【医学课件】细胞增生与凋亡的分子机制

1、 细胞增生和凋亡的分子机制细胞增生和凋亡的分子机制 Fate of cells nUndergoing cell cycle proliferationndifferentiating to specific cellnDeathn细胞分裂增生的研究细胞分裂增生的研究 20 20世纪世纪6060年代年代 细胞周期分子机制的研究细胞周期分子机制的研究 Hartwell L, Nurse P, Hunt T 2001 Nobel prize for physiology and medicinen细胞增生proliferationn 细胞在严密调控下有序进入细胞周期而分裂繁殖。n细胞增殖的意义n

2、细胞增殖为细胞分化提供来源n 补充因死亡而消失的细胞n细胞凋亡的研究始于上世纪60年代，n上世纪80年代在线虫首次说明n2002年诺贝尔医学和生理学奖Nobel Prize for Physiology and Medicine 2002nFor “genetic regulation of organ development and programmed cell deathnSydney Brenner (English)nH. Robert Horvitz (American)nJohn Sulston (English)Sydney Brenner H. Robert Horvitz

3、John Sulston细胞凋亡的概念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机体细胞在生理或病理状态下发生的自发性的程序性死亡性的程序性死亡细胞凋亡的意义细胞凋亡的意义去除错误细胞去除错误细胞去除多余细胞，使各组织的细胞到达平衡去除多余细胞，使各组织的细胞到达平衡第一节 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 c

4、ells prepare for and then undergo cytokinesis. During mitosis the chromosomes are paired and then divided 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 quies

5、cent or to continue dividing. G0 phase Quiescent and terminally differentiated cells are identified 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

6、, the nucleoli disappear and two microtubule organizing centers begin polymerizing tubulins for eventual production of the spindle poles. T Two transitions (两个转折点)： G1-S transition G2-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n细胞周期蛋白

7、n周期蛋白依赖性激酶n周期蛋白-周期蛋白依赖性激酶抑制因子nRB-DP1转录因子n调节CDK的蛋白激酶和磷酸酶n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nCdk4/6和Cdk2的活化限制点nCdk1活化G2M checkpointnAPC介导的多泛素化蛋白降解有丝分裂中期checkpointnDNA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Why is dying so important?Physiologically: embyro stage, CNS development, thymus

8、atrophy, endometrium desquamatingPathologically: tumor, Parkinsons disease, Alzheimers diseaseProgrammed Cell Death in EukaryotesCaenorhabditis elegans:The Perfect Model C. eleganss complexity but simplicitynA nematode approximately one mm long containing blood, muscle, heart, nervous, as well as ot

9、her tissuesnFrom fertilization to adult in three daysnLife span of two to three weeksnAdult organism comprised of 959 cellsnDuring embryological development will form 1090 cells nApproximately 40 percent of the worms genes are also found in humansnResponds to taste, smell, temperature, touch, and po

10、ssibly lightnSo, where did the other 131 cells go?The Fundamental Genes Being ExaminedEgl-1 Ced9 Ced4 Ced3 apoptosisnEGL-1initiates apoptosis by inhibiting the normal restraining action of CED-9 on CED-4nCED-3triggered by CED-4 resulting in highly destructive proteases acting upon cell structurenCED

11、-4acted upon by EGL-1; required in cell deathnCED-9 protects against cell death egl-1 egg laying defective-1 ced cell death abnormalnEGL-1has multiple mammalian killer gene counterpartsnCED-3human counterparts are called caspases which initiate apoptosis; protein ICEnCED-4human counterpart called Ap

12、af1 which promotes caspase activationnCED-9comparable to the human oncogene BCL-2 which blocks cell suicideMajor Players in Apoptosis-caspasenCaspasesnCysteine proteasesnRecognize tetrapeptide motifs and cleaves at the carboxyl side of an aspartate reside (caspase = cysteine aspartate-specific prote

13、ase)nSynthesized as zymogens (“procaspases) that are activated by caspase-mediated cleavagen Procaspase: n Nprodomain-p20 -p10 domain-C nInitiator caspases (e.g. caspase-8 and caspase-9) start a cascade of increasing caspase activity by processing andnactivating downstream effector caspases (e.g. ca

14、spase-3, -6 and -7) activated effector caspases cleave and inactivate vital cellular proteins and induces morphological changes that are characteristic of cells undergoing apoptosisnPlays an integral role in regulating mitochondrial outer membrane permeabilization, and thus the release of key effect

15、or proteins including cyto c and Smac/DIABLO from the mit intermembrane spacenAt least 20 Bcl-2 related proteins identified in mammalian cellsnBcl-2 family members share one or more Bcl-2 homology (BH) domains and are divided into two main groups whether they promote or inhibit apoptosisnAnti-apopto

16、tic members such as Bcl-xL, Bcl-w and Boo/Diva share at least three or four regions of extensive amino acid sequence similarity with the prototypical Bcl-2 (BH1 BH4 regions)nPro-apoptotic members usually posses only a BH3 region e.g. Bad, Bik/Nbk/Blk, and BidnBax-Bak examples of pro-apoptotic multid

17、omain proteinsMajor Players in Apoptosis-Bcl-2 familyMajor Players in Apoptosis-adaptor proteinnForm bridges between cell death effectors (caspases) and the cell death regulators (death receptors and Bcl-2 family members)nDeath receptors of the TNF-R family interact with adaptor proteins via the dea

