shock & signal transduction

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Southern MedicalUniversity SHOCK & SIGNAL TRANSDUCTION YONG JIANG Key Laboratory of Proteomics of Guangdong Province, Southern Medical University

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SHOCK & SIGNAL TRANSDUCTION. YONG JIANG Key Laboratory of Proteomics of Guangdong Province, Southern Medical University. 炎症( inflammation ) 是高等动物对损伤性刺激的一种防御反应 。 是伴随多种疾病或病理过程的一种共有的病理现象,涉及感染、创伤、高温、辐射损伤、动脉粥样硬化、缺血 / 再灌流、休克、自身免疫性疾病、支气管哮喘、糖尿病、老年性痴呆( Alzheimer's disease )等。 - PowerPoint PPT Presentation

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Page 1: SHOCK &  SIGNAL TRANSDUCTION

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SHOCK & SIGNAL TRANSDUCTION

YONG JIANG

Key Laboratory of Proteomics of Guangdong Province, Southern Medical University

Page 2: SHOCK &  SIGNAL TRANSDUCTION

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y炎症( inflammation )是高等动物对损伤性刺激的一种防御反应。是伴随多种疾病或病理过程的一种共有的病理现象,涉及感染、创伤、高温、辐射损伤、动脉粥样硬化、缺血 / 再灌流、休克、自身免疫性疾病、支气管哮喘、糖尿病、老年性痴呆( Alzheimer's disease )等。

早在 1 世纪, Celsius 总结出炎症反应的共同特征:●红 (Rubor)---Redness

●肿 (Tumor)---Swelling

●热 (Calor)---Heat

●痛 (Dolor)---Pain

●功能丧失 (Functio laesa)---Loss of function

Page 3: SHOCK &  SIGNAL TRANSDUCTION

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y 炎症反应研究取得的重要进展 :● 微循环在炎症反应中的作用;● 细胞因子( cytokines )的生成和调控作用;● 补体( complement )和激肽( kinin )的激活和作用;● 白细胞的激活和趋化因子( chemokine )的反应;● 白细胞和内皮细胞粘附分子( adhesion molecules )的

鉴定和功能研究;● 前列腺素( prostaglandins )和白三烯( leukotriene

s )生成机制的研究 ;● NSAIDs ( nonsteroidal anti-inflammatory drugs )的合

成和临床应用;● NO ( nitricoxide )调节血管紧张性的发现及其机制的

研究;● 炎症反应的免疫调控,如抗原呈递( antigen presentati

on )

Page 4: SHOCK &  SIGNAL TRANSDUCTION

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y 炎症反应研究的焦点问题 :

致炎刺激( proinflammatory stimuli)致炎刺激( proinflammatory stimuli)

细胞内信号转导系统细胞内信号转导系统

细胞炎症反应细胞炎症反应

Page 5: SHOCK &  SIGNAL TRANSDUCTION

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MAPK—哺乳动物细胞功能调控的重要信号系统 :

● 1991 年:首先在哺乳动物细胞鉴定出 ERK (extracellular-signal regulated protein kinase) 。

● 1993 年: JNK(c-Jun amino-terminal kinase)/SAPK ( stress-activated protein kinase )

● 1994 年: p38/RK/CSBP

● 1995 年: ERK5/BMK1(big MAP kinase 1)

Page 6: SHOCK &  SIGNAL TRANSDUCTION

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y MAPK 通路的细胞内信号转导过程

MKKK

MKK

MAPK

MEK1/2

ERK1/2

MEKK1

MKK4

Raf1

JNKs

应激

炎症反应生长 凋亡

生长信号

ASK1/MLK

MKK3/6

p38s

LPS

MEK5

ERK5

?

