医学课件细胞增生和凋亡的分子机制

1、 细胞增生和凋亡的分子机制 Fate of cells Undergoing cell cycle proliferationdifferentiating to specific cellDeath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80年代

2、在线虫首次说明2002年诺贝尔医学和生理学奖Nobel Prize for Physiology and Medicine 2002For “genetic regulation of organ development and programmed cell deathSydney 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亡细胞凋亡的意义去除错误细胞

3、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 cells prepare for and then undergo cytokinesis. During mitosis the chromosomes are paired and then divided prior to cell

4、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 as being in G0 phase

5、. 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 eventual production of t

6、he spindle poles. 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活化G2M checkpointAPC介导的多

7、泛素化蛋白降解有丝分裂中期checkpointDNA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 desquamatingPathologically: tumor, Parkinsons disease, Alzheimers diseaseProgrammed Cell Death in Euk

8、aryotesCaenorhabditis elegans:The Perfect Model C. eleganss complexity but simplicityA nematode approximately one mm long containing blood, muscle, heart, nervous, as well as other tissuesFrom fertilization to adult in three daysLife span of two to three weeksAdult organism comprised of 959 cellsDur

9、ing embryological development will form 1090 cells Approximately 40 percent of the worms genes are also found in humansResponds to taste, smell, temperature, touch, and possibly lightSo, where did the other 131 cells go?The C. elegans OrganismThe Fundamental Genes Being ExaminedEgl-1 Ced9 Ced4 Ced3

10、apoptosisEGL-1initiates apoptosis by inhibiting the normal restraining action of CED-9 on CED-4CED-3triggered by CED-4 resulting in highly destructive proteases acting upon cell structureCED-4acted upon by EGL-1; required in cell deathCED-9 protects against cell death egl-1 egg laying defective-1 ce

11、d cell death abnormalEGL-1has multiple mammalian killer gene counterpartsCED-3human counterparts are called caspases which initiate apoptosis; protein ICECED-4human counterpart called Apaf1 which promotes caspase activationCED-9comparable to the human oncogene BCL-2 which blocks cell suicideMajor Pl

12、ayers in Apoptosis-caspaseCaspasesCysteine proteasesRecognize 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 caspase-mediated cleavage Procaspase: Nprodomain-p20

13、-p10 domain-C Initiator caspases (e.g. caspase-8 and caspase-9) start a cascade of increasing caspase activity by processing andactivating downstream effector caspases (e.g. caspase-3, -6 and -7) activated effector caspases cleave and inactivate vital cellular proteins and induces morphological chan

14、ges that are characteristic of cells undergoing apoptosisPlays 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 spaceAt least 20 Bcl-2 related proteins identified in

15、 mammalian cellsBcl-2 family members share one or more Bcl-2 homology (BH) domains and are divided into two main groups whether they promote or inhibit apoptosisAnti-apoptotic members such as Bcl-xL, Bcl-w and Boo/Diva share at least three or four regions of extensive amino acid sequence similarity

16、with the prototypical Bcl-2 (BH1 BH4 regions)Pro-apoptotic members usually posses only a BH3 region e.g. Bad, Bik/Nbk/Blk, and BidBax-Bak examples of pro-apoptotic multidomain proteinsMajor Players in Apoptosis-Bcl-2 familyBcl-2 familyMajor Players in Apoptosis-adaptor proteinForm bridges between ce

17、ll 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 domain (DED) of the adaptor.e.g. the DD of the CD95 effector is as

18、sociated 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 activationSuppress apoptosis triggered by wide variety of stimuli e.g. viral infection, chemotherapeutic drugs and components

19、 of the TNF-a/Fas signaling pathwayCharacterized 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 SurvivinMost of IAP family members have been shown to interact with caspases, inhi

20、biting their activityPlay 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 all human tumors but is rarely present in adult tissuesMajor Players in

21、Apoptosis-IAPApoptosis-inducing factor (AIF)Flavoprotein that is normally located in the intermembrane space of mitochondria. When cells receive a signal for apoptosisAIF is released from the mitochondria AIF translocates into the nucleus and causesnuclear fragmentation and cell deathDNA destruction

22、 mediated by AIF is not blocked by caspase inhibitors and is thus considered a caspase-independent pathwayOther molecules of ApoptosisSmac: The second mitochondria-derived activator of caspase, 239aa, N-terminal 55aa as mitochondria signal. It normally resident in mitochondria but is released into t

23、he cytosol when cell undergo apoptosis. Mechanism: binding to IAP Smac: second mitochondria-derived ativator of caspase DIABLO: direct IAP-binding protein with low pIOther molecules of ApoptosisOther molecules of ApoptosisOmi: most recently discovered proapoptotic protein released from mitochondria

24、and shows much similarity to Smac.Cell death process three phasesInduction or initiation phase 起始Effector or decision phase效应 activating hydrolase (protease and nuclease)Degradation phase降解 digestion of protein, fragmentation of DNATwo main apoptotic pathwaysThe activation of death receptors 死亡受体途径M

25、itochondria pathway 线粒体途径 common pathway: activation of caspase cascadeMajor Apoptotic Pathways in Mammalian CellsHengartner, M.O. 2000. Nature. 407:770.Green, D. and Kroemer, G. 1998. Trends Cell Biol. 8:267.Mitochondrial PathwayDeath Receptor PathwayFasLCaspase 3DDDDFas/Apo1/CD95FADDProcaspase 8DI

26、SCCaspase 8BIDoxidantsceramideothersBcl-2DCytochrome cdATPProcaspase 9Apaf -1dATPApaf -1Caspase 9Procaspase 3apoptosomeDNA damageCellular targetsApoptosisOxygen Society Education Program Tome & Briehl 3DISC: death inducing signal complexFADD: Fas associated protein with death domain外源性的死亡受体途径Fas：单跨膜

27、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 DomainsDeath receptor: Fas, TNFR1, TNFR2, DR3, DR4, DR5, DcR1,DcR2TRADD: TNF receptor-associated death domainThe extrinsic or deat

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

29、mplementary death activator (FasL and TNF-a, respectively) transmits a signal (via an adaptor protein) to the cytoplasm that leads toactivation of caspase-8Caspase-8 (like caspase-9) initiates a cascade of caspase activation leading to cell deathExample: when cytotoxic T-cells recognize (bind to) th

30、eir targets:they produce more FasL at their surfacethis binds with Fas on the surface of the target cells and starts the cascade that leads to its death by apoptosisMechanisms of ApoptosisCell death receptorsmembers of TNFR family, can have pleiotropic action depending on cell type and signals recei

31、ved i.e., can trigger cell proliferation, differentiation or deathActivated by structurally-related ligands of the TNF ligand familye.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 caspases4 TRA

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

33、eath receptor Recruitment of adaptor molecules (FADD) Activating caspase 8 directly activating caspase 3 cleave Bid (tBid) and caspase 7 translocate to mit bcl-2 cyto C releaseFas 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

34、caspase8活化 DISC 复合物1FLIP: Fas associated death domain-like interleukin beta converting enzyme inhibitory proteinTNF Signaling PathwayMitochondria pathway1. The stimuli leading to cell death (growth factor deprivation, ionizing radiation 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 nucleas