细胞凋亡线粒体途径的调控Regulation of mittochondrial

apoptotic pathways

高方远 马欣荣 康海岐

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 Smac

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

DISC

( 死亡诱导信号复合体 )

Death-signaling pathway

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.

Disassaciation of Bim

mit

cytosol

Bim

Microtubule

complexes

LC8

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

Two steps in the Bax apoptotic pathway

Bcl-2

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?