H2O2

? 细胞反应

刺激

Page 7: SHOCK &  SIGNAL TRANSDUCTION

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y MAPK的作用底物 :

转录因子: ATF2、 c-Jun、 Chop10、

MEF2C、 ELK1

胞内蛋白激酶:MAPKAPK2/3、MNK1/2、

PRAK、MSK1/2骨架相关蛋白:MAPs、 Tau、 sHSP

离子通道蛋白:

Page 8: SHOCK &  SIGNAL TRANSDUCTION

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y Cellular response mediated by MAP kinases

MAPkinasesMAPkinases

ProliferationProliferation

DifferentiationDifferentiation

StressresponseStressresponse ApoptosisApoptosis

CellcycleCellcycle

InflammationInflammation

SurvivalSurvival

DevelopmentDevelopment

Page 9: SHOCK &  SIGNAL TRANSDUCTION

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y 炎症反应过程中的二次打击 :

原始损伤

细菌毒素

高温

辐射

病毒

细胞因子

粘附蛋白

NOS

二次打击

MAPK 通路

Page 10: SHOCK &  SIGNAL TRANSDUCTION

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MAPK

MKK

Substrates

Loop-12 is a key structure to determine the selection of substrate

(...TXY...)---

Page 11: SHOCK &  SIGNAL TRANSDUCTION

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1. 发现和克隆了三个新的 p38 亚型和三个新的 p38上游激酶

Page 12: SHOCK &  SIGNAL TRANSDUCTION

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JNK2

JNK3

JNK1

p38

p38p38p38ERK1

ERK2

ERK5

The relationship between

the members of MAPK family

Page 13: SHOCK &  SIGNAL TRANSDUCTION

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He

art

Bra

in

Pla

ce

nta

Lu

ng

Liv

er

Skel

etal

Mus

cle

Kid

ney

Pa

nc

rea

s

p38

p38

p38

p38

kb

3.5-

2.5-

1.8-

2.0-

Tissue distribution of mRNA of p38 isoforms

Page 14: SHOCK &  SIGNAL TRANSDUCTION

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p38

p38

p38

p38

38-

39-

38-

39-

Ju

rke

t

293

He

pG

2

U9

37

He

la

kDa

The protein expression of p38 isoforms

Page 15: SHOCK &  SIGNAL TRANSDUCTION

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77

154

308

400

500

700

MBP

p38

MBP

p38

77

154

308

400

500

700Osmolarity

(mOsmol/L)

A B

Activation of p38 and p38 by different osmolarity medium

Page 16: SHOCK &  SIGNAL TRANSDUCTION

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Co

ntr

ol

An

iso

.

Ars

enit

e

TN

F-80kDa

-47kDa-39kDa

-80kDa

-47kDa

kDa97.4-68.0-43.9-

29.0-

18.4-

14.3-C

on

tro

l

An

iso

.

Ars

enit

e

TN

F

A B

The major kinases of p38 and p38

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2. 鉴定了受 p38特异性调控的蛋白激酶 PRAK

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y The ancestral relationships among PRThe ancestral relationships among PRAK related kinasesAK related kinases

45 40 35 30 25 20 15 10 5 0

47.9

Rsk1

Rsk3

Rsk2

Mnk1

Mnk2

Mapkapk2

Mapkapk3

PRAK

Page 19: SHOCK &  SIGNAL TRANSDUCTION

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293

A5

49

Hep

G2

He

la

Jark

etB

-54kD

he

art

bra

in

pla

ce

nta

lun

g

live

r

skel

etal

mus

cle

kid

ne

y

pa

nc

rea

s

A

-2.4kb

-4.4kb-7.5kb-9.5kb

-1.5kb

The distribution of PRAK

Page 20: SHOCK &  SIGNAL TRANSDUCTION

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y Activation of endogenous PRAK Activation of endogenous PRAK by various stimuli by various stimuli in vivoin vivo

HSP27

Ca

2+ Io

no

pho

re

Co

ntr

ol

PM

A

EG

F

IL-6

TN

F

H2O

2

An

iso

.

