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 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 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 quiescent 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, the nucleoli disappear and two microtubule organizing centers begin polymerizing tubulins for eventual production of the 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 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

Why is dying so important?

Physiologically: embyro stage, CNS development, thymus atrophy, endometrium desquamatingPathologically: tumor, Parkinson’s disease, Alzheimer’s disease

Programmed Cell Death in Eukaryotes

Caenorhabditis elegans:The Perfect Model

C. elegans’s complexity but simplicity

A 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 Adult organism comprised of 959 cells During embryological development will form

1090 cells

Approximately 40 percent of the worm’s genes are also found in humans

Responds to taste, smell, temperature, touch, and possibly light

So, where did the other 131 cells go?

The The C. elegansC. elegans Organism Organism

The Fundamental Genes Being Examined

Egl-1 Ced9 Ced4 Ced3 apoptosis

EGL-1…initiates apoptosis by inhibiting the normal restraining action of CED-9 on CED-4

CED-3…triggered by CED-4 resulting in highly destructive proteases acting upon cell structure

CED-4…acted upon by EGL-1; required in cell death

CED-9… protects against cell death egl-1 egg laying defective-1 ced cell death abnormal

EGL-1…has multiple mammalian killer gene counterparts

CED-3…human counterparts are called caspases which initiate apoptosis; protein ICE

CED-4…human counterpart called Apaf1 which promotes caspase activation

CED-9…comparable to the human 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 caspase-mediated cleavage

Procaspase: N—prodomain---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 vital 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 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 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 familyBcl-2 family

Major 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 domain (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 stimuli – e.g. viral infection, chemotherapeutic drugs and components of the TNF-/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 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 all 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 into 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 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 pI

Other molecules of Apoptosis

Other molecules of Apoptosis

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, 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. 8:267.

Mitochondrial PathwayDeath Receptor Pathway

FasL

Caspase 3

DDD D

Fas/Apo1/CD95

FADD

Procaspase 8

DISC

Caspase 8

BID

oxidants ceramide others

Bcl-2D

Cytochrome c

dATP

Procaspase 9

Apaf -1

dATP

Apaf -1

Caspase 9

Procaspase 3

apoptosome

DNA damage

Cellular targets

Apoptosis Oxygen Society Education Program Tome & Briehl 3

DISC: death inducing signal complexDISC: death inducing signal complex

FADD: Fas associated protein with death domainFADD: 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 domain

DD and DED (death effector domain)

DISC ： FasL—Fas—FADD

Fas and Related Proteins with Death Domains

Death receptor: Fas, TNFR1, TNFR2, DR3, DR4, DR5, Death receptor: Fas, TNFR1, TNFR2, DR3, DR4, DR5,

DcR1,DcR2DcR1,DcR2

TRADD: TNF receptor-associated death domainTRADD: 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 death 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 leads 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 cascade 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 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 decoys 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 receptor

Recruitment of adaptor molecules (FADD)

Activating caspase 8

directly activating caspase 3 cleave Bid (tBid)cleave Bid (tBid) and caspase 7 translocate to mit bcl-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 1

FLIP: Fas associated death domain-like interleukin beta converting enzyme inhibitory protein

TNF 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 C formation of apoptosome(Apaf-1,cyto C, dATP) （ apoptotic proteonase activating factor ）3. Activating caspase 9 by Apaf1 CARD （ caspase recruitment domain ）

4. Activating caspase-3, -7,-6, cleave 45KD subunit of the DFF

5. 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 CytC

1.Bax 和 Bak 在 mit外膜打孔 2.VDAC在外膜形成通道 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 release 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 and 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