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1、1细胞质细胞质细胞质细胞质cytoplasmcytoplasm2I.Cytoplasm(chapter6,7,8)Structure and Function of OrganellesII.Cytoplasm(chapter9)Cytoskeletonchapter 6 Structure and Function of Organelles involved in protein synthesis, processing and transportchapter 7 Structure and Function of Organelles involved in protein degr
2、adationchapter 8 Structure and Function of mitochondrion and peroxisome33for their discoveries concerning the structural and functional organization of the cell.MLAstyle:TheNobelPrizeinPhysiologyorMedicine1974.Nobelprize.org.NobelMediaAB2014.Web.18Mar2015.AlbertClaudeChristiandeDuveGeorgeE.PaladeThe
3、NobelPrizeinPhysiologyandmedicine,1974“method”Lysosome, peroxisome“ribosome”4Tostudytheprocessofproteinsecretionwithpancreaticepithelialcells.GeorgePaladeCellBiologyfinallymakespossibleacenturyolddream:thatofanalysis of diseases, at the cellular level - the first step towards their final control.We
4、never truly touch or see these wonderful tiny devices that keep every cell and every being alive - since they are far beyond what our senses can perceive unaided. But for us they are alive in our minds, close to our hearts, very much parts of the real world, just like the galaxies with their neutron
5、 stars and their pulsars are at the other end of the spectrum of dimensions of matter for our colleagues, the radio astronomers.5AlbertClaude,workingduringthe1930sand40sattheRockefellerInstitutehadadominatingrolebothfortheapplicationoftheelectronmicroscopeforthestudyofanimalcellsandforthedevelopment
6、ofthedifferentialcentrifugation.InparticularPaladeandcoworkerstudiedanetworkofsubmicroscopicmembranes,calledtheendoplasmic reticulum,originallydiscoveredbyClaudeandPorter.Theyshowedthatthereticulumcanbedescribedasamultiplyfolded,moreorlessdeflatedsackoccupyingmostofthecytoplasm.Paladediscoveredandde
7、scribedsmallgranularcomponentsnowknownunderthenameofribosomescoveringtheoutsideofthemembranesandheshowed,withothergroups,thattheribosomescarryouttheproteinsynthesisinthecell.Lysosomeshavenowbeenshown,bydeDuveandothers,tobeengagedinaseriesofcellularactivitiesduringwhichbiologicalmaterialmustbedegrade
8、d.deDuvehasnotonlyahighlydominatingroleinlysosomeresearch,heisalsothediscovererofanothercellcomponent,the peroxisome,thefunctionofwhichisstillenigmaticbutwhichmayverywellofferastoryasfascinatingasthatofthelysosomesinthefuture.6TheNobelPrizeinChemistry2004“for the discovery of ubiquitin-mediated prot
9、ein degradation 7HowdoProteinsfunction?(1)Quantity:synthesis(geneexpressionfromDNA-RNAtoprotein)(2)Quality:three-dimensionalstructure(conformation)(correctedfoldingandassembly)(3)RightLocalization：transportation(proteinsortingandothermechanisms)properactivity* Quantity *Quality *Right Localization8H
10、owareproteinregulated?（3）Translocationregulation:（4）ActivityRegulation：(alterationsofpost-translationalmodificationsalterationsofandinteractionwithothermolecules)otherway?（2）Qualitycontrol:（1）Quantityregulation：*up-regulationofgeneexpressionandproteinsynthesis*ERexportmisfoldedproteinandthen*down-re
11、gulationofgeneexpressionandproteinsynthesisHow?9Whatisproteindegradation?bywhichproteinisdigestedtoshortpeptidesoraminoacidbyaseriesofproteases.DegradationofproteinisaproteolyticprocessRemovalofproteinBreakdownofproteinDigestionofProteinTurnoverofprotein:degradationandgenerationGVYVECHS-L-Y-Qproteas
