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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。

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SHOCK & SIGNAL TRANSDUCTION

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Shock signal transduction

SHOCK &

SIGNAL TRANSDUCTION

YONG JIANG

Key Laboratory of Proteomics of Guangdong Province,

Southern Medical University


Shock signal transduction

  • 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


Shock signal transduction

  • 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(leukotrienes)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 presentation)


Shock signal transduction

致炎刺激(proinflammatory stimuli)

细胞内信号转导系统

细胞炎症反应

炎症反应研究的焦点问题:


Shock signal transduction

  • 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)


Shock signal transduction

MAPK通路的细胞内信号转导过程

生长信号

应激

LPS

H2O2

刺激

Raf1

MEKK1

ASK1/MLK

?

MKKK

MEK1/2

MKK4

MKK3/6

MEK5

MKK

JNKs

ERK5

p38s

MAPK

ERK1/2

生长

凋亡

炎症反应

?

细胞反应


Shock signal transduction

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:


Shock signal transduction

Inflammation

Apoptosis

Stressresponse

Development

Cellcycle

MAPkinases

Survival

Proliferation

Differentiation

Cellular response mediated by MAP kinases


Shock signal transduction

原始损伤

二次打击

细菌毒素

细胞因子

粘附蛋白

NOS

高温

辐射

病毒

炎症反应过程中的二次打击:

MAPK通路


Shock signal transduction

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

MKK

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

MAPK

Substrates


Shock signal transduction

1. 发现和克隆了三个新的p38 亚型和三个新的p38上游激酶


Shock signal transduction

JNK2

JNK3

JNK1

p38

p38

p38

p38

ERK1

ERK2

ERK5

The relationship between

the members of MAPK family


Shock signal transduction

Heart

Brain

Placenta

Lung

Liver

Skeletal Muscle

Kidney

Pancreas

kb

p38

3.5-

2.5-

p38

p38

2.0-

1.8-

p38

Tissue distribution of mRNA of p38 isoforms


Shock signal transduction

Jurket

293

HepG2

U937

Hela

kDa

38-

p38

39-

p38

39-

p38

38-

p38

The protein expression of p38 isoforms


Shock signal transduction

77

154

308

400

500

700

77

154

308

400

500

700

B

A

Osmolarity

(mOsmol/L)

MBP

MBP

p38

p38

Activation of p38 and p38 by different

osmolarity medium


Shock signal transduction

The major kinases of p38 and p38

B

A

Control

Aniso.

Arsenite

TNF

Control

Aniso.

Arsenite

TNF

kDa

97.4-

68.0-

43.9-

29.0-

18.4-

14.3-

-80kDa

-47kDa

-80kDa

-47kDa

-39kDa


Shock signal transduction

2. 鉴定了受p38特异性调控的蛋白激酶PRAK


The ancestral relationships among prak related kinases

Rsk1

Rsk3

Rsk2

Mnk1

Mnk2

Mapkapk2

Mapkapk3

PRAK

47.9

The ancestral relationships among PRAK related kinases

45 40 35 30 25 20 15 10 5 0


Shock signal transduction

skeletal muscle

A

lung

pancreas

placenta

kidney

liver

brain

heart

-9.5kb

-7.5kb

-4.4kb

-2.4kb

HepG2

Jarket

293

A549

Hela

-1.5kb

B

-54kD

The distribution of PRAK


Activation of endogenous prak by various stimuli in vivo

Ca 2+ Ionophore

Control

Aniso.

Arsenite

H2O2

PMA

EGF

TNF

IL-6

Fold

1.0 4.0 2.5 1.0 0.88 8.4 10.6 20.8 18.7

Activation of endogenous PRAK by various stimuli in vivo

HSP27


Effect of individual mapk pathways on prak activity in intact cells

Effect of individual MAPK pathways on PRAK activity in intact cells

MKK1(E)

MKK7(D)

MKK6(E)

