shock & signal transduction
DESCRIPTION
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 PresentationTRANSCRIPT
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SHOCK & SIGNAL TRANSDUCTION
YONG JIANG
Key Laboratory of Proteomics of Guangdong Province, Southern Medical University
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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
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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 )
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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
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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)
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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
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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:
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y Cellular response mediated by MAP kinases
MAPkinasesMAPkinases
ProliferationProliferation
DifferentiationDifferentiation
StressresponseStressresponse ApoptosisApoptosis
CellcycleCellcycle
InflammationInflammation
SurvivalSurvival
DevelopmentDevelopment
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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
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MAPK
MKK
Substrates
Loop-12 is a key structure to determine the selection of substrate
(...TXY...)---
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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
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JNK2
JNK3
JNK1
p38
p38p38p38ERK1
ERK2
ERK5
The relationship between
the members of MAPK family
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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...
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MAPK
MKK
Substrates
Loop-12 is a key structure to determine the selection of substrate
(...TXY...) ---
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4. p38 通过磷酸化MEF2C,调节
c-Jun的基因表达
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yIdentification of MEF2C as a substrate for p38
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yT293,T300 and S387 of MEF2C is p38 phosphorylat
ion sites
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y Induction of c-Jun through MEF2C phosphorylation by p38
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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
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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
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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
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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
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LPS、 UV刺激心肌细胞共聚焦显微镜大体扫描
LPS ControlUV
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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
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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)
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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
?
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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
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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
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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
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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
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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
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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
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THANKS
FORYOURATTENTION
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y ACKNOWLEDGEMENT