12、es10Protein synthesis and processingribosomeERGolgi ApparatuslysosomeProtein degradationproteasomeCellularorganellesworkforprotein11LevelA1.Concept:proteindegradation;ubiquitin-proteasomesystem.2.Thestructureandfunctionofthelysosome.LevelB1.Thebiogenesisofautophagosome2.Theformationoflysosome.3.Thew
13、orkingprincipleoftheproteasome.LevelC1.Thefunctionalconnectionoflysosome,endoplasmicreticulumandGolgiapparatus.2.Thebiologicalimplicationofautophagy.SYLLABUS12DegradationofProteinsTwo ways*Proteasomal degradation via ubiquitin-proteasome syetem *Lysosomal degradationvia autophagy and endocytosis13I、
14、LysosomeChapter 7.Structure and function of organelles involved in degradation of proteins14*singlemembraneenclosed*heterogeneityinshapeandsize*highelectrondensityClassicalmorphology:dense,round,andsmallcompartment.Shape and structure of lysosomeEM*0.2-0.5M15acid hydrolases40typeshydrolyticenzymes*M
15、embraneglucosylated membraneproteinsprotectthemselvesfromproteaseinthelumenH+ pumpATPhydrolysisandpumpH+intothelysosomes*Lumenacidenvironment,pH=4-516*Sortingofnewlysynthesizedlysosomalhydrolasesfromthetrans Golginetwork(TGN)tolysosomeviaendosome.*Deliveringdigestedmaterialsfromextracellularfluidbye
16、ndocytosistolysosmeviaendosome.MultivesicularbodyLysosomalenzymesendocytosedmaterialsFormation of lysosome (misterious)1717a.PhagocytosisEndocytosis(胞吞） in lysosome formationb.Pinocytosis Receptor-mediated endocytosisSpecialformofendocytosisinwhichacelluseslargeendocyticvesiclestoingestlargeparticle
17、ssuchasmicroorganismsanddeadcells.*Inprofessionalphagocytes-macrophagesandneutrophils.Aformofendocytosisinwhichacellcontinuallyingestsbitsoftheirplasmamembraneandextracellularfluidintheformofsmallpinocyticvesicles.*Inalleucaryoticcells.Aformofendocytosisinwhichacelltakesupspecificmacromoleculesbound
18、withplasmamembranereceptorsfromtheextracellularfluidsuchascholesterolandsomekindsofsignalingmolecules.*Inmostofanimalcells.18multivesicularbodylateendosomeTransportvesiclesEndosome（内体）EarlyendosomeSmall,irregularlyshapedorganells,containingendocytosedmolecules.Thesiteswherethelysosomalenzymestranspo
19、rttofromTGN.LateendosomeMildlyacidicinterior.Thesiteswherethehydrolyticdigestionofendocytosedmoleculesbegin.Endosomeisaseriesofspecialorganellescorrelatedwithendocytosisandextracellularproteinandplasmamembraneproteindegradation.Maturation19MultivesicularbodyLysosomalenzymesendocytosedmaterialsendocy
20、tosisLysosomalproteinsorting+2020multivesicularbodylateendosomeTransportvesicles Degration of proteins from endocytosis in lysosomedegradedprotein:*extracellularmaterials;*plasmamembraneproteinsFunction of lysosome21Theprocessbeginswiththeenclosureoftheorganellessuchasmitochondrion,ERbyadoublemembra
21、neofunknownorigin,called autophagosome.Autophagyisoneofthemajorintracellulardegradationprocessinwhichcytoplasmicmaterialsaredeliveredtoanddegradedinlysosometogetcellularrenovationandhomeostasis. Degration of intracellular proteins by autophagy in lysosome22MitochondriondamagedOxidativestressCellfate
22、Example1:degradationofdamagedmitochondrionExample2:degradationofcytosolicproteinp62P62degradationUbiquitouslyexpressedcellularproteinAutophagyimpairmentDiseaseExample3:Autophagyinnutrient-starvedcellsautolysosomeautophagosomeERfragmentsmouseembryonicfibroblastautophagyapoptosisnecrosis23The function
23、al implications of autophagyEliminationofcytoplasmicmaterialstogetcellularrenovationandhomeostasis.a.Removeofsenescentanddamagedorganells;b.Producenewbuildingblocksandenergyforturnoverofcytoplasmiccomponents.Steadystateofthecell(basallevel)c.Saveenergyandgetnutrientsfromitselfforcellsurvivalundersta
