During this phase cells make a decision to either exit the cell cycle and become quiescent or to continue dividing. G0 phase Quiescent and terminally differentiated cells are identified
5、as being in G0 phase. S phase is the phase of the cell cycle during which the DNA is replicated. G2 phase is reached following completion of DNA replication. During G2 the chromosomes begin condensing, the nucleoli disappear and two microtubule organizing centers begin polymerizing tubulins for even
6、tual production of the spindle poles.,Two transitions (两个转折点)：G1-S transitionG2-M transition Four checkpoints (细胞周期中的四个关卡)G1 晚期的限制点G1-S转折的DNA损伤关卡G2-M转折的DNA损伤关卡有丝分裂中期的关卡,二、参与调控细胞周期进程的蛋白质,细胞周期蛋白 周期蛋白依赖性激酶 周期蛋白-周期蛋白依赖性激酶抑制因子 RB-DP1转录因子 调节CDK的蛋白激酶和磷酸酶 泛素和使蛋白质泛素化的酶,三、调控蛋白协同作用调控细胞周期,Cdk4/6和Cdk2的活化限制点 Cdk1
7、活化G2M checkpoint APC介导的多泛素化蛋白降解有丝分裂中期checkpoint DNA损伤关卡与G1及G2期停滞相关,四、生长因子等通过信号转导调控细胞周期,1. G0期进入细胞周期 2. G1期细胞也需要生长因子,Apoptosis:,Why is dying so important?,Physiologically: embyro stage, CNS development, thymus atrophy, endometrium desquamating Pathologically: tumor, Parkinsons disease, Alzheimers dis
8、ease,Programmed Cell Death in Eukaryotes,Caenorhabditis elegans: The Perfect Model,C. eleganss complexity but simplicityA nematode approximately one mm long containing blood, muscle, heart, nervous, as well as other tissues From fertilization to adult in three days Life span of two to three weeks Ad
9、ult organism comprised of 959 cells During embryological development will form 1090 cells,Approximately 40 percent of the worms genes are also found in humans Responds to taste, smell, temperature, touch, and possibly lightSo, where did the other 131 cells go?,The C. elegans Organism,The Fundamental
10、 Genes Being Examined,Egl-1 Ced9 Ced4 Ced3 apoptosisEGL-1initiates apoptosis by inhibiting the normal restraining action of CED-9 on CED-4 CED-3triggered by CED-4 resulting in highly destructive proteases acting upon cell structure CED-4acted upon by EGL-1; required in cell death CED-9 protects agai
11、nst cell deathegl-1 egg laying defective-1ced cell death abnormal,EGL-1has multiple mammalian killer gene counterparts CED-3human counterparts are called caspases which initiate apoptosis; protein ICE CED-4human counterpart called Apaf1 which promotes caspase activation CED-9comparable to the human
12、oncogene BCL-2 which blocks cell suicide,Major Players in Apoptosis-caspase,Caspases Cysteine proteases Recognize tetrapeptide motifs and cleaves at the carboxyl side of an aspartate reside (caspase = cysteine aspartate-specific protease) Synthesized as zymogens (“procaspases”) that are activated by
13、 caspase-mediated cleavageProcaspase: Nprodomain-p20 -p10 domain-C,Initiator caspases (e.g. caspase-8 and caspase-9) start a cascade of increasing caspase activity by processing and activating downstream effector caspases (e.g. caspase-3, -6 and -7)activated effector caspases cleave and inactivate v
14、ital cellular proteins and induces morphological changes that are characteristic of cells undergoing apoptosis,Plays an integral role in regulating mitochondrial outer membrane permeabilization, and thus the release of key effector proteins including cyto c and Smac/DIABLO from the mit intermembrane
15、 space At least 20 Bcl-2 related proteins identified in mammalian cells Bcl-2 family members share one or more Bcl-2 homology (BH) domains and are divided into two main groups whether they promote or inhibit apoptosis Anti-apoptotic members such as Bcl-xL, Bcl-w and Boo/Diva share at least three or
