Apoptosis and Diseases

1. Concept2. Apoptotic process and changes3. Key molecules and Major pathways4. Techniques to detect apoptosis

5. Apoptosis-related diseases• Insufficient apoptosis in diseases• Excessive apoptosis in diseases• Coexistence of insufficient and

excessive apoptosis in diseases6. Principles of treatment

What is Apoptosis ? Apoptosis refers to the process in which the dying

procedures that have been in advance deposited in cell are triggered by various causes from in vitro and in vivo, and eventually cause cell death.

Programmed cell death（ PCD ）

Initiation

Regulation

Execution

Phagocytosis

Physiological: GFs, estrogen, etc;Pathological: virus; chemicals, etc.

Inhibitory Factors Stimulatory FactorsPhysiological: FasL;Pathological: glutamate, free radicals; therapeutics.

Conserved

Causes and Process of Apoptosis

Apoptotic changes ---Morphological changes in apoptosis

---Biochemical Changes in Apoptosis

Morphological changes in apoptosis Cell membrane Cytoplasm Cell nucleus Apoptotic body Phagocytose Normal Cell

Apoptotic Cell

condensation

margination

Apoptotic BodiesBudding

Changes of Cell membrane

Apoptosis NecrosisNature Physiological or

pathological; specificPathological, accidental

Stimulus Mild Strong

Biochemistry Active, energy-dependent, new protein synthesis

Passive, energy-independent, no protein synthesis

DNA

Specific degradation, ladder (180-200 bp)

Random degradation

Morphology Intact, shrinkage, condensation

Lysis, swelling

Inflammation No Yes

Apoptotic body Yes No

Gene regulation Yes No

Apoptosis and Necrosis

Morphological differences in apoptosis and necrosis

Biochemical Changes in Apoptosis

Caspase activationEndonuclease activation

Most apoptotic proteolytic cleavage results from the action of caspases

Caspases are activated by proteolytic cleavage

Removal of prodomain and linker region

Assembly of the large and small subunits into an active enzyme complex

Two heterodimers interacting via the small subunits to form a tetramer with two catalytic sites

Family members>14

Caspases: (cysteine-containing aspartate-specific

proteases)

Caspase functions and structure

Classification of Caspases

半胱天冬酶的特征前功能域 活性 优先作用

酶原 别名 长度 亚单位 四肽(kDa) 和基序 kDa 序列

a

凋亡启动因子 ICH-1样 半胱天冬酶-2 亚家族 半胱天冬酶-2 (51) ICH-1 长,CARD 20//12 RAIDD DXXD半胱天冬酶-8 (51) FLICE, MACH, Mch5 长,DED 18//11 FADD (I/V/D)EXD

半胱天冬酶-9 (45) ICE-LAP 6, Mch 6 长,CARD 17//10 APAF-1 (I/V/L)EHD 半胱天冬酶-10 (55) Mch4 长,DED 17//12 FADD 不明

凋亡执行因子 CPP32样 半胱天冬酶-3 亚家族 半胱天冬酶-3 (32) CPP32,Yama, apopain 短 17//12 不需 DEXD 半胱天冬酶-6 (34) Mch2 短 18//11 不需 (V/T/I)EXD 半胱天冬酶-7 (35) Mch3,ICE-LAP3, CMH-1 短 20//12 不需 DEXD

细胞因子加工因子 ICE样 半胱天冬酶-1 亚家族 半胱天冬酶-1 (45) ICE 长,CARD 20//10 ?CARDIAK (W/Y/F)EHD 半胱天冬酶-4 (43) ICErel-Ⅱ , TX, ICH-2 长,CARD 20//10 不明 (W/L/F)EHD 半胱天冬酶-5 (48) ICErel-Ⅲ , TY 长 20//10 不明 (W/L/F)EHD m半胱天冬酶-11(42) 长 20//10 不明 不明 m半胱天冬酶-12(50) 长 20//12 不明 不明 半胱天冬酶-13(43) 长 20//12 不明 不明 m半胱天冬酶-14(30) 短 20//12 不需 不明无脊椎动物半胱天冬酶 CED-3 (56) 长,CARD 17//14 CED-4 DEXD

