分子医学信号调控柯越海

1、Molecular Medicine VIISignal transduction信号调控概论KE, Yuehai 柯越海, School of Basic Medical Sciences (BMS-ZJU)浙江大学医学院al Profile 个人信息栏目 基本信息 教学工作(14年春夏学期)医学 ()组学与系统生物学概论 3月18日教408 周二下午 13：15-15：40 ；3月19日 紫金港东1A-403 周三上午 9：50-12：15 学术交流（讲座报告） 科研资助 招生信息 课题组成员 科研伙伴(PMCB等) 学校服务与学术 NewsCsmg ReadingCsmg English

2、细胞信号传导 4月8日教408 周二下午 13：15-15：40 ；4月9日 紫金港东1A-403 周三上午 9：50-12：15课件注意事项：本网页所有课件(PDF)本校本专业学员教学复习参考，它用、提供任何第；除特殊注明之外所有课件文字、数据、图表等均来自公开物，请尊重原作者；部分课件有删除或技术处理，教学内容以课堂授课为准。Overview-Facts of cellular communications (Intral & Intercellular)Pathway componentsSignal modificationEssential domainsSignal net

3、working and common pathwaysPost-genomic era: Variation and RegulationGenetic variation:Regulation geneticsSignificances of genetic variation1. A genetic resource for biological diversity2. A power of evolution3. A compromised pattern by genetic-environmental interaction4. A genetic basis for regulat

4、ion (How does interaction work?)5. A powerful tool to understand human diseases (genetic markers)Genetic variation:Regulation geneticsModification （修饰）: Essential for regulation DNA-based modification：DNA/Histone RNA-based modification： RNA geneticsProtein-base modification:Posttranslational modific

5、ationPosttranslational modificationSignal transductionWhat is Signal transduction?is the cellular regulatory process by which an extracellular signaling molecule activates a membrane receptor that in turn alters intracellular molecules, eventually causes a variety of biological responses1.2.A signal

6、ing molecule activates a specific receptor protein on the cell membrane.A second messenger transmits the signal into the cell, eliciting a physiological response.Inter & Intral cellular communicationssignal events, molecular events, cell signaling, signal relay, cellular communication, signal pa

7、thway/cascadeIntercellular signaling: Local and Distant (Hormonal signaling)Paracrine, autocrine, and endocrineIntercellular signaling:Direct intercellular connection and signalingThree Major junctional complexes1.Tight junction / zonula occludens (紧密连接/闭合带)An impermeable junction in which membranes

8、 of adjacent cells fuse together2.Desmosome (桥粒)hold cells together and provide strength without affecting the passage of material between cells.3.Gap junction (隙缝连接)connect cells by a protein channel that allows cells to exchange chemical signalsIntracellular signalingSignal regulation and human di

9、seases1. A power tool for understanding neural disorders molecular pathology scientific language /molecular language for human diseases2. Targeting human diseases at the molecular level Fundamental for molecular medicine3. Providing biomarkers for Diagnosis and Prognosis4. Offering potentials for th

10、erapeutic optionTips for studying signal transduction in human diseases.Modification is essential for signal transductionSignal is temporally- and spatially regulated in a finely-tuned manner Understanding signal transduction in more pathophysiological relevancies Impossible is nothing, while

11、 finding solid evidence is the only way to goExample ： Complexity of signal modulator Shp2 in stem cell and brain system-Facts of cellular communicationsGenetic variation and regulation Intral & Intercellular signalingSignal transduction and human diseases Tips for studying signal transduction-S

12、ignal components and modificationEssential domainsSignal networking and common pathwaysPathway componentsFirst messages->Receptors->Secondary messages->ResponsesComponents of signaling pathway:1.First messagers (Chemical stimulus):(1) Endocrine hormones: GH(2) Neurotransmitters: Ach, GABA(3

13、) Growth factors/cytokines: ILs, IFN, FGF, EGF2.Receptors/Ligand:(1)G-protein-coupled receptors(GPCR): Rhodopsin(2)(3)Receptor enzymes: EGFRIon channels (ligand-gated, voltage-gated): 5-HT, nAChR3.Secondary messagers:(1)Kinase/Phosphatase/Adaptors(2)(3)(4)(5)cAMPG-protein Ca2+Inositol phosphate4.Res