Control

HSP27

PRAK

GST-ATF2

GST-ELK1


Shock signal transduction

MAPKAPK2

PRAK

MIX

Chromatography

Chromatography

Chromatography

+

+

+

Electrophoresis pH1.9

Electrophoresis pH1.9

Electrophoresis pH1.9

Phosphopeptide map of HSP27 phosphorylated by PRAK in vitro


Effects of sb203580 and pd98059 on endogenous prak activity

HSP27

-

+

+

+

-

-

-

-

-

-

-

-

TNF

-

-

-

-

-

+

+

+

-

-

-

-

Arsenite

-

-

-

-

-

-

-

-

-

+

+

+

PMA

-

-

+

-

-

-

+

-

-

-

+

-

SB203580

_

-

-

-

+

-

-

-

+

-

-

+

PD98059

Effects of SB203580 and PD98059 on endogenous PRAK activity


Shock signal transduction

p38-PRAK(182A)

B

A

GST-PRAK(182D 212D)

GST-PRAK(212A 214A)

GST-PRAK(182D)

GST-PRAK(WT)

GST-PRAK(186A)

GST-PRAK(182A)

GST-PRAK(93A)

GST-PRAK(WT)

+

-

-

-

-

+

+

+

p38

-

-

-

-

+

+

+

+

+

p38-PRAK(182D)

HSP27

20

15

+

Fold of Activation

10

p38-PRAK(wt)

5

0

GST-PRAK(WT)

GST-PRAK(93A)

GST-PRAK(186A)

GST-PRAK(182A)

GST-PRAK(182D)

GST-PRAK(182D 212D)

GST-PRAK(212A 214A)

+

Electrophoresis pH 8.9

T182 is the regulatory phosphorylation site of PRAK


Shock signal transduction

3. 提出MAPK L-12磷酸化环状结构影响底物特异性的观点


Shock signal transduction

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

MAPKDural Phosphorylation Sites L-12 Length

* *

hp38DFGLARHTDD-------------EMTGYVATRWYRAPE25

hP38bDFGLARQADE-------------EMTGYVATRWYRAPE25

hp38gDFGLARQADS-------------EMTGYVVTRWYRAPE25

hp38dDFGLARHADA-------------EMTGYVVTRWYRAPE25

hJNK1DFGLARTAGTS-----------FMMTPYVVTRYYRAPE27

hJNK2DFGLARTACTN-----------FMMTPYVVTRYYRAPE27

hJNK3DFGLARTAGTS-----------FMMTPYVVTRYYRAPE27

hERK1DFGLARIADPEHDH-------TGFLTEYVATRWYRAPE31

hERK2DFGLARVADPDHDH-------TGFLTEYVATRWYRAPE31

hBMK1 DFGMARGLCTSPAEH------QYFMTEYVATRWYRAPE32

YHOG1DFGLARIQDP-------------QMTGYVSTRWYRAPE 25

YSMK1DFGLARGIHAGFFKCHS--TVQPHITNYVATRWYRAPE36

YMPK1DFGLARGYSENPVEN------SQFLTEYVATRWYRAPE32

YKSS1DFGLARCLASSSDSRET---LVGFMTEYVATRWYRAPE35

YFUS3DFGLARIIDESAADNSEPTGQQSGMTEYVATRWYRAPE38

domain VII VIII


Shock signal transduction

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...


Shock signal transduction

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

MKK

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

MAPK

Substrates


Shock signal transduction

4. p38 通过磷酸化MEF2C,调节c-Jun的基因表达


Shock signal transduction

Identification of MEF2C as a substrate for p38


Shock signal transduction

T293,T300 and S387 of MEF2C is p38 phosphorylation sites


Shock signal transduction

Induction of c-Jun through MEF2C phosphorylation by p38


Shock signal transduction

LPS

p38

JNK

MKK3

MKK6

MKK4

p

p

JNK

p

p38

p

MEF2C

c-Jun

p

p

p

c-Jun Gene

Expression

MEF2C

MEF2 site

p

Cytokine Genes

Expression

p

c-Jun

TNF,IL-1,etc.