Ars

enite

1.0 4.0 2.5 1.0 0.88 8.4 10.6 20.8 18.7 Fold

Page 21: SHOCK &  SIGNAL TRANSDUCTION

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yEffect of individual MAPK pathways on PREffect of individual MAPK pathways on PRAK activity in intact cellsAK activity in intact cells

MK

K7

(D)

GST-ATF2GST-ELK1

MK

K1

(E)

MK

K6

(E)

Co

ntr

ol

HSP27

PRAK

Page 22: SHOCK &  SIGNAL TRANSDUCTION

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y Phosphopeptide map of HSP27 phosphoryPhosphopeptide map of HSP27 phosphorylated by PRAK lated by PRAK in vitroin vitro

Ch

rom

ato

gra

ph

y

Electrophoresis pH1.9

+

MAPKAPK2

Ch

rom

ato

gra

ph

y

Electrophoresis pH1.9

+

PRAK

Ch

rom

ato

gra

ph

y

Electrophoresis pH1.9

MIX

+

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Effects of SB203580 and PD98059 on Effects of SB203580 and PD98059 on endogenous PRAK activityendogenous PRAK activity

HSP27

TNF

Arsenite PMA

SB203580 PD98059

Page 24: SHOCK &  SIGNAL TRANSDUCTION

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+ + + +- - - - + -

HSP27

p38

GST-PRAK(93

A)

GST-PRAK(WT)

GST-PRAK(186A

)

GST-PRAK(212A

214A

)

GST-PRAK(182A

)

GST-PRAK(WT)

GST-PRAK(182D

)

GST-PRAK(182D

212D

)

+ - + - + -

Fold

of A

ctiv

atio

n

05

1015

20

GST-PRAK(182A

)

GST-PRAK(182D

)

GST-PRAK(WT)

GST-PRAK(93A)

GST-PRAK(186A

)

GST-PRAK(182D

212D

)

GST-PRAK(212A

214A

)

BA

Electrophoresis pH 8.9

+

+

p38-PRAK(182A)

+

p38-PRAK(182D)

p38-PRAK(wt)

T182 is the regulatory phosphorylation site of PRAK

Page 25: SHOCK &  SIGNAL TRANSDUCTION

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3. 提出MAPK L-12磷酸化环状

结构影响底物特异性的观点

Page 26: SHOCK &  SIGNAL TRANSDUCTION

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MAPK Dural Phosphorylation Sites L-12 Length * *

hp38 DFGLARHTDD-------------EMTGYVATRWYRAPE 25hP38 DFGLARQADE-------------EMTGYVATRWYRAPE 25hp38 DFGLARQADS-------------EMTGYVVTRWYRAPE 25hp38 DFGLARHADA-------------EMTGYVVTRWYRAPE 25hJNK1 DFGLARTAGTS-----------FMMTPYVVTRYYRAPE 27hJNK2 DFGLARTACTN-----------FMMTPYVVTRYYRAPE 27hJNK3 DFGLARTAGTS-----------FMMTPYVVTRYYRAPE 27hERK1 DFGLARIADPEHDH-------TGFLTEYVATRWYRAPE 31hERK2 DFGLARVADPDHDH-------TGFLTEYVATRWYRAPE 31hBMK1 DFGMARGLCTSPAEH------QYFMTEYVATRWYRAPE 32

YHOG1 DFGLARIQDP-------------QMTGYVSTRWYRAPE 25YSMK1 DFGLARGIHAGFFKCHS--TVQPHITNYVATRWYRAPE 36YMPK1 DFGLARGYSENPVEN------SQFLTEYVATRWYRAPE 32YKSS1 DFGLARCLASSSDSRET---LVGFMTEYVATRWYRAPE 35YFUS3 DFGLARIIDESAADNSEPTGQQSGMTEYVATRWYRAPE 38domain VII VIII

Loop-12(T-Loop) sequence of MAP kinases

Page 27: SHOCK &  SIGNAL TRANSDUCTION

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Construction of p38 loop-12 to ERK like structure

p38 ...DFGLARHTDDE------MTGYVATRWYRAPE...p38(E) ...DFGLARHTDDE------MTEYVATRWYRAPE...p38(6+) ...DFGLARHTDDEHDHTGFMTGYVATRWYRAPE...p38(6+E) ...DFGLARHTDDEHDHTGFMTEYVATRWYRAPE...p38(VAP) ...DFGLARVADPE------MTGYVATRWYRAPE...p38(DL) ...DFGLARHTDDD------LTGYVATRWYRAPE...p38(VAPD6+LE) ...DFGLARVADPDHDHTGFLTEYVATRWYRAPE...ERK2 ...DFGLARVADPDHDHTGFLTEYVATRWYRAPE...

