取得学位：博士(薬学)，学位授与番号：博甲第473号，学位授与年月日：平成14年3月22日,学位授与年：200

Nishida, Jun

西田 純

Kanazawa University Repository for Academic Resources

西田純氏名生年月日本籍学位の種類学位言Ｂ番号学位授与の日付学位授与の要件学位授与の題目三重県博士(薬学）博甲第４７３号平成１４年３月２２曰課程博士(学位規則第４条第１項）Structuralchangeofribosomesduringapoptosis：degradationandtranslocationofribosomalproteinsindoxorubicin-treatedJurkatcells(アポトーシス過程におけるリポゾームの構造変化：ドキソルビシンで処理したJurkat細胞でのリボソームタンパク質の分解と局在変化）松永司(薬学部・助教授）二階堂修(薬学部・教授）正宗行人(薬学部・教授）中西義信(医学系研究科・教授）平山明子(医学系研究科・講師）論文審査委員(主査）論文審査委員(副査）学位論文要 旨ＳＵＭＭＡＲＹChangesintlleamountandlocalizationofhumanlibosomalproteinsduringapoptosisweredeterminedWhentotallysatesofJurkatcellswhichhadbeeninducedtounde1狸oapoptosisbydoxorubicinwereanalyzedbyWesternblotting,degradationofthreeribosomalproteins,Ｓ18,Ｌ５ａｎｄＬ１４,ｗａｓdetectedat48haftertlleinductionofapoptosis、Decreasesinthealnountsofthesethreeribosomalproteinswelealsoobservedwhenribosome-enrichedfractionswereexalnined、Thesechangeswelepartlyabolishedbytlleadditionofthepan-caspaｓｅｉ血ibitorz-VAD-fink，Moreover，fbnnationofthe80Sribosomecolnplexappearedtobeinhibitedat48hafterapoptosisinduction・Ontlleotherhand,theextentofproteinsynthesis，assessedbymeasunng３５theincolporationof［Ｓ]MetmtobullKproteins,decreasedaseallyaslZhaftertlleadditionofdoxorubicin，Theseresultsindicatethatchangesintheamountofribosomalproteinsandtheoverallstrucmreofribosolne,occurinapoptosingcellsafterproteinsynthesisdeclines、Finallyうanalysesbyflowcytometrylinmunofluolescence，andWesternblottingshowedthatsixribosomalproteinsS15,ＰＯ,Ｌ5,Ｌ6,Ｌ7,L36aandL4I1wererelocalizedandexpressedatthecellsurfacedulingapoptoSis・TheaboveresultscollectivelyindicatetllatribosonlesarestructurallyaltelCdinapoptoticcellsfmlowinginactivationofproteinsynthesis．mＴＲＯＤＵＣＴＩＯＮDegradationofcllrolnosomalDNAshouldresultinthecompletecessationofgeneexpressionincellsfateｄｔｏｄｉｅ、However,therateofproteinsynthesisdecreasesevenbeforechromosomalDNAisdegraded，suggestingtheplesenceofanactivｅ-262-DmechanislnofinhibitinggeneexpressionattllelevelofProteinsyntllesisinapoptoticcells・Avarietyofproteinsandnucleicacidsareinvolvedinproteinsynthesis,andtlleribosomestandsattllecenteroftlletranslationallnachineryltisreasonabletoexpecttllattlleinllibitionofproteinsyntllesisinapoptoticcellsistheconsequenceofsomestructurallnodificatiOn(s）ofthesemolecules．’、fact,the28SribosomalRNAandtranslationalfactorshavebeenshowntoundergodegradationinapoptoticcells・mis，howeveLnotclearwhetllertllesechangesleadtostructuralchangesofribosomesortlleinllibitionofproteinsynthesis・Incontrast,changesinribosomalproteinsappeartobemorecomplex・Therea]resolnereportedchangesofribosomalproteinsinapoptoticcells；