DCP-1b (36) 短 22//13 不需 不明

激活作用接头蛋白

Caspase-deficient mice Knockout Phenotype

Caspase-1 Viable; impaired processing of IL-1; resistant to endotoxic shock. Caspase-2 Viable; excess numbers of female germ cells; oocytes resistant to chemotherapeutic drugs; B lymphoblasts resistant to granzyme B; accelerated death of facial neurons during development and of sympathetic neurons deprived of NGF.Caspase-3 Lethality at 3–5 weeks of age; defective neuronal apoptosis; T cells resistant to antigen-induced death; abnormal apoptotic morphology in dying cells.Caspase-8 Lethality around E12.5; hyperemia and abnormal heart muscle development; MEFs resistant to TNF, Fas and DR3 but sensitive to UV irradiation, etoposide, staurosporine, serum deprivation.Caspase-9 Perinatal lethal; impaired neuronal apoptosis; ES cells, MEFs and thymocytes generally resistant to intrinsic death stimuli such as DNA damage, though resistance depends on cell type.Caspase-11 Viable; impaired processing of caspase-1, IL-1; resistant to endotoxic shock. Caspase-12 Viable; embryonic fibroblasts are resistant to ER stress.

Caspases activation •Death receptor pathway: caspase 8•Mitochondrial pathway: caspase 9•ER stress pathway: caspase 12

(CAD: caspase-activated deoxyribonulease)

Caspase substrates:•ICAD： DNA 降解•PARP ：破坏 DNA 修复•破坏细胞骨架： gelsolin, laminA •灭活凋亡抑制蛋白： Bcl-2• etc

Cleavage of death substrates

Role of Endonuclease:degrade DNA

180-200 bp

H1 ZnZn2+CaCa2+ MgMg2+

Endonuclease

Signaling activation

Apoptotic substrates

( DNA-PKCS, DNA protein kinase catalytic subunit; HnRNP, heteronuclear ribonucleoproteins; ICAD,inhibitor caspase activated deoxyribonuclease; FAK,focal adhesion kinase; GAS,growth arrest specific gene-2; GDI, GDP dissociation inhibitor; NuMA,nuclear mitotic apparatus; PAK,p21 activated kinase;PARP, poly(ADP-ribose) polymerase; cPLA2, cytoplasmic phospholipase A2; RFC-140, replication factor C; SAF-A,scaffold attachment factor-A; U1-70kDa, U1-specific 70-kDa protein; )

凋亡性底物 底物类别 预计功能 底物举例

信号放大 抑制因子半胱天冬酶激活脱氧核糖核酸酶抑制因子(ICAD),肌动蛋白,胶溶蛋白(gelsolin), p21激活的激酶-2(PAK2),丝裂原激活的蛋白激酶/细胞外信号调节激酶的激酶-1 (MEKK1),蛋白激酶Cδ (PKCδ )

细胞完整性解体细胞包装

破坏大分子合成和细胞修复机制终止存活信号

不明？凋亡诱导

促-和抗凋亡蛋白 前-半胱天冬酶, Bcl-2, Bcl-XL, Bid, p28Bap31凋亡机器成员 诱发凋亡表型

其它 Huntingtin蛋白,早老蛋白(presenilin), 萎缩素-1(atrophin-1),ataxin-3, 胞浆磷脂酶A2

结构蛋白和结合分子 核纤层蛋白，核有丝分裂器蛋白(nuclear mitoticapparatus protein), 支架附着因子-A (scaffoldattachment factor-A),胞衬蛋白(fodrin),Gas2[生成停顿特异基因-2(growth arrest specific gene-2)编码蛋白],角蛋白,肌动蛋白,Rabaptin-5,β -连环蛋白,粘着斑激酶(focal adhesion kinase, FAK),

稳态蛋白 DNA-依存性蛋白激酶酶促亚单位(DNA dependentprotein kinase catalytic subunit DNA-PKcs),多聚(ADP-核糖)聚合酶[poly(ADP-ribose)polymerase,PARP],U1-70-kDa蛋白,复制因子C(replication factor C,RFC-140),不均一核核糖核蛋白(HnRNP),D4-GDP解离抑制因子(GDPdissociation inhibitor, GDI),转录因子

Regulators of Apoptosis

Bcl2 family proteinsIAP (Inhibitors of Apoptosis Proteins)

Bcl2 family: killers and protectors Two groups (>15 members) to keep the balance between

apoptosis and survival:---Suppressors of apoptosis: Bcl2, BclXL, BclW, Bag1,

Mcl1, A1, etc---Activators of apoptosis: Bax, Bok, Hrk, Bnip3, Bim,

Bik, BclXs, Bik, Blk, Bid, Bak, Bad, etc.