18、th domain (DD) of the receptor and the death effector domain (DED) of the adaptor.ne.g. the DD of the CD95 effector is associated with the adaptor molecule designated FADD (Fas-associating death domain protein)ninteractions between the DD of CD95 and FADD results in pro-caspase 8 aggregation and act

19、ivationnSuppress apoptosis triggered by wide variety of stimuli e.g. viral infection, chemotherapeutic drugs and components of the TNF-a/Fas signaling pathwaynCharacterized by one or more repeats of highly conserved 70 amino acid domain termed baculoviral IAP repeat (BIR)nCurrently six human IAP mem

20、bers c-IAP1, c-IAP2, XIAP, NIAP, Livin and SurvivinnMost of IAP family members have been shown to interact with caspases, inhibiting their activitynPlay 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

21、cancer cell lines; Survivin is overexpressed in nearly all human tumors but is rarely present in adult tissuesMajor Players in Apoptosis-IAPnApoptosis-inducing factor (AIF)nFlavoprotein that is normally located in the intermembrane space of mitochondria. nWhen cells receive a signal for apoptosisnAI

22、F is released from the mitochondria nAIF translocates into the nucleus and causesnnuclear fragmentation and cell deathnDNA destruction mediated by AIF is not blocked by caspase inhibitors and is thus considered a caspase-independent pathwayOther molecules of ApoptosisnSmac: The second mitochondria-d

23、erived activator of caspase, 239aa, N-terminal 55aa as mitochondria 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 p

24、IOther molecules of ApoptosisOther molecules of ApoptosisnOmi: most recently discovered proapoptotic protein released from mitochondria and shows much similarity to Smac.Cell death process three phasesnInduction or initiation phase 起始nEffector or decision phase效应n activating hydrolase (protease and

25、nuclease)nDegradation phase降解n digestion of protein, fragmentation of DNATwo main apoptotic pathwaysnThe activation of death receptorsn 死亡受体途径nMitochondria pathwayn 线粒体途径n common pathway: n activation of caspase cascadeMajor Apoptotic Pathways in Mammalian CellsHengartner, M.O. 2000. Nature. 407:770

26、.Green, D. and Kroemer, G. 1998. Trends Cell Biol. 8:267.Mitochondrial PathwayDeath Receptor PathwayFasLCaspase 3DDDDFas/Apo1/CD95FADDProcaspase 8DISCCaspase 8BIDoxidants ceramideothersBcl-2DCytochrome cdATPProcaspase 9Apaf -1dATPApaf -1Caspase 9Procaspase 3apoptosomeDNA damageCellular targetsApopto

27、sisOxygen Society Education Program Tome & Briehl 3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，在细胞外表形成三聚体，细胞毒T细胞外表FADD：Fas-associated death domain DD and DED (death effector domain) DISC：FasLFasFADDFas and Related Proteins with Death DomainsnThe extrinsic or death receptor pathwaynInitiated

28、by binding of a death-inducing ligand to a Cys-rich repeat region in the extracellular domain of a death receptornDeath receptors such as Fas and the TNF receptor are integral membrane proteins with their receptor domains exposed at the surface of the cellnBinding of the complementary death activato

29、r (FasL and TNF-a, respectively) transmits a signal (via an adaptor protein) to the cytoplasm that leads tonactivation of caspase-8nCaspase-8 (like caspase-9) initiates a cascade of caspase activation leading to cell deathnExample: when cytotoxic T-cells recognize (bind to) their targets:nthey produ

30、ce more FasL at their surfacenthis binds with Fas on the surface of the target cells and starts the cascade that leads to its death by apoptosisMechanisms of ApoptosisnCell death receptorsnmembers of TNFR family, can have pleiotropic action depending on cell type and signals received i.e., can trigg

31、er cell proliferation, differentiation or deathnActivated by structurally-related ligands of the TNF ligand familyne.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 caspasesn4 TRAIL/APO-2L recept

32、ors identified 2 of them, DcR1 and DcR2 lack the death domain and cannot induce apoptosis acts as decoys to inhibit TRAIL/APO-2L-mediated apoptosisnDecoy receptor for FasL (DcR3) found overexpressed in lung and colon tumorsSchematic for death receptor TNF or Fas ligand interact with death receptor R

33、ecruitment of adaptor molecules (FADD) Activating caspase 8 directly activating caspase 3 and caspase 7 translocate to mit bcl-2 cyto C releaseFas Signaling Pathway TNFR-TNFa 凋亡途径nTNFR1单跨膜受体，分布广泛nTNFa由活化的巨噬细胞和淋巴细胞产生nTNFR1胞内DD募集TRADD, 后者与TRAF2和RIP形成复合物，nRIP活化NFkB，通过FLIP抑制caspase8活化 DISC 复合物复合物1FLIP:

34、Fas associated death domain-like interleukin beta FLIP: Fas associated death domain-like interleukin beta converting enzyme inhibitory proteinconverting enzyme inhibitory proteinTNF Signaling PathwayMitochondria pathway1. The stimuli leading to cell death (growth factor deprivation, ionizing radiati

35、on and several chemical agents)2. mitochondrial membrane permeabilization release of cytochrome C formation of apoptosome(Apaf-1,cyto C, dATP) apoptotic proteonase activating factor3. Activating caspase 9 by Apaf1 CARD caspase recruitment domain4. 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：