24、rvation.24Autophagy in health and diseaseStressstateofthecellImpairmentoractivationofautophagycontributestopathogenesisofdiversediseases.fromneuron-degenerativediseasesuchasParkinsondiseasetoinflammatorydisorderssuchasCrohnsdisease.The functional implications of autophagy2525Ref:Physiologicalandpath
25、ologicalofAutophagy26Take-homemessages:http:/www.ncbi.nlm.nih.gov/pmc/articles/PMC3252826/2.Autophagiccelldeath?1.Howcanweinterfereautophagytotreatdisease?Potentialinferencemethod:smallmolecules,geneticmethodsoranythingelse?27* cytoplasmic components* the organelles proteinWhichkindofproteinsaredegr
26、adedinlysosomebyautophagy?Whichkindofproteinsaredegradedinlysosomebyendocytosis?* extracellular proteins* plasma membrane proteins28Notjust“CellularGarbageCan”Eliminationofextracellularmaterials;Productionofnutrients;Productionofnewbuildingblocksforcellrenovation;Regulationofhormonesecretion（crinoph
27、agy）The functional implications of lysosomal degradation system for cell 29Pathology of Lysosome*Pneumoconiosis(尘肺）、RheumatoidArthritis（类风湿）*Lysosomestoragedisease(LSD）esp.Inclusioncelldisease（I细胞病）;Gaucherdiseasehttp:/ Lysosomes arespecializedfortheintracellulardigestionofmacromolecules.2. Lysosome
28、scontainuniquemembraneproteinsandawidevarietyofsolublehydrolyticenzymesthatoperatebestatpH5,whichistheinternalpHoflysosomes.AnATP-drivenH+pumpinthelysosomalmembranemaintainsthislowpH.3.NewlysynthesizedlysosomalhydrolasesaretransferredintothelumenoftheER,transportedthroughtheGolgiapparatus,andthenfro
29、mthetransGolginetworktoearlyendosomesbytransportvesicles.4.Autophagyisthemajorintracellulardegradationsystembywhichcytoplasmicmaterialsaredeliveredtoanddegradedinlysosometogetcellularrenovationandhomeostasis.Muchofthematerialthatisendocytosedisdeliveredtoendosomesandthentolysosomes,whereitisdegraded
30、byhydrolyticenzymes.Essential Concepts31II、ProteasomeChapter 7.Structure and function of organelles involved in degradation of proteins3232Q1.WhyproteindegradationisATP-dependent?TheCell-ateemingmini-workshopInthecell,proteinsarebeingbuiltupandbrokendownallthetime.Foreverythingtofunctionoptimally,th
31、ecellalsohasanintegralcheckpointwherethecompositionofvariousproteinsiscontrolled.Unlikeinthespontaneousproteinbreakdownthatfoodundergoesinourintestines,breaking down proteins inside cells requires energy.Thiswaslongaresearchmystery.3333*size：20S、26SinvisibleunderLMorEM*non-membraneenclosedComparasio
32、n:Ribosome(EM)Ribosome：80S，15-20nmMorphology of proteasome*inbothofthenucleusandcytoplasmStainingwithproteasomefluoresencemarker（Sun X，Yan S et al. unpublished）3434Chemicalcomposition：protein complexesStructure：barrel-like(cylinder)20Scorecoreparticles“proteasesactivesites”19Scapregulatoryparticles“
33、recognizationsites”Structure of proteasome3535Example 1：Cyclin G1cyclinSynthesisinG1,DegradationinSphaseExample 2 ：p53 p53p53*non-stress(Resting):lowlevelP53(unneede,degradation)MDM2MDM2MDM2TorapidlydegradeunneededordamagedproteinsFunctions of proteasomes36Example 3 ：damaged protein misfolded protei
34、nsProtein quality control in ER and proteasomeTorapidlydegradeunneededordamagedproteinsFunctions of proteasomesER lumenproteasome37TheCell-ateemingmini-workshop.ThankstothisyearsNobelLaureates,however,weknowthatthisformofbreakdownisanextremelydetailedcontrolprocessinwhichtheproteintobedestroyedismar
35、kedwithaspeciallabel.Thishappensthroughaseriesofchemicalreactions,asshownbelow.TheNobelPrizeinChemistry2004http:/www.nobelprize.org/nobel_prizes/chemistry/laureates/2004/illpres/2_cell.htmlproteins degradation in proteasomeQ2.Who decide certain protein need to be degraded?38ubiquitin:asmallpeptideof
36、76aa,ubiquitousandconserved.Ubiquitin-linked protein: ubiquitinated protein, ubiquitination modification of proteinAsatag,atleastfourubiquitinpeptidescovalentlybindtothetarget(substrate)proteinsfordegradation.proteins degradation in proteasome(ubiquitin-proteasomesystem,UPS)proteinubiquitin-linked p
37、roteins 39391.Proteinsarelinkedbypolyubiquitin(polyubiquitinated)E1:ActivatingenzymeE2:ConjugatingenzymeE3:ubiquitinLigase2.Recognizationby“cap”3.Translocationintothecore4.Digestiontoshortpeptidesoraminoacidbyproteases.proteins degradation in proteasomeATP-dependentATP-dependent40* ATP consumption *
38、 Ubiquitin recognization * A1(why).ATP dependent protein degradation* A2(who).Ubiquitin-mediated protein degradationproteins degradation in proteasome4141To rapidly degrade unneeded or damaged proteinshttp:/www.nobelprize.org/nobel_prizes/chemistry/laureates/2004/animation.htmlproteins degradation i
39、n proteasome for cellsBiological function4242Resting:p53degradation,lowlevelP53Stress:decreaseofp53degradation,highlevelp53DNAdamageExample2：p53,“stresssensor”p53-UbSubstrateprotein：MDM2,p53E3ligaseforp53MDM2P53accumulationCellcyclearrestDNArepairGeneexpressionp53p53MDM2MDM2MDM2P53degradationMDM2deg
40、radationUbUb4343Bortezomib(Velcade),inhibitorofproteasomebecomesanticancerdrug.1.Clinicaltreatmentofmultiplemyeloma.(approvedbyFDAin2003)2.Preclinicaltrial:B-cellcancers3.Animaltrial:pancreaticcancerLightBlue:Bortezomib，Bindingtosubunitsof20Scoreproteins degradation in proteasome in disease4444TheNo
41、belPrizeinChemistry2004“ThankstotheworkofthethreeLaureatesitisnowpossibletounderstandatmolecularlevelhowthecellcontrolsanumberofcentralprocessesbybreakingdowncertainproteinsandnotothers.Examplesofprocessesgovernedbyubiquitin-mediatedproteindegradationarecelldivision,DNArepair,qualitycontrolofnewly-p
42、roducedproteins,andimportantpartsoftheimmunedefence.Whenthedegradationdoesnotworkcorrectly,wefallill.Cervicalcancerandcysticfibrosisaretwoexamples.Knowledgeofubiquitin-mediatedproteindegradationoffersanopportunitytodevelopdrugsagainstthesediseasesandothers.”“forthediscoveryofubiquitin-mediatedprotei
43、ndegradation45Two major organelles for protein degradation in cellsLysosomeProteasome*Extracellularmaterials*Plasmamembrane*Cytoplasmiccomponentsorganells,long-liveproteins*Stressresponseproteins*Short-liveproteins*misfoldedproteinsUbiquitin-linkedsubstratesEndocytosis-relatedAutophagy-relatedSynthe
44、sisofLysosomalenzyme-relatedTissueandcellRenovationHomeostasisCellcycle,geneexpression,stressresponsesandsoonCharacristics eventsSubstratesImplications46Howareproteinregulated?*otherway:degradationofprotein（2）Qualitycontrol:（1）Quantityregulation：*up-regulationofgeneexpressionandproteinsynthesis*ERex
45、portmisfoldedproteinandthen*down-regulationofgeneexpressionandproteinsynthesisdegradationProteasomal degradation in the cellsQuantity and quality control system4747Protein synthesis and processingribosomeERGolgi ApparatuslysosomeendosomeProtein degradationProteasome48RibosomeUbiquitin-proteasomeLysosomeEndosomeEndoplasmicReticulumGolgiApparatusMitochondrionPeroxisomeNon-membrane-enclosedMembrane-enclosedCytoplasm-organelles
