1、Two-component regulatory system 三、三、Cyclic Nucleotide Second Messengers in Bacteria 细胞通讯是细胞间或细胞内通过高度精确和高细胞通讯是细胞间或细胞内通过高度精确和高效地发送与接收信息的通讯机制，对环境作出效地发送与接收信息的通讯机制，对环境作出综合反应的细胞行为。综合反应的细胞行为。 细胞信号转导细胞信号转导细胞通过位于胞膜或胞内的受体感受胞外信息分子的刺激，经复杂的细胞通过位于胞膜或胞内的受体感受胞外信息分子的刺激，经复杂的细胞内信号转导系统的转换来影响细胞的生物学功能。细胞内信号转导系统的转换来影响细胞的生
2、物学功能。 What is popular in research today？信号转导（细胞周期调控；细胞凋亡；细胞衰老；信号转导（细胞周期调控；细胞凋亡；细胞衰老；DNA损伤与修复）损伤与修复）美国科学情报研究所根据美国科学情报研究所根据SCISCI收录及引用论文检索，收录及引用论文检索，全世界自然科学研究中论文发表最集中的三个领域分全世界自然科学研究中论文发表最集中的三个领域分别别细胞信号转导细胞信号转导, ,细胞凋亡细胞凋亡, ,基因组与蛋白组学研基因组与蛋白组学研究究 细胞信号转导研究的细胞信号转导研究的3 3部分部分接收信号的特定受体接收信号的特定受体受体后的信号转导通路受体后的信
3、号转导通路信号的生物学效应信号的生物学效应细胞通讯的一般过程细胞通讯的一般过程信号分子的产生信号分子的产生 信号分子信号分子细胞识别（细胞识别（Cell recognition） 受体蛋白受体蛋白信号转导（信号转导（Signal transduction） 胞内信号胞内信号 特定基因表达特定基因表达 应答反应应答反应 细胞将胞外信号细胞将胞外信号(分子分子A)转变成胞内的信号转变成胞内的信号(分子分子B)电话接收器将电信号转换成声信号电话接收器将电信号转换成声信号细胞通讯与信号传递引起的反应细胞通讯与信号传递引起的反应酶活性的变化酶活性的变化基因表达的变化基因表达的变化细胞骨架构型细胞骨架构型
4、通透性的变化通透性的变化DNADNA合成活性的变化合成活性的变化细胞死亡程序变化等。细胞死亡程序变化等。细细胞胞通通讯讯的的作作用用 细胞间的通讯对于多细胞生物体的发生和组织的构建，协调细胞的功能，控制细胞细胞间的通讯对于多细胞生物体的发生和组织的构建，协调细胞的功能，控制细胞的生长、分裂、分化和死亡是必须的。的生长、分裂、分化和死亡是必须的。CELL COMMUNICATIONSection A: An Overview of Cell Signaling1. Cell signaling evolved early in the history of life2. Communicatin
5、g cells may be close together or far apart3. The three stages of cell signaling are reception, transduction, and responsenCell-to-cell communication is absolutely essential for multicellular organisms.nCells must communicate to coordinate their activities.nCommunication between cells is also importa
6、nt for many unicellular organisms.nBiologists have discovered some universal mechanisms of cellular regulation, involving the same small set of cell-signaling mechanisms.nCells may receive a variety of signals: chemical signals, electromagnetic signals, and mechanical signals.IntroductionnOne topic
7、of cell “conversation” is sex.nThe yeast Saccharomyces cerevisiae, the yeast of bread, wine, and beer, identifies its mates by chemical signaling.nThere are two sexes, a and alpha, each of which secretes a specific signaling molecule, a factor and alpha factor respectively.nThese factors each bind t
8、o receptor proteins on the other mating type.1. Cell signaling evolved early in the history of lifenOnce the mating factors have bound to the receptors, the two cells grow toward each other and experience other cellular changes.nTwo opposite cells fuse, or mate.nThe a/alpha cell contains the genes o
9、f both cells. nThe process by which a signal on a cells surface is converted into a specific cellular response is a several steps in a signal-transduction pathway.nThe molecular details in both yeast and animal cells are strikingly similar, even though their last common ancestor was over a billion y
10、ears ago.nSignaling molecules evolved first in ancient prokaryotes and were then adopted for new uses by single-celled eukaryotes and multicellular descendents.nCell signaling has remained important in the microbial world.nMyxobacteria, soil-dwelling bacteria, use chemical signals to communicate nut