14、ponses(1) Proliferation(2) Differentiation(3) Apoptosis(4) Migration/AdhesionSignal networking5.Chemical modification for signal componentsCommon types for post-translational modification.-Phosphorylation (磷酸化） Methylation (甲基化） Acetylation （乙酰化） Ubiquitination （泛素化） Sumoylation （类泛素化） Glycosylation

15、 （糖基化） Sulfation (硫化） Nitrosylation （亚硝基化）Modifications enable signal transaction in a reversible mannerPost-translational modification for proteinStructural modification and location (protein)Single residual modification (amino acid)MethylationMethylation contributing to epigenetic inheritance can

16、occ through either DNA methylation or protein methylation， methylation is catalyzed by enzymes; regulation of gene expression, regulation of protein function, and RNA processing.DNA methylation in vertebrates typically occurs at CpG sites (cytosine-phosphate-guanine sites, that is, where a cytosine

17、is directly followed by a guanine in the DNA sequence). This methylation results in the conversion of th cytosine to 5-methylcytosine，by DNA methyltransferaseProtein methylation typically takes place on arginine or lysine amino acid residues in the protein sequence, by peptidylarginine methyltransfe

18、rases (PRMTs), methylation has been most studied in the histones. The transfer of methyl groups from S-adenosyl methionine to histones is catalyzed by enzymes known as histone methyltransferase Histones that are methylated on certain residues can act epigenetically to repress or activate gene expres

19、sion.Cancer and methylation imbalanceAcetylationdescribes a reaction that introduces an acetyl functional group into a chemical compound. (Deacetylation is the removal of the acetyl group.)Acetylation occurs as a co-translational and post- translational modification of proteins, for example, histone

20、s, p53, tubulins and STAT3. Among these proteins,chromatin proteins and metabolic enzymes are highly represented, indicating that acetylation has a considerable impact on gene expression and metabolism.N-terminal Acetylation： is one of the most common co- translational covalent modifications， an imp

21、ortant role in the synthesis, stability and localization of proteins.UbiquitinationUbiquitination is a post-translational modification where ubiquitin is attached to a substrate protein. The addition of ubiquitin can affect proteins in many ways: It can signal for their degradation via the proteasom

22、e, alter their cellular location, affect their activity, and promote or prevent protein interactions.Ubiquitin is a small (8.5 kDa) regulatory protein that has been found in almost all tissues (ubiquitously) of eukaryotic organisms, was discovered in 1975.Ubiquitination is carried out in three main

23、steps: activation, conjugation, and ligation, performed by ubiquitin-activating enzymes (E1s), ubiquitin- conjugating enzymes (E2s), and ubiquitin ligases (E3s).SumoylationSmall Ubiquitin-like Modifier (or SUMO) proteins are a family of small proteins that are covalently attached to and detached fro

24、m other proteins in cells to modify their functionSUMOylation is a post-translational modification involved in various cellular processes, such asnuclear-cytosolic transport, transcriptional regulation, apoptosis, protein stability, response to stress, and progression through the cell cycle.SUMO pro

25、teins are similar to ubiquitin, and SUMOylation is directed by an enzymatic cascade analogous to that involved in ubiquitination.In contrast to ubiquitin, SUMO is not used to tag proteins for degradation. Mature SUMO is produced when the last four amino acids of the C-terminus have been cleaved off

26、to allow formation of an isopeptide bond between the C-terminal glycine residue of SUMO and an acceptor lysine on the target protein.PhosphorylationPhosphorylation: A ubiquitous regulatory mechanism in control of molecular events Addition/removal of a phosphate (PO4) group to a protein (Signal regul

27、ator), commonly occurs on serine, threonine and tyrosine. Kinases, Phosphatase and Docking proteinsWhy is Reversible phosphorylation?1.Most essential chemical reaction leads to alternative conformational switches in enzymes and receptors, causing activated or deactivated action.2.Most flexible biolo

28、gical response in under less one minute or over hours3.Most efficient amplified activation, a single kinase molecule can result in the phosphorylation of many proteins.Putative phosphorylation sitesand physiological relevanciesPutative phosphorylation sitesA signal regulator (protein) have thousand

29、phosphorylation sites, providing conformational potentials to change, may or may not (mostly).Each phosphorylation event may have different effects on protein activity, increase, decrease, no effect (mostly).A protein may have diversity of regulatory action for the distinct phosphorylation sites in