16、four regions of extensive amino acid sequence similarity with the prototypical Bcl-2 (BH1 BH4 regions) Pro-apoptotic members usually posses only a BH3 region e.g. Bad, Bik/Nbk/Blk, and Bid Bax-Bak examples of pro-apoptotic multidomain proteins,Major Players in Apoptosis-Bcl-2 family,Bcl-2 family,Maj
17、or Players in Apoptosis-adaptor protein,Form bridges between cell death effectors (caspases) and the cell death regulators (death receptors and Bcl-2 family members) Death receptors of the TNF-R family interact with adaptor proteins via the death domain (DD) of the receptor and the death effector do
18、main (DED) of the adaptor. e.g. the DD of the CD95 effector is associated with the adaptor molecule designated FADD (Fas-associating death domain protein) interactions between the DD of CD95 and FADD results in pro-caspase 8 aggregation and activation,Suppress apoptosis triggered by wide variety of
19、stimuli e.g. viral infection, chemotherapeutic drugs and components of the TNF-a/Fas signaling pathway Characterized by one or more repeats of highly conserved 70 amino acid domain termed baculoviral IAP repeat (BIR) Currently six human IAP members c-IAP1, c-IAP2, XIAP, NIAP, Livin and Survivin Most
20、 of IAP family members have been shown to interact with caspases, inhibiting their activity Play a role in pathological conditions e.g. NIAP gene originally identified in patients with spinal muscular atrophy; XIAP and c-IAP1 are found in most cancer cell lines; Survivin is overexpressed in nearly a
21、ll human tumors but is rarely present in adult tissues,Major Players in Apoptosis-IAP,Apoptosis-inducing factor (AIF) Flavoprotein that is normally located in the intermembrane space of mitochondria. When cells receive a signal for apoptosis AIF is released from the mitochondria AIF translocates int
22、o the nucleus and causes nuclear fragmentation and cell death DNA destruction mediated by AIF is not blocked by caspase inhibitors and is thus considered a caspase-independent pathway,Other molecules of Apoptosis,Smac: The second mitochondria-derived activator of caspase, 239aa, N-terminal 55aa as m
23、itochondria signal.It normally resident in mitochondria but is released into the cytosol when cell undergo apoptosis.Mechanism: binding to IAP Smac: second mitochondria-derived ativator of caspase DIABLO: direct IAP-binding protein with low pI,Other molecules of Apoptosis,Other molecules of Apoptosi
24、s,Omi: most recently discovered proapoptotic protein released from mitochondria and shows much similarity to Smac.,Cell death process three phases,Induction or initiation phase （起始） Effector or decision phase（效应）activating hydrolase (protease and nuclease) Degradation phase（降解）digestion of protein,
25、fragmentation of DNA,Two main apoptotic pathways,The activation of death receptors（死亡受体途径） Mitochondria pathway（线粒体途径）common pathway: activation of caspase cascade,Major Apoptotic Pathways in Mammalian Cells,Hengartner, M.O. 2000. Nature. 407:770. Green, D. and Kroemer, G. 1998. Trends Cell Biol. 8:
26、267.,Apoptosis Oxygen Society Education Program Tome & Briehl 3,DISC: death inducing signal complex FADD: Fas associated protein with death domain,外源性的死亡受体途径,Fas：单跨膜受体，N端在胞外，DD位于胞内，分布广泛 FasL:单跨膜受体，在细胞表面形成三聚体，细胞毒T细胞表面 FADD：Fas-associated death domainDD and DED (death effector domain)DISC：FasLFasFADD,
27、Fas and Related Proteins with Death Domains,Death receptor: Fas, TNFR1, TNFR2, DR3, DR4, DR5, DcR1,DcR2 TRADD: TNF receptor-associated death domain,The extrinsic or death receptor pathway Initiated by binding of a death-inducing ligand to a Cys-rich repeat region in the extracellular domain of a dea