11、rient availability.nWhen food is scarce, cells secrete a signal to other cells leading them to aggregate and form thick-walled spores.Fig. 11.2Individual rod-shaped cellsSpore-formingstructure(fruiting body)Aggregation inprocessFruiting bodies0.5 mm132nA plasma membrane is essential for a cellnIt do
12、es isolate the cell from its surroundingsnA cell cannot survive if it cannot sense changes in the extracellular environment and respond to themnCell communication is the process of cells detecting and responding to signals in the extracellular environment nNeeded to coordinate cellular activities in
13、 a multicellular organismnOne important reason for cell communication is to promote cell divisionnCell communication or cell signaling involves incoming and outgoing signalsnSignals are agents that influence the properties of cellsnSignals affect the conformation of a receptor leading to a response
14、in the cellWhy do cells need to respond to signals?1.Need to respond to a changing environmentnAdaptation or a cellular response is critical for survivalnGlucose acts as a signal to yeast cells to increase number of glucose transporters and enzymes allowing efficient uptake and use of glucoseSignal-
15、Transduction EmphasisThis chapters emphasis is on signals that are released from one cell and allowed to freely diffuse to a second (or more) recipient cell(s)These communications are deliberately initiated, received, and interpreted in order to increase the physiological coordination of the cells i
16、n multicellular organismsWe will consider in particular those events that follow the reception of chemical signalsThree Stages of Signal Transduction1. Reception of extracellular signal by cell2. Transduction of signal from outside of cell to inside of celloften multi-stepped Note not necessarily tr
17、ansduction of ligand 3. Cellular Response Response is inititiated and/or occurs entirely within receiving cellThree Stages of Signal TransductionThree Stages2a. Transduction2b. Transduction2c. Transduction2d. Transduction1. Reception3. ResponseResponses usually involve increasing or decreasing some
18、Proteins FunctionThree Stages2a. Transduction2b. Transduction1. Reception3. Response物理信号：物理信号：光、热、电流光、热、电流化学信号：化学信号：最广泛的信号是化学信号。短肽、蛋白质、气体分子（最广泛的信号是化学信号。短肽、蛋白质、气体分子（NO、CO）以及氨基酸、核苷酸、脂类和胆固醇衍生物等。以及氨基酸、核苷酸、脂类和胆固醇衍生物等。信号分子的共同特点：信号分子的共同特点：特异性特异性，只能与特定的受体结合；，只能与特定的受体结合；高效性高效性，几个分子即可发生明显的生物学效应，这一特性，几个分子即可发生明
19、显的生物学效应，这一特性 有赖于细胞的信号逐级放大系统；有赖于细胞的信号逐级放大系统；可被灭活可被灭活，完成信息传递后可被降解或修饰而失去活性，完成信息传递后可被降解或修饰而失去活性，保证信息传递的完整性和细胞免于疲劳。保证信息传递的完整性和细胞免于疲劳。信号分子信号分子 非营养物非营养物 非能源物质非能源物质 非结构物质非结构物质 不是酶不是酶 主要是用来在细胞间和细胞内传递信息主要是用来在细胞间和细胞内传递信息 2、表面受体、表面受体受体受体(ion-channel-linked receptors)G-蛋白蛋白 (trimeric GTP-binding regulatory prote
20、in)配体结合区配体结合区胞质区胞质区穿膜区穿膜区催化中心催化中心调节序列调节序列递质门离子通道递质门离子通道(transmitter-gated ion channels)细胞表面细胞表面与与细胞内受体细胞内受体: : 表面受体表面受体(surface receptor) 细胞质膜上：主要是同大的信号分子或小细胞质膜上：主要是同大的信号分子或小的亲水性的信号分子作用的亲水性的信号分子作用 细胞内受体细胞内受体(intracellular receptor) 胞质溶胶、核基质中：同脂溶性的小信号胞质溶胶、核基质中：同脂溶性的小信号分子作用分子作用信号分子信号分子受体受体第二信使的概念第二信使的