Page 28: SHOCK &  SIGNAL TRANSDUCTION

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MAPK

MKK

Substrates

Loop-12 is a key structure to determine the selection of substrate

(...TXY...) ---

Page 29: SHOCK &  SIGNAL TRANSDUCTION

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4. p38 通过磷酸化MEF2C,调节

c-Jun的基因表达

Page 30: SHOCK &  SIGNAL TRANSDUCTION

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yIdentification of MEF2C as a substrate for p38

Page 31: SHOCK &  SIGNAL TRANSDUCTION

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yT293,T300 and S387 of MEF2C is p38 phosphorylat

ion sites

Page 32: SHOCK &  SIGNAL TRANSDUCTION

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y Induction of c-Jun through MEF2C phosphorylation by p38

Page 33: SHOCK &  SIGNAL TRANSDUCTION

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MKK4MKK4MKK3MKK6

MKK3MKK6

Cytokine GenesExpression

LPS

p38

p38 JNK

JNK

MEF2C

c-Jun

MEF2Cc-Jun

MEF2 site

c-Jun GeneExpression

TNF,IL-1,etc.

Inflamatory Reaction

AP1

pp

pp

pp

p p

p

Page 34: SHOCK &  SIGNAL TRANSDUCTION

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5. p38在 LPS诱导 TNF基因表达中起着重要的调控作用

Page 35: SHOCK &  SIGNAL TRANSDUCTION

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0

2

4

6

8

10

12

14

0 5 10 20 30 60 90 120

Time (min)

Re

lati

ve

kin

ase

ac

tivi

ty

Dynamic processes of p38 activation by LPS in RAW cells

Page 36: SHOCK &  SIGNAL TRANSDUCTION

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0

5

10

15

Re

lati

ve

luc

ife

ras

e a

ctiv

ity

Co

ntr

ol

LP

S

EG

F

LP

S+

FH

PI

p38 is involved in the enhancement of TNF- promoter transactivity

Page 37: SHOCK &  SIGNAL TRANSDUCTION

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LPS、 UV刺激心肌细胞共聚焦显微镜大体扫描

LPS ControlUV

Page 38: SHOCK &  SIGNAL TRANSDUCTION

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y p38 p38

p38 p38

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y p38 p38

p38 p38

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yRed fluorescence protein ( RFP ) expression vector

Page 41: SHOCK &  SIGNAL TRANSDUCTION

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pDsRed1-N1 空载体在 HeLa 细胞中的表达 (800)

p38RFP 融合载体在 HeLa 细胞中的表达 (800)

Page 42: SHOCK &  SIGNAL TRANSDUCTION

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p38的 ATP结合位点在激活后移位的作用

DTKTGLRVAVKKLSRPFQSIIHA

50 60

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p38 、、、

ATF2

MEF2C

CHOP10

SAP1

TNF- mRNA etc.

monocytemacrophage

Rt

LPS

MKK3 / 6

MKKKs

nucleus

TK

CD

14

PRAK

sHSP

Cytoskeleton

?