AP1

Inflamatory

Reaction


Shock signal transduction

5. p38在LPS诱导TNF基因表达中起着重要的调控作用


Shock signal transduction

1

4

1

2

1

0

8

Relative kinase activity

6

4

2

0

0 5 10 20 30 60 90 120

Time (min)

Dynamic processes of p38 activation by LPS in RAW cells


Shock signal transduction

1

5

1

0

Relative luciferase activity

5

Control

LPS

EGF

LPS+FHPI

0

p38 is involved in the enhancement of TNF- promoter transactivity


Shock signal transduction

LPS

UV

Control

LPS、UV刺激心肌细胞共聚焦显微镜大体扫描


Shock signal transduction

p38

p38

p38

p38


Shock signal transduction

p38

p38

p38

p38


Shock signal transduction

Red fluorescence protein(RFP)expression vector


Shock signal transduction

pDsRed1-N1空载体在HeLa细胞中的表达(800)

p38RFP融合载体在HeLa细胞中的表达(800)


Shock signal transduction

p38的ATP结合位点在激活后移位的作用

DTKTGLRVAVKKLSRPFQSIIHA

50

60


Shock signal transduction

LBP

LPS

monocyte

macrophage

CD14

Rt

TK

MKKKs

MKK3 / 6

p38a、b、g、d

PRAK

ATF2

MEF2C

sHSP

?

nucleus

CHOP10

SAP1

TNF-a mRNA

etc.

Cytoskeleton


Shock signal transduction

本研究的病理学和临床意义:

TLR4

虎杖4号

MAPKs

依据不同的靶点选择炎症抑制剂

转录因子

炎症因子

红 、肿 、热 痛、功能丧失

炎症反应

抑制炎症反应


Shock signal transduction

细胞信号网络

刺激

刺激B

刺激A

刺激C

反应A

反应C

反应B

反应


Shock signal transduction

细胞信号的反馈调节

刺激

刺激

+

-

反应

反应


Shock signal transduction

Serumdepletion

ASK1

MKK7

MKK3/6

JNK

p38

Transcriptionfactors,suchasc-Jun

GSTexpression

Celldeathrelatedgeneexpression

Celldeath


Types of interactions courtesy of k kohn

Types of Interactions(courtesy of K. Kohn)


Shock signal transduction

目标基因X克隆到DNA-BD载体

随机克隆cDNAs(Y)到AD载体

基因Y

基因X

DNA-BD载体

DNA-AD载体

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

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

-半乳糖苷酶活性检测,

证实蛋白之间的相互作用

进一步分析阳性克隆

减少假阳性克隆

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

His-TLR4

His-TLR4ic


Systems biology a new paradigm

Proteomics

Genomics

Metabolomics

Transcriptomics

Functional Proteomics/Genomics

Systems Biology

Systems Biology: A New Paradigm


Shock signal transduction

What is the “-OMICS” Revolution?

Proteomics/

Functional Proteomics

Genomics

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


Shock signal transduction

Functional Scales of Systems

Process Hierarchy (GO)

Purpose and Diseases

Pathways / Networks

Roles and Controls

Molecular Interactions (GO)

Mechanisms


Understanding from systems biology

Understanding from Systems Biology

Genomics

Gene Sequences; SNPs

DNA

RNA

Transcriptomics

mRNA Levels

Protein

Entire Protein Compliment

Proteomics

Proteins Present/Absent in

Affected versus Normal System

Cell

Differential Proteomics

Tissue

Protein Modifications; Protein/DNA,

Protein/RNA, Protein/Ligand,

Protein/Protein Interactions

Functional Proteomics

Organ

Protein Function; Cell, Tissue,

Organ, Individual Interactions

Metabolomics

Individual

Pharmacogenomics


Shock signal transduction

Systems Biology

Drug Discovery

Discovery Target validation Lead Generation Lead Optimization Clinical Trials

Bioinformatics

HTS

QSAR

Proteomics

Pharmacogenomics

Combi-Chem

Structural Proteomics

Functional Genomics

Animal Models

Rational Design


Shock signal transduction

Systems Biology can be used for…

  • Target identification

  • Target validation

  • Diagnostic and prognostic biomarkers

  • Disease progression models

  • Pharmacology and toxicology

  • Inclusion/exclusion criteria for clinical trials

  • Drug response profiles


Platform for systems biology

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

metabolite index

protein index

gene index

9

8

7

6

5

4

3

2

1

0

ppm

Platform for Systems Biology

Gene

Quantitative

Comparisons

Protein

Complex Cellular Samples

bodyfluids, tissue

Dynamics

i.e. environmental + time

BioSystematicsTM

Metabolite

Targets

Biomarkers


Shock signal transduction

THANKS

FORYOURATTENTION


Shock signal transduction

ACKNOWLEDGEMENT


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