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Execution of mitochondrial apoptosis signals
Release of cytochrome c Release of Smac Release of apoptosis-inducing factor Release of endonuclease G
Release of cytochrome c
Cyto c Apaf--1 Cas-9
Cas-3Cleave intracelluar
substrates
Chr 浓缩
DNA 分解
核膜裂解等
+dATP/ATP
Release of apoptosis-inducing
Factor(AIF)
57KD 黄素蛋白 AIF
(Mit) NucleusChr 浓缩
DNA 降解
Independent of caspase activation and oxidoreductases activity of AIF
Release of endonuclease G
EndoG release
DNA fragment
apoptosis
Independent of caspase activation
Bcl-2 family
Features of mitochondria-initiated apoptosis
Multiple factors function to trigger cell death in conjunction and in parallel ways.
the pathway is able to feed-forward and amplified the apoptotic signal. Even when caspase-dependent and caspase-independent pathway cannot function properly, mitochondrial dysfunction may lead to cell death.
The best way to prevent cell death is to block apoptotic signals before mitochondrial damage occurs.
Features of mitochondria-
initiated apoptosis
1. Disruption of electron transport and energy metabolism.
2. Release of caspase-activating proteins.
3. Alteration of cellular reduction-oxidation potential
Three general mechanisms
Regulation of mittochondrial
apoptotic pathways
Regulation of mitochondrial apoptotic signals.
Mitchondrial response to apoptotic signals.
Loss of mitchondrial functions during apoptosis.
Regulation of mitochondrial apoptotic signals
Tanslocation of the BH3-only family of proteins to mitochondrail Cleavage of Bid Phosphorylation of Bad Disassaciation of Bim Transcriptional regulation of BH3-only proteins Tanslocation of other proteins to mitochondra during apoptosis
Tanslocation of the BH3-only family
of proteins to mitochondrail
Other cellular compartments
mit
BH3-only proteins
(Bid, Bad, Bim, Noxa, Puma)
Bcl-2 famillyCause mit damage
Release cyto c, Smac, AIF,EndoG etc.
Cleavage of Bid
Cell surface death receptors
Fas/CD95
TNFR1
DR-3,4
TRAIL-R1,DR5
TRAIL-R2
FasL
TNF
Apo3L
Apo2L/TRAIL
Receptors
ligand
extra
cytoplasmic
Cys-rich
Death domain tail
Type I cell Type II cell
(肝细胞)
Cas-8Bcl-2
apoptosis Bid
tBid
Amlified signals
enough Not enough
cleave
translocation
Cleavage of Bid
mit
mit mit
Bid (cytocolic
In living cells )tBid(cyto)
tBid(mit)
Cyto C
realease
Domain: helicese 4-6
Specificity: mit-specific lipid, casdiolipin( 心磷脂 )
Efficiency: enhanced by myristorylation ( 豆蔻酰化 ) of N-terminal glycine residue of tBid exposed after cas-8 cleavage
Cas-8 cleave
signals
Fas/FasL
apoptosis
Other regulation pathway
Other cas (cas-3) cas-8
And protases
( 粒酶 B, Bid (cyto) other BH3-only proteins
溶原体蛋白酶 ) (inactive Bad)
tBid
active Bad
tBid (mit)
rease
apoptosis
Phosphorylation of Bad
apoptosis
Bad (In cytosol)
14-3-3 pro
Bcl-2(active)
inactive
- Survival signals
+ trophic factors
(In cytosol)
(In mit)
Akt, PKA
The critical site of phosphrylation By survival factors
BH3 domain of Bad
Ser112 Ser136 Ser155
apoptosis
Ser155 kinases
Electrostatic (静电)
and 空间限制
Ser112 136 155
Bcl-xl
14-3-3
p70S6 kinase 纳巴霉素
Phosphatases (In vitro)
Bad-p dephosphorulated Bad
Calcineurin( 钙调磷酸酶 )
Protein phosphatase 1a
Protein phosphatase 2A
Protein phosphatase 1a is a Ras-activated Bad phosphatase that regulates interleukin-2 deprivation-induced apoptosis
EMBO J. 19:2237-2246
Materials: interleukin-2 (IL-2)-dependent murine T-cell line Methods:Using the yeast two-hybrid system, glutathione S-transferase (GST) fusion proteins and co-immunoprecipitation techniques,Results: Bad interacts with protein phosphatase 1a (PP1 a ).
Bad+IL-2
Activated kinases
Akt, Psk, PKA etc.
Bad phosphorylation
- IL-2
PP1a Ras
Bad dephosphorylation
Akadaic acid
( 冈田酸 )
apoptosis
How these phosphatases are regulated in vivo by apoptotic signals remains to be invested??