LBP

Page 44: SHOCK &  SIGNAL TRANSDUCTION

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y 本研究的病理学和临床意义 :

TLR4

虎杖 4 号

红 、肿 、热 痛、功能丧失

炎症反应

依据不同的靶点选择炎症抑制剂

MAPKs

转录因子

炎症因子

抑制炎症反应

Page 45: SHOCK &  SIGNAL TRANSDUCTION

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刺激

反应 B

反 应A

反应 C

刺激 B

反应

刺 激A

刺激 C

细胞信号网络

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y 细胞信号的反馈调节

+

刺激

反应

刺激

反应

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ASK1

MKK7 MKK3/6

JNK p38

Serumdepletion

Transcriptionfactors,suchasc-Jun

GSTexpression

Celldeath

Celldeathrelatedgeneexpression

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y Types of InteractionsTypes of Interactions(courtesy of K. Kohn)(courtesy of K. Kohn)

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His-TLR4

His-TLR4ic

目标基因 X 克隆 到 DNA-BD载体

随机克隆 cDNAs( Y )到 AD 载体

基因 X

共转染质粒到酵母宿主菌

在合适的培养基上选择生长的共转染菌落

进一步分析阳性克隆

基因 Y

减少假阳性克隆

-半乳糖苷酶活性检测,证实蛋白之间的相互作用

两个杂交蛋白在同一细胞的表达

DNA-BD/ 蛋白 X 杂交蛋白

AD/蛋白 Y杂交蛋白

DNA-AD载体

DNA-BD载体

+

图 7.用 Two-hybridsystem筛选与 TLR4相互作用蛋白的实验策略

Page 51: SHOCK &  SIGNAL TRANSDUCTION

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y Systems Biology: A New ParadigmSystems Biology: A New Paradigm

HO

R

MetabolomicsGenomics Proteomics

Functional Proteomics/Genomics

Transcriptomics

Systems Biology

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y What is the “-OMICS” Revolution?What is the “-OMICS” Revolution?

Genomics Proteomics/Functional Proteomics

Metabolomics

1. Analysis of whole organism, cell or organelle

2. Driven by platform technologies

3. Characterized by rapid parallel throughput analyses

4. Generation of massive complex data blocks

Page 53: SHOCK &  SIGNAL TRANSDUCTION

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y Functional Scales of SystemsFunctional Scales of Systems

Molecular Interactions Molecular Interactions (GO)(GO)

MechanismsMechanisms

Pathways / NetworksPathways / NetworksRoles and ControlsRoles and Controls

Process Hierarchy Process Hierarchy (GO)(GO)

Purpose and DiseasesPurpose and Diseases

Page 54: SHOCK &  SIGNAL TRANSDUCTION

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y Understanding from Systems BiologyUnderstanding from Systems Biology

DNA

RNA

Protein

Cell

Tissue

Organ

Individual

Genomics

Transcriptomics

Proteomics

Metabolomics

Differential Proteomics

Functional Proteomics

Gene Sequences; SNPs

mRNA Levels

Entire Protein Compliment

Proteins Present/Absent in Affected versus Normal System

Protein Modifications; Protein/DNA,Protein/RNA, Protein/Ligand,Protein/Protein Interactions

Protein Function; Cell, Tissue, Organ, Individual Interactions

Pharmacogenomics

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Pharmacogenomics

Discovery Target validation Lead Generation Lead Optimization Clinical Trials

Drug DiscoveryDrug Discovery

Proteomics Combi-Chem

Bioinformatics

Functional Genomics

HTS

Rational Design Structural

Proteomics Animal Models

Systems Biology

QSAR

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Target identification

Target validation

Diagnostic and prognostic biomarkers

Disease progression models

Pharmacology and toxicology

Inclusion/exclusion criteria for clinical trials

Drug response profiles

Systems Biology can be used for…

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y Platform for Systems BiologyPlatform for Systems Biology

ppm0123456789

Complex Cellular Samplesbodyfluids, tissue

Dynamicsi.e. environmental + time

Gene

Protein

Metabolite

• Objective is to link gene response, protein activity, metabolite dynamics to disease and interventions

QuantitativeComparisons

QuantitativeComparisons

TargetsBiomarkers

TargetsBiomarkers

BioSystematicsTMBioSystematics

TM

gene

inde

x

prot

ein

inde

x

metabolite index

Page 58: SHOCK &  SIGNAL TRANSDUCTION

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y

THANKS

FORYOURATTENTION

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y ACKNOWLEDGEMENT