医学PPT课件】信号传导的病理生理学.ppt

Southern MedicalUniversity SHOCK 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） Southern MedicalUniversity 炎症反应研究的焦点问题 : 致炎刺激（proinflammatory stimuli ） 细胞内信号转导系统 细胞炎症反应 Southern MedicalUniversity 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) Southern MedicalUniversity 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 Southern MedicalUniversity 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： Southern MedicalUniversity Cellular response mediated by MAP kinases MAPkinases Proliferation Differentiation StressresponseApoptosis Cellcycle Inflammation Survival Development Southern MedicalUniversity 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 Southern MedicalUniversity MAPK MKK Substrates Loop-12 is a key structure to determine the selection of substrate (.TXY.)- Southern MedicalUniversity 1. 发现和克隆了三个新的p38 亚 型和三个新的p38上游激酶 Southern MedicalUniversity JNK2 JNK3 JNK1 p38 p38 p38 p38 ERK1 ERK2 ERK5 The relationship between the members of MAPK family Southern MedicalUniversity Heart Brain Placenta Lung Liver Skeletal Muscle Kidney Pancreas p38 p38 p38 p38 kb 3.5- 2.5- 1.8- 2.0- Tissue distribution of mRNA of p38 isoforms Southern MedicalUniversity p38 p38 p38 p38 38- 39- 38- 39- Jurket 293 HepG2 U937 Hela kDa The protein expression of p38 isoforms Southern MedicalUniversity 77 154 308 400 500 700 MBP p38 MBP p38 77 154 308 400 500 700 Osmolarity (mOsmol/L) AB Activation of p38 and p38 by different osmolarity medium Southern MedicalUniversity Control Aniso. Arsenite TNF -80kDa -47kDa -39kDa -80kDa -47kDa kDa 97.4- 68.0- 43.9- 29.0- 18.4- 14.3- Control Aniso. Arsenite TNF AB The major kinases of p38 and p38 Southern MedicalUniversity 2. 鉴定了受p38特异性调控的蛋白 激酶PRAK Southern MedicalUniversity The ancestral relationships among PRAK related kinases 45 40 35 30 25 20 15 10 5 0 47.9 Rsk1 Rsk3 Rsk2 Mnk1 Mnk2 Mapkapk2 Mapkapk3 PRAK Southern MedicalUniversity 293 A549 HepG2 Hela Jarket B -54kD heart brain placenta lung liver skeletal muscle kidney pancreas A -2.4kb -4.4kb -7.5kb -9.5kb -1.5kb The distribution of PRAK Southern MedicalUniversity Activation of endogenous PRAK by various stimuli in vivo HSP27 Ca 2+ Ionophore Control PMA EGF IL-6 TNF H2O2 Aniso. Arsenite 1.0 4.0 2.5 1.0 0.88 8.4 10.6 20.8 18.7Fold Southern MedicalUniversity Effect of individual MAPK pathways on PRAK activity in intact cells MKK7(D) GST-ATF2 GST-ELK1 MKK1(E) MKK6(E) Control HSP27 PRAK Southern MedicalUniversity PhosphopeptidePhosphopeptide map of HSP27 map of HSP27 phosphorylatedphosphorylated by PRAK by PRAK in vitroin vitro Chromatograph y Electrophoresis pH1.9 + MAPKAPK2 Chromatography Electrophoresis pH1.9 + PRAK Chromatography Electrophoresis pH1.9 MIX + Southern MedicalUniversity Effects of SB203580 and PD98059 on endogenous PRAK activity HSP27 TNF+- Arsenite+- PMA+- SB203580 _ -+-+-+PD98059 -+-+-+- Southern MedicalUniversity +- - - + - HSP27 p38 GST-PRAK(93A) 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 Activation 0 5 10 15 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 Southern MedicalUniversity 3. 