30、a given cell at a given time,Distinctive phosphorylation sites at FAK kinase131.Growth factors or integrin clustering promotes FAK autophosphorylation at Tyr397, which creates a binding site for SH2 domain of Src.Src-mediated p-Tyr576/577 of22.FAK results in catalytic activity.al FAK3.FAK phosphoryl

31、ation at Tyr925 results in Grb2 binding and Erk activaiton-Facts of cellular communicationsSignal components and modificationMethylation (甲基化） Acetylation （乙酰化） Ubiquitination （泛素化） Sumoylation （类泛素化） Phosphorylation (磷酸化）-Essential domainsSignal networking and common pathwaysEssential domainsUseful

32、 tools for studying signal transductionFunctional domains and structural motifsA protein domain is a part of protein sequence and structure that can evolve, function, and exist independently of the rest of the protein chain. Signal molecule proteins may consist of several structural domains.Example:

33、 SH2, SH3, PH, PTPA structural motif is a three-dimensional structural element or fold within the chain, which appears also in a variety of other molecules. Motif is sometimes used interchangeably with "structural domain," although a domain need not be a motif nor, if it contains a motif,

34、need not be made up of only one.Example: helix-loop-helix, zinc finger, leucine zipperAnthony 'Tony' James Pawson, British-born Canadian scientist whose research has revolutionized the understanding of signal transduction, the molecular mechanisms by which cells respond to external cues, and

35、 how they communicate with each other.He identified the phosphotyrosine-binding Src homology 2 (SH2 domain) as the prototypic non-catalytic interaction module. SH2 domains serve as amfor a large family of protein modules that act together to controlmany aspects of cellular signaling. Since the disco

36、very of SH2 domains, hundreds of different modules have been identified in many proteins.Anthony 'Tony' James Pawson (1952- 2013 )University of TorontoAnthony 'Tony' Rex Hunter (1943- )Salk Institute/UCSDDr. Hunter is one of the foremost recognized leaders in the field of cell growth

37、 control, growth factor receptors and their signal transduction pathways.KinaseKingHe is well known for discovering that tyrosine phosphorylation is a fundamental mechanism for transmembrane-signal transduction in response to growth factor stimulation and that disregulation of such tyrosine phosphor

38、ylation, by activated oncogenic protein tyrosine kinases, is a pivotal mechanism utilized in the malignant transformation of cells. His work is important in signaling pathways and their disorders.He was a founder of Signal Pharmaceuticals.Essential domains and motifs in signal moleculesSH2: Adaptor,

39、 Binding with phosphotyrosineSH3: Adaptor: Binding wtih proline-rich motifsPDZ: Membrane targetingPH:Signaling; phospholipid bindingTubby: DNA bindingThe building blocksmodular interaction domains in signal transduction.SH2 domain1.Src-homology 2 (SH2) domains are modules of 100 amino acids that bin

40、d to specific phospho (pY)- containing peptide motifs.2.Conventional SH2 domains have a conserved pocket that recognizes pY, and a more variable pocket that binds 3-6 residues C-terminal to the pY and confers specificity.SH3 domain1.The basic fold of SH3 domains contains five anti-parallel beta-stra

41、nds packed to form two perpendicular beta-sheets.2.The ligand-binding site consists of a hydrophobic patch that contains a cluster of conserved aromatic residues and is surrounded by two charged and variable loops.Src kinase (SH2-SH3-TK)The first TYROSINE KINASE was discovered from aviansarcoma(RSV)

42、 by Peyton Rous (1911), winner of 1966Nobel Prize in Physiology or Medicine.J. Michael Bishop and Harold Varmus identified cellular counterpart of src oncogene (1978-1981), sharing Nobel Prize in Physiology or Medicine in 19899 Src kinases exist in the human genome, including c-Src, c- Yes, Fyn, Lyn

43、, Lck, Hck, Blk, Fgr, and YrkInactivation of human Src occurs when its C-terminal Tyr530 is phosphorylated and binds to the SH2 domain. Dephosphorylation at Tyr530 by protein tyrosine phosphatases, such as PTPa, PTPN11/SHP2, PTP1BActivation of human c-Src occurs when its C-terminal Tyr530 is dephosp

44、horylated and fails to bind the SH2 domain, opening of the c-Src molecular structure.Full activation involves phosphorylation of Tyr419 in the kinase domain.Inactivation and ActivationFunctional relevancies of common signal domainsDomainFunction al relevanciesSH2Src Homology 2, Adaptor, specific pho