Forms heterodimers

On the cytoplasmic face of the outer mitochondrial membrane, endoplasmic reticulum, and nuclear envelope

In hematopoietic cell, epithelial cell, lymphocyte, nerve cell, and various cancer cells

Bcl-2: Regulate the release of pro-apoptotic molecules from mitochondria

Structure of Bcl-2 family TM: transmembrane region; BH: Bcl-2 homology

TM

Bax:Apoptotic stimuli induce translocation of Bax from cytosol to mitochondria

create pores in the outer membrane of mitochondria of sufficient size to allow cytochrome C to escape

IAPs---family members: c-IAP1,c-IAP2,XIAP,NAIP,survivin;---preventing some procaspases activation, or inhibiting caspase activity.

Apoptosis pathways and related genesDeath Receptor induced apoptosisMitochondria – Integrator of ApoptosisER stress pathway Others

Death Factor and Death Receptor Family

Death receptor induced apoptosis

Fas ( factor associated suicide):

Homologous cytoplasmic domain: death domain (DD)

Interacts with each other through DD

Anti-apoptotic pathway: NF-kB pathway

TNF rarely induces apoptosis unless protein synthesis is inhibited

Decoy receptors

Death Receptor Signaling

Apoptosis signaling by CD95, TNFR1, and DR3

Apoptosis signaling by DR4 and DR5 and its modulation by decoy receptors

Three Types of Killing by the Fas and FasL System

A. Activation-induced suicide of T cellsB. CTL-mediated killing of target cellsC. Killing of inflammatory cells in immune

privilege sites and killing of CTL by tumor cells

Mitochondria – Integrator of Apoptosis

Current models of the intracellular pathways leading to trophic factor mediated cell survival in mammalian cells

Current models of the intracellular pathways leading to apoptosis induced by withdrawal of trophic factor

ER and Apoptosis

Cross-talking among Organelles and Molecules in Apoptosis

p53 Mediated Apoptosis

p53-Inducible Apoptosis Related Genes Scotin: localized to the ER and the nuclear membrane

PERP： similarity to PMP-22/gas3 tetraspan membrane

protein NOXA: A member of Bcl-2 family BAX KILLERS/DR5 FAS P53AIP1: p53-regulated apoptosis-inducing protein 1, leads

to apoptosis via dissipation of mitochondrial ψ△ m PIDD: A new death domain containing protein PIG: P53 induced genes,related to ROS production IGFBP

The Mammalian DNA Damage Checkpoint

Four patterns of death: from apoptosis to necrosis

Apoptosis is observed almost exclusively when caspases, in particular caspase-3, are activated.

Apoptosis-like PCD chromatin condensation less compact without other apoptotic features “caspase-independent apoptosis”

Necrosis-like PCD no chromatin condensation with chromatin clustering to speckles. Usually involves specialized caspase-independent signalling pathways. “aborted apoptosis”

Accidental necrosis/cell lysis associated with cellular oedema (organelle swelling) and devoid of zeiosis

Techniques to detect apoptosisMorphological studiesDNA ladderTUNELFlow cytometryExternalization of PhosphatidylserineActivation of caspases and cleavage of

their substrates

Ultrastructural feathers of Normal and Apoptotic Cell

Induced Apoptosis of Cultured Rat Hepatocytes

DNA Ladder Pattern Seen in Diospyrin diethyl ether Induced Apoptotic Cell

Fragmented DNA can be labeled by Terminal deoxynucleotidyl transferase (TdT) mediated deoxyUridine Nucleotide (dUTP)-End Labeling (TUNEL)

Flowcytometric Analysis of Cellular DNA Content

Externalization of Phosphatidylserine

Phosphatidylserine on the surface of apoptotic cells[stained with annexin V (green)]

Due to: Caspase-3-mediatedcleavage and activationof scramblase

PKC activation

Inactivated amino- phospholipidtranslocase

Activation of Caspase 3 and Cleavage of Its Substrates, PARP and D4-GDI