28、th receptor Death receptors such as Fas and the TNF receptor are integral membrane proteins with their receptor domains exposed at the surface of the cell Binding of the complementary death activator (FasL and TNF-a, respectively) transmits a signal (via an adaptor protein) to the cytoplasm that lea
29、ds to activation of caspase-8 Caspase-8 (like caspase-9) initiates a cascade of caspase activation leading to cell death Example: when cytotoxic T-cells recognize (bind to) their targets: they produce more FasL at their surface this binds with Fas on the surface of the target cells and starts the ca
30、scade that leads to its death by apoptosis,Mechanisms of Apoptosis,Cell death receptors members of TNFR family, can have pleiotropic action depending on cell type and signals received i.e., can trigger cell proliferation, differentiation or death Activated by structurally-related ligands of the TNF
31、ligand family e.g. CD95 (also called Fas or APO-1) contains a cytoplasmic region called the death domain which transmits the signals via an adaptor protein to initiator caspases 4 TRAIL/APO-2L receptors identified 2 of them, DcR1 and DcR2 lack the death domain and cannot induce apoptosis acts as dec
32、oys to inhibit TRAIL/APO-2L-mediated apoptosis Decoy receptor for FasL (DcR3) found overexpressed in lung and colon tumors,Schematic for death receptor,TNF or Fas ligand interact with death receptorRecruitment of adaptor molecules (FADD)Activating caspase 8directly activating caspase 3 cleave Bid (t
33、Bid)and caspase 7 translocate to mitbcl-2 cyto C release,Fas Signaling Pathway,TNFR-TNFa 凋亡途径,TNFR1单跨膜受体，分布广泛 TNFa由活化的巨噬细胞和淋巴细胞产生 TNFR1胞内DD募集TRADD, 后者与TRAF2和RIP形成复合物， RIP活化NFkB，通过FLIP抑制caspase8活化DISC 复合物1FLIP: Fas associated death domain-like interleukin beta converting enzyme inhibitory protein,TNF
34、 Signaling Pathway,Mitochondria pathway,1. The stimuli leading to cell death(growth factor deprivation, ionizing radiation and several chemical agents)2. mitochondrial membrane permeabilization release of cytochrome Cformation of apoptosome(Apaf-1,cyto C, dATP) （apoptotic proteonase activating facto
35、r） 3. Activating caspase 9 by Apaf1 CARD （caspase recruitment domain）4. Activating caspase-3, -7,-6, cleave 45KD subunit of the DFF5. Release DFF40 (CAD mouse homolog) with nuclease activity,内源性凋亡的诱因：失去赖以生存的生长因子或激素支持脱离原来的生长环境DNA损伤等,Three models of translocation for CytC1.Bax和Bak在mit外膜打孔2.VDAC在外膜形成通道
36、3.外膜上ATP-ADP转运蛋白孔道保持开 放,The intrinsic or mitochondrial pathway,In a healthy cell, the outer membrane of mit express the protein Bcl-2 on its surface Bcl-2 is bound to Apaf-1 (“apoptotic protease activating factor-1) Internal damage to cells (e.g. from reactive oxygen species) causes: Bcl-2 to releas
37、e Apaf-1 a related protein, Bax, to penetrate mitochondrial membranes causing cyto c and other proteins such as Smac/DIABLO and AIF to leak out The released cyt c and Apaf-1 binds to molecules of caspase-9 forming an aggregate called the apoptosome This stimulates/amplifies activation of caspase-9 a
38、nd downstream apoptotic events Smac/DIABLO neutralizes IAP (“inhibitor of apoptosis”) proteins and allows caspase activation to proceed AIF induces chromatin condensation and DNA fragmentation,Mitochondria in Apoptosis,三、Positive and negative regulation for apoptosis and crosstalk (凋亡途径的正负调节和串话),Positive regulation：caspase 激活和 caspase级联放大作用 Negative regulation：FLIP抑制caspase8活化Bcl-2抑制凋亡IAP含有BIR（baculovirus IAP repeat）BIR2抑制caspase3 和7；BIR3抑制caspase 9,
