Download Pathophysiology: apoptosis

Apoptosis and Diseases
1. Concept
2. Apoptotic process and changes
3. Key molecules and Major pathways
4. Techniques to detect apoptosis
5. Apoptosis-related diseases
• Insufficient apoptosis in diseases
• Excessive apoptosis in diseases
• Coexistence of insufficient and
excessive apoptosis in diseases
6. 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）
Causes and Process of Apoptosis
Inhibitory Factors
Stimulatory Factors
Physiological:
GFs, estrogen, etc;
Pathological:
virus; chemicals, etc.
Physiological:
FasL;
Pathological:
glutamate, free radicals; therapeutics.
Initiation
Regulation
Conserved
Execution
Phagocytosis
Apoptotic changes
---Morphological changes in apoptosis
---Biochemical Changes in Apoptosis
Morphological changes in apoptosis





Cell membrane
Cytoplasm
Cell nucleus
Apoptotic body
Phagocytose
Normal Cell
condensation
margination
Budding
Changes of Cell membrane
Apoptotic Cell
Apoptotic Bodies
Apoptosis and Necrosis
Apoptosis
Necrosis
Nature
Physiological or
pathological; specific
Pathological, 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
Intact, shrinkage,
condensation
Lysis, swelling
Inflammation
No
Yes
Apoptotic body
Yes
No
Gene regulation
Yes
No
Morphology
Morphological differences
in apoptosis and necrosis
Biochemical Changes in Apoptosis

Caspase activation
Endonuclease
activation
Caspases:
(cysteine-containing aspartate-specific proteases)
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
Caspase functions and structure
Classification of Caspases
半胱天冬酶的特征
前功能域
长度
和基序
活性
亚单位
kDa
激活作用
接头蛋白
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
半胱天冬酶-8 (51)
FLICE, MACH, Mch5
长,DED
20//12
18//11
RAIDD
FADD
DXXD
(I/V/D)EXD
长,CARD
长,DED
17//10
17//12
APAF-1
FADD
(I/V/L)EHD
不明
短
短
短
17//12
18//11
20//12
不需
不需
不需
DEXD
(V/T/I)EXD
DEXD
酶原
(kDa)
半胱天冬酶-9 (45)
半胱天冬酶-10 (55)
别名
ICE-LAP 6, Mch 6
Mch4
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
半胱天冬酶-6 (34)
Mch2
半胱天冬酶-7 (35)
Mch3,ICE-LAP3, CMH-1
细胞因子加工因子 ICE样 半胱天冬酶-1 亚家族
ICE
半胱天冬酶-1 (45)
长,CARD
半胱天冬酶-4 (43)
长,CARD
ICErel-Ⅱ, TX, ICH-2
半胱天冬酶-5 (48)
ICErel-Ⅲ, TY
长
m半胱天冬酶-11(42)
长
m半胱天冬酶-12(50)
长
半胱天冬酶-13(43)
长
m半胱天冬酶-14(30)
短
无脊椎动物半胱天冬酶
CED-3 (56)
DCP-1b (36)
长,CARD
短
20//10
20//10
20//10
20//10
20//12
20//12
20//12
17//14
22//13
优先作用
四肽
序列a
?CARDIAK (W/Y/F)EHD
不明
(W/L/F)EHD
不明
(W/L/F)EHD
不明
不明
不明
不明
不明
不明
不需
不明
CED-4
不需
DEXD
不明
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
Signaling
activation
Endonuclease
Ca2+ Mg2+
H1
180-200 bp
Zn2+
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
促-和抗凋亡蛋白
凋亡机器成员
预计功能
信号放大 抑制因子
诱发凋亡表型
底物举例
前-半胱天冬酶, Bcl-2, Bcl-XL , Bid, p28Bap31
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)
核纤层蛋白，核有丝分裂器蛋白(nuclear mitotic
apparatus protein), 支架附着因子-A (scaffold
attachment 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),
结构蛋白和结合分子
细胞完整性解体
细胞包装
稳态蛋白
破坏大分子合成和细胞修
复机制
终止存活信号
DNA-依存性蛋白激酶酶促亚单位(DNA dependent
protein 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(GDP
dissociation inhibitor, GDI),转录因子
其它
不明
？凋亡诱导
Huntingtin蛋白,早老蛋白(presenilin), 萎缩素-1
(atrophin-1),ataxin-3, 胞浆磷脂酶A2
Regulators of Apoptosis
Bcl2 family proteins
IAP (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 genes
Death Receptor induced apoptosis
Mitochondria – Integrator of Apoptosis
ER 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 cells
B. CTL-mediated killing of target cells
C. 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△ψm
 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 “caspaseindependent 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 apoptosis
 Morphological
 DNA
studies
ladder
 TUNEL
 Flow cytometry
 Externalization of Phosphatidylserine
 Activation 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-mediated
cleavage and
activation
of scramblase
PKC activation
Inactivated aminophospholipid
translocase
Activation of Caspase 3 and Cleavage
of Its Substrates, PARP and D4-GDI