45、spho (pY)-containing peptide motifsSH3Src Homology 3, Adaptor, binds Proline-rich domain to a hydrophobic sitesPHPleckstrin-homology(PH), Adaptor, respond to lipid messengers by cellular localization to membranes and transmitting signals to downstream, widely involved intracellular trafficking, cell

46、ular signaling and cytoskelremingPDZNearly always found in cytoplasmic proteins, involved in membrane target.14-3-3cellular processes such as apoptosis, stress response and cell cycleIgImmunoglobulin domain, extracellular, protein interactionsFnIIIFibronectin type III domain, extracellular, protein

47、interactionsSAMSterile alpha motif, dimerization domainPTPEnzymatic domain, protein tyrosine phosphatasePTKEnzymatic domain, protein tyrosine kinaseL domainReceptor-Ligand domainCRDCysteine rich domain, related with ligand bindingLRDLeucine-rich domain, Ligand binding, protein interactionPEST-likepo

48、lypeptide sequences enriched in proline (P), glutamic acid (E), serine (S) and threonine (T) target proteins for rapid destruction, proteolysis.FERMnamed for the four proteins in which this domain was originally described, involved in the linkage of cytoplasmic proteins to the membranePRDProline ric

49、h domain, binds to a hydrophobic sitesPWWPInvolved in DNA methylation, DNA repair, DNA binding and regulation of transcription.SrcCombination of SH2-SH3-PTK domainFunctional relevancies of common signal domainsFunctionsDomainsExtracellular domainIg, FnIII, L, CRD, LRDTrans-membrane (Dimerization )SA

50、MMembrane-binding/targeting/recognitionSH3, PH, PDZ, PRD, FERMCytoplasmic protein interactionSH2, PH, 14-3-3Nuclear location (DNA-binding)14-3-3, PWWPEnzymatic activitiesPTP, PTKSpecific pY-containingSH2Apoptosis, Destruction14-3-3, PEST-likePredicting function of signal molecules based on domainIgT

51、KFGFRPHPRDGab1, Gab2FERMTKSH2JAK1, JAK2FERMPTPPDZPTP1SH2PTPSH2Shp2IgFnIIITK SAMCRDEphrinA-Facts of cellular communicationsSignal components and modificationPhosphorylation (磷酸化）-Essential domainsTony Pawson and SH2 domain Common domains-Signal networking and common pathwaysRTK/RAS/MAPK JAK/STATs/SOC

52、S TLR/NF-kBRTK/RAS/MAPK pathwayRTK: Receptor Tyrosine KinaseRAS: Rat Sarcoma(GTPase)MAPK: Mitogen-Activated Protein KinaseKeywords of functions:1.2.3.Growth factorsProliferation and development Cancer signalingReceptor tyrosine kinases (RTKs)Approx. 60 in 90-110 totalNon-receptor tyrosine kinases (N

53、RTKs)Approx. 35 in 90-110 totalGeneral Action of RTKsEGFR / ErbB familyInsufficient ErbB signaling in humans is associated with the development Excessive ErbB signaling is associated with malignancy of these tumorsEGFR and its ligands are cell signaling molecules involved in diverse cellular functio

54、ns, including cell proliferation, differentiation, motility, and survival, and in tissue development.ErbB family of receptors, a subfamily of four closely related receptor tyrosine kinases:EGFR (ErbB-1),HER2/c-neu (ErbB-2), Her 3 (ErbB-3)Her 4 (ErbB-4).Mutations affecting EGFR expression or activity

55、 could result in cancerEGFR / ErbB family: Ligand and ReceptorEGFR dimers (homo or hetero) have multiple effects on cellular signalingEffects of HER receptor dimerization and subsequent activation. After ligand binding, receptors dimerize, or form pairs. Upon dimerization, the intracellular tyrosine kinase domains of the receptors are phosphorylated, activating the receptors and initiating downstream signalingPotential Strategies for Inhibiting erbB Receptor Activity试题分析最近一项基于临床数据的大规模遗传学研究显示x与乳HER2阳性患的耐药性密切相关，后续研究发现x者的某靶向具有一个磷酸激酶催化中心，生化分析提示是一个编码EGFR膜受体的关键信号蛋白，新入学就读的同学接手该课题，导师提供同学