提出MAPK L-12磷酸化环状结 构影响底物特异性的观点 Southern MedicalUniversity MAPK Dural Phosphorylation Sites L-12 Length * * hp38 DFGLARHTDD-EMTGYVATRWYRAPE25 hP38 DFGLARQADE-EMTGYVATRWYRAPE25 hp38 DFGLARQADS-EMTGYVVTRWYRAPE25 hp38 DFGLARHADA-EMTGYVVTRWYRAPE25 hJNK1 DFGLARTAGTS-FMMTPYVVTRYYRAPE27 hJNK2 DFGLARTACTN-FMMTPYVVTRYYRAPE27 hJNK3 DFGLARTAGTS-FMMTPYVVTRYYRAPE27 hERK1 DFGLARIADPEHDH-TGFLTEYVATRWYRAPE31 hERK2 DFGLARVADPDHDH-TGFLTEYVATRWYRAPE31 hBMK1 DFGMARGLCTSPAEH-QYFMTEYVATRWYRAPE32 YHOG1 DFGLARIQDP-QMTGYVSTRWYRAPE 25 YSMK1 DFGLARGIHAGFFKCHS-TVQPHITNYVATRWYRAPE36 YMPK1 DFGLARGYSENPVEN-SQFLTEYVATRWYRAPE32 YKSS1 DFGLARCLASSSDSRET-LVGFMTEYVATRWYRAPE35 YFUS3 DFGLARIIDESAADNSEPTGQQSGMTEYVATRWYRAPE38 domain VII VIII Loop-12(T-Loop) sequence of MAP kinases Southern MedicalUniversity 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. Southern MedicalUniversity MAPK MKK Substrates Loop-12 is a key structure to determine the selection of substrate (.TXY.) - Southern MedicalUniversity 4. p38 通过磷酸化MEF2C，调节c- Jun的基因表达 Southern MedicalUniversity Identification of MEF2C as a substrate for p38 Southern MedicalUniversity T293,T300 and S387 of MEF2C is p38 phosphorylation sites Southern MedicalUniversity Induction of c-Jun through MEF2C phosphorylation by p38 Southern MedicalUniversity MKK4MKK3 MKK6 Cytokine Genes Expression LPS p38 p38 JNK JNK MEF2C c-Jun MEF2C c-Jun MEF2 site c-Jun Gene Expression TNF,IL-1,etc. Inflamatory Reaction AP1 p p p p p p p p p Southern MedicalUniversity 5. p38在LPS诱导TNF基因表达中 起着重要的调控作用 Southern MedicalUniversity 0 2 4 6 8 10 12 14 0 5 10 20 30 60 90 120 Time (min) Relative kinase activity Dynamic processes of p38 activation by LPS in RAW cells Southern MedicalUniversity 0 5 10 15 Relative luciferase activity Control LPS EGF LPS+FHPI p38 is involved in the enhancement of TNF- promoter transactivity Southern MedicalUniversity LPS、UV刺激心肌细胞共聚焦显微镜大体扫描 LPSControlUV Southern MedicalUniversity p38p38 p38p38 Southern MedicalUniversity p38p38 p38p38 Southern MedicalUniversity Red fluorescence protein（RFP） expression vector Southern MedicalUniversity 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) Southern MedicalUniversity p38的ATP结合位点在激活后移位的作用 DTKTGLRVAVKKLSRPFQSIIHA 5060 Southern MedicalUniversity p38、 ATF2 MEF2C CHOP10 SAP1 TNF- mRNA etc. monocyte macrophage Rt L P S MKK3 / 6 MKKKs nucleus TK CD14 PRAK sHSP Cytoskeleton ? LBP ？ Southern MedicalUniversity 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 Southern MedicalUniversity 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 Southern MedicalUniversity 细胞信号的反馈调节 +- 刺激 反应 刺激 反应 Southern MedicalUniversity ASK1 MKK7MKK3/6 JNKp38 Serumdepletion Transcriptionfactors,suchasc-Jun GSTexpression Celldeath Celldeathrelatedgeneexpression Southern MedicalUniversity Southern MedicalUniversity Types of Interactions (courtesy of K. Kohn) Southern MedicalUniversity 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 Southern MedicalUniversity Systems Biology: A New Paradigm MetabolomicsGenomicsProteomics Functional Proteomics/Genomics Transcriptomics Systems Biology Southern MedicalUniversity 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 Southern MedicalUniversity Functional Scales of SystemsFunctional Scales of Systems Molecular Interactions (GO)Molecular Interactions (GO) MechanismsMechanisms Pathways / NetworksPathways / Networks Roles and ControlsRoles and Controls Process Hierarchy (GO)Process Hierarchy (GO) Purpose and DiseasesPurpose and Diseases Southern MedicalUniversity Understanding 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 Southern MedicalUniversity Pharmacogenomics Discovery Target validation Lead Generation