21、概念(second messenger) 大多数激素类信号分子不能直接进入细胞，大多数激素类信号分子不能直接进入细胞，只能通过同膜受体结合后进行信息转换，只能通过同膜受体结合后进行信息转换，通常把细胞外的信号称为第一信使，而把通常把细胞外的信号称为第一信使，而把细胞内最早产生的信号物质称为第二信使。细胞内最早产生的信号物质称为第二信使。 第二信使第二信使第二信息至少有两个特征第二信息至少有两个特征: : 是第一信息同其膜受体结合后最早在细胞膜内侧是第一信息同其膜受体结合后最早在细胞膜内侧或胞浆中出现，仅在细胞内部起作用的信息分子；或胞浆中出现，仅在细胞内部起作用的信息分子； 能启动或调节细胞内
22、稍晚出现的反应。能启动或调节细胞内稍晚出现的反应。 目前公认的第二信息有目前公认的第二信息有cAMP、DG、IP3、cGMP和和Ca2+。Two-component regulatory system Life in the microbial world is no picnicEnviromental fluctucationsScarcity of NutritionCompetitors lurking at every turnStrong selective pressure How to survive?Therefore, There must be mechanisms by
23、 which bacteria receive signals from the environment and transmit them to the specific target to be regulated. 1、 What is signal transduction微生物必须对生存条件的变微生物必须对生存条件的变化迅速作出反应；必须与其化迅速作出反应；必须与其它个体或群体进行竞争，获它个体或群体进行竞争，获取并有效地利用营养物质。取并有效地利用营养物质。必须能够产生、感应、传必须能够产生、感应、传递信号，同时对多个相关递信号，同时对多个相关的操纵子进行系统性调控。的操纵子进行系
24、统性调控。Cellular responsenTwo-component regulatory systemnSensor kinasenEnzyme-linked receptornHydrolyzes ATP and phosphorylatesnPhosphate group transferred to response regulatornResponse regulatornRegulates expression of many genesWhere are 2-Component Systems Found? （2CS）nBacteria (absent in parasiti
25、c bacteria)nPlantsnFunginSlime moldsnNote: They are not found in mammals!The signal transduction modules via transmembrane receptorsnHisKin- histidine kinase (HK)nACyc- adenylate cyclasen STYK- serine-threonine-tyrosine kinasenGGDEF, HD-GYP, EAL nMCP- Methyl-accepting chemotaxis proteinsASM News 71
26、(2005):326-Not all sensor sense environmental signalsnAlmost 1/3 HK and close to 1/2 of the ACyc, DDGEF-, EAL-, STYK proteins are not membrane boundnThe cytoplasmic sensor domains are modulated by intracellular signalsCellular targets nSTYKn Regulate the expression or function of sigma, anti-sigma,
27、and anti-anti-sigma factors nHisK RR (response regulator)nTCS or 2CS (two component system)nc-di-GMP nAcycn Pseudomonas aeruginosa Acyc regulates the expression of type III secretion system genesn MCPs flagellar motorn CCW or CW rotationBacterial 2-Component Signal Transduction Systems1. Component 1
28、 : Sensor kinasei) Substrate receptor, signal recognition domain, input domain (periplasmic)ii) Signal transduction domain, membrane spanning regioniii) Autokinase domain, phosphorylation domain (cytoplasmic)a) ATP binding (sub) domainb) phosphorylation-phosphotransfer (sub)-domain2. Component 2 : R
29、esponse regulatori) Phosphorylation domainii) DNA binding domainSimplest case: transcriptional activator when phosphorylatedFirst component is typically (auto)-phosphorylated on a His residue and transfers to a Asp group on the response regulator (second component).细胞信号转导（细胞信号转导（cellular signal tran
30、sduction) 细胞通过位于胞膜或胞内的受体细胞通过位于胞膜或胞内的受体感受胞外信息分子的刺激，经复杂的感受胞外信息分子的刺激，经复杂的细胞内信号转导系统的转换来影响细细胞内信号转导系统的转换来影响细胞的生物学功能。胞的生物学功能。 双组分信号转导系统又名双组分磷酸接力系双组分信号转导系统又名双组分磷酸接力系统，是一种通过磷酰基团的转移来转导信号，并统，是一种通过磷酰基团的转移来转导信号，并控制基因转录的调节系统。控制基因转录的调节系统。 双组分是：传感蛋白激酶、应答调节蛋白。双组分是：传感蛋白激酶、应答调节蛋白。 双组分系统广泛存在于细菌中，也存在于真双组分系统广泛存在于细菌中，也存在于
31、真核微生物中；高等真核生物也利用磷酸化这一机核微生物中；高等真核生物也利用磷酸化这一机制转导信号。制转导信号。 Two-component signal transduction system (2CS/TCS)HPSignalDHTHPInput domainTransmitter domainReceiver domainoutput domainATPADPTranscription factorcytoplasminner membraneSensor kinase-HKResponse regulator- RR2CS phosphorelay* N-transmembrane do
32、main for signaling input* Transmitter domain (conserved histidine residue) nBifunctional as kinase or phosphatase (unphosphorylated form) * A conserved N-receiver domain (Aspartate residue)* A variable C-terminal output domainDifferent types of kinase domainn Micro Mol Biol Rev (2006) DecDifferent t
33、ypes of response regulatorsnA variable C-terminal output domainnLack C- effector domainnCheY associated with FliM (flagellar motor) nDNA-binding domainnEnzymenCheB (chemotaxis methylesterase)nRegA (cAMP phosphodiesterase)nC-di-GMP cyclase or phosphodiesterase Response RegulatorvN-terminal Receiver o
34、r Regulatory domainvC-terminal Effector domain:DNA-binding transcriptional regulatorenzymatic activity (CheB or RegA)protein-protein interactionsvCatalyze the transfer of phosphryl group from phospho-HK to conserved aspartic acid: phosphorylation results in conformational change of response regulato
35、r.vMany also catalyze auto-dephosphorylation.AspPClassical systemUnorthodox systemHybrid systemInput Transmitter Receiver Hpt Receiver OutputPPPPN G1 F G2HHDDPPPPN G1 F G2HHDDPPHN G1 F G2D(Rodrigue et al., 2000) HTHHTHHTHPhosphorelay Obligate phosphorelayHisAspHisATPADPPPPAspPBvgSBvgAAspPEvgAAspPBvg
36、ABordetella pertussis-(百日咳桿菌百日咳桿菌 ) BvgAS- a central regulator of virulence genes (pertussis toxin genes ptx and the genes for secretion pts)E. coli 2CS for virulence genes expressionOptional phosphorelayHisAspHisATPADPPPPAspPArcBArcAAspPAspPOmpRCheYAspPArcAArcAB- response to anaerobic environment (
37、 control 20 genes )Two-component systemTwo-component systemEssentials of phosphotransfer reactions1. Autophosphorylation: HK-His + ATP HK-HisP +ADP2. Phosphotransfer: HK-HisP + RR-Asp HK-His + RR-AspP3. Dephosphorylation: RR-AspP + H20RR-Asp + PiAlternative mechanisms1. HK Phosphatases: HK-HisP + PP
38、HK-His + Pi2. RR Phosphatases: RR-AspP + PP RR-Asp + PinProtein kinases can be divided into two groupsnMembrane-associated proteinsnSense environmental signalsnCytoplasmic proteinsnUsually part of a pathway that is more complex than a simple two-component mechanismnRespond to intracellular signalsHi
39、stidine kinase*Approximately 250 amino acids in length*Contains several conserved sequence motifsResponse regulatorMinimum of 120 aaConserved spacing of hydrophobic residues二元信号组分含有组氨酸激酶和反应调节子，有的简单，有的复杂，叫多步骤二元信号转导，即磷酸基团的转移过程中多几个步骤。From Lohrmann & Harter,2002HPt:His-containing phosphotransferModifica
40、tion of the 2-Component Pathway: The PhosphorelayModular Organization of tcsE. coliosmoregulationE. coliAnoxic RedoxRegulationE. colichemotaxisB. subtilissporulationHisPAspPN-G1-F-G2HisPN-G1-F-G2HisPN-G1-F-G2AspPAspPHisPHisPAspPN-G1-F-G2AspPEnvZOmpRHisPAspPHisPN-G1-F-G2AspPArcBArcASpoOASpoOBKinAKinB
41、SpoOFCheACheYCheBTwo component systemsnEarly 80snNtrB/NtrC - nitrogen assimilation nEnvZ/OmpR - osmoregulationnPhoR/PhoB - phosphate scavengingnDctB/DctD - dicarboxylate transportnVirA/VirG - virulence P. aeruginosa 135135个个Synechocystis PCC6803 82 82个个Anabaena PCC7120 211211个个双组份系统基因双组份系统基因 E. coli
42、 62个个Methanobacterium thermoautotrophicum 24个个 蛋白激酶蛋白激酶 n n NTP NTP n n NDP NDP 蛋白质蛋白质 蛋白质蛋白质- -n n Pi Pi n n Pi Pi H H2 2O O 蛋白磷酸酶蛋白磷酸酶 信号转导中的蛋白质信号转导中的蛋白质可逆磷酸化可逆磷酸化蛋白磷酸酶蛋白磷酸酶(protein phosphatase (protein phosphatase ，PPPP） 蛋白磷酸酶的分类与蛋白激酶相对蛋白磷酸酶的分类与蛋白激酶相对应，分为丝氨酸应，分为丝氨酸/ /苏氨酸型蛋白磷酸酶苏氨酸型蛋白磷酸酶和酪氨酸型蛋白磷酸酶。
43、有些酶具有双和酪氨酸型蛋白磷酸酶。有些酶具有双重底物特异性。重底物特异性。 对蛋白磷酸酶的研究还不如蛋白激对蛋白磷酸酶的研究还不如蛋白激酶那样深入。但两者的协同作用在细胞酶那样深入。但两者的协同作用在细胞信号转导中的作用是不言而喻的。信号转导中的作用是不言而喻的。Responses Mediated by 2-Component SystemsnBacterianAdaptation to changing osmolarity nSporulationnChemotaxisnN2 metabolismnRedox status of cellnLightnVirulence and many
44、 more!Two Component SystemsnRegulate:nnutrient acquisitionnNitrogennPhosphatenCarbonTwo Component SystemsnRegulate:nEnergy metabolismnElectron transport systemsnUptake machinerynCatabolic machineryTwo Component SystemsnRegulate:nVirulencenPlasmid transfernDegredative secretionsnToxin productionnAdhe
45、rence factorsnIntracelleular survival factorsTwo Component SystemsnRegulate:nAdaptation to physical or chemical aspects of the environmentnpHnOsmolaritynLight qualityTwo Component SystemsnRegulate:nComplex developmental pathwaysnSporulationnFruiting body formationnSwarmer cell production蓝细菌种类蓝细菌种类双组
46、份系统双组份系统可能的功能（突变株表型）可能的功能（突变株表型）Synechocystis PCC6803slr0474光信号传导光信号传导pisJl(s110041)向光性向光性ctrl(slrl044)滑移运动能力，对厚菌毛的生物发生是必要的滑移运动能力，对厚菌毛的生物发生是必要的slr2100参与蓝光信号传导途径参与蓝光信号传导途径slr2104参与控制，扩增，整合环核苷酸信号参与控制，扩增，整合环核苷酸信号s110337，slr0081突变株阻止在磷限制条件下碱性磷酸酶活性的诱导突变株阻止在磷限制条件下碱性磷酸酶活性的诱导cph2cph2表达显示暗诱导，由光负调节表达显示暗诱导，由光负
47、调节cph1 rcp1在暗中，在暗中，cphl，rcplmRNA增加，照光就降低增加，照光就降低plpA突变株不能在蓝光下生长突变株不能在蓝光下生长dspADpsA是一个参与化学感受的是一个参与化学感受的HPKslll905，s110698调节调节desB基因表达也参与调节基因表达也参与调节crh和和desD的表达的表达(低温适应低温适应)Synechoccus PCC 7942sphS sphR直接参与调节直接参与调节phoA转录转录sphs sphK突变株阻止在磷限制下碱性磷酸酶的诱导突变株阻止在磷限制下碱性磷酸酶的诱导sasA突变降低突变降低KaiBC表达。对维持有力的昼夜节奏是必须的表
48、达。对维持有力的昼夜节奏是必须的nblR参与整合细胞对营养缺失和光强应答，对胁迫条件下细胞存活起必要的作用参与整合细胞对营养缺失和光强应答，对胁迫条件下细胞存活起必要的作用cikA提供环境输入给昼夜节律振荡器途径中的一个关键因子提供环境输入给昼夜节律振荡器途径中的一个关键因子nblSNbls蛋白质参与控制应对高光和胁迫条件的光合器官改变过程蛋白质参与控制应对高光和胁迫条件的光合器官改变过程Anabaena PCC 7120hepK突变阻止异型胞外膜多糖合成和突变阻止异型胞外膜多糖合成和hepA的诱导（异型胞发育）的诱导（异型胞发育）patA突变株在固氮条件下长得很慢，且只分化出末端异形胞突变株
49、在固氮条件下长得很慢，且只分化出末端异形胞hetR在发育的异形胞中聚集，调节异形胞发育中起关键作用在发育的异形胞中聚集，调节异形胞发育中起关键作用Calothrix PCC 7601cphA，cphB, rcpA, rcpB光依赖的信号传导光依赖的信号传导Spirulina platensiscyac腺苷酸环化酶腺苷酸环化酶Synechoccus WH 7803devR固定空气中的氮。固定空气中的氮。devR基因产物对成熟异形胞发育是必须的。参与不直接与细胞氮状态起应基因产物对成熟异形胞发育是必须的。参与不直接与细胞氮状态起应答的感受途径答的感受途径nNarQ/ NarLnTwo-compon
50、ent regulatory systemnSenses presence of nitrate (NO3-) and nitrite (NO2-)nBinding phosphorylates NarQnTransfers phosphate to NarL (response regulator)nActivates genes involved in nitrate and nitrite metabolism and transportlegume plant RhizobiumsuccinateLmalatefumarateaspartateOxalacetateC4-dicarbo