Interaction of Bad and PP1 phosphatase.
Interaction of Bad and the catalytic subunit of PP1 phosphatase in the two-hybrid system.
Bim-deficient lymphocytes are resistant to certain apoptotic stimuli.
Cytokine deprivation (细胞因子缺失)
Calcium ion flux (钙离子流出)
Microtubule pertubation( 微管干扰 )
Abundance of BimBe regulated at the level of transcription
?
Transcriptional regulation of BH3-only proteins
Apoptosis requires new proteins synthesis
transcriptional regulation of BH3-only protein may be important for apoptosis
newly generated protein mitDirectly target
AKT
FKHR-L1( 叉头转录因子 )
Bim transcription
Neuron and hematopoietic
progenitors( 造血干细胞 ) - Cytokine
显性失活的 C-Jun
HRK transcription
①
②
-NGF ( In neurons)③
④
apoptosis
X-irradiation DNA damag
e
P53
Noxa, Puma
(BH3-only member)
Transcriptional regulation of BH3-only protein play a major role in DNA damage apoptosis
BH3 domain
triggerCytochrome c release
Caspases activation
Noxa, a BH3-Only Member of the Bcl-2 Family and Candidate Mediator of p53-Induced Apoptosis 2000, science 288:1053
Tanslocation of other proteins to
mitochondra during apoptosis
mit
P53 TR3 P53AIP
LKB1
Mit damage
apoptosis
Cyto c etc.
? ? ? ?
. Targeting p53 to mitochondria of p53-deficient cells is sufficient t
o induce apoptosis.
Role of mitochondrial p53 in regulating apoptosis.
Cytochrome c Release and Apoptosis Induced by Mitochondrial Targeting of Nuclear Orphan Receptor TR3
2000 , science 289:1159-1164.
Mitochondrial targeting of TR3 regulates mitochondrial activity.
(A) TR3 expression is required for cytochrome c release in response to apoptosis inducers. (B) Mitochondrial targeting of TR3 is associated with cytochrome c release. (C) TR3/ 1 blocks cytoplasmic localization of TR3. (D) TR3/ 1 inhibits MM11453-induced apoptosis. Quantification of apoptotic cells in 400 TR3/ 1-transfected or nontransfected cells is shown in (E).
Mitchondrial response to apoptotic signals
Bax and Bak
Bcl-2 and other antiapoptotic members
VDAC and ANT
Bax and Bak
Bax and Bak double KO mice
most
Die during embryonic development
few
A persistence of 指间蹼
An imperforate vaginal canal
An accumulation of excess cells in nervous and 造血系统
Resistant to multiple apoptotic stimuli
( overexpression of tBid, Bim and Bad )
BH3-only proteins
Bax and Bak (mit)
Conformayional changes and oligomerization(Bax and Bak)
Form a big pore
Apoptogenic proteins(cytochrome c, Smac, EndoG )pass through
?
Destabilize the mitchondrial outer membrane
Bcl-2 and other antiapoptotic members
BH3-only proteins
Bax and Bak
oligomerization
X Bcl-2, Bcl-xL
Bcl-2, Bcl-xL
apoptosis
VDAC and ANT
VDAC and ANT
(outer and inner membranes of mit)
Bax+
VDAC-Bax ANT-Bax
Change of VDAC permeability
apoptosis
Form protein pore
ATP/ADP exchage
Small metabolites and nucleotides across
Export 肌酸磷酸
- cytokine
?Cyto c release
Loss of mitchondrial functions
during apoptosis
Suffer special damage (mit)
Apoptotic stimuli
Loss of mit function
apoptosis
Loss of mitochondrial function
Release of cyto c
Electron transfer
Maintain membrane potential
ATP synthesis
BH3-only proteins
mit ADP 和内膜肌 酸磷酸脂 浓度 ATP
Overexpression of Bcl-xL
tBidAbolish the ability to buffer cacium, important mit function for cellular homeostasis
+cyto c in early stage
Cyto c release
apoptosis
+ caspases
activation
AIF, EndoG release
- caspases
activation
ATP synthesis interruption
Mit dysfunction
代谢变化( 1 ) Mit matrix alkalinization ;
2 )谷胱肝肽 -S 转移酶; 果糖 1 , 6- 二磷酸;脂肪酸结合蛋白; UPC-2 ; VDAC 活性
perspectives
How are the signals from either developmental cues or damage signals transduced to and integratedin the mit? Are the BH3-only proteins the major signal transducers? Or are they only part of a more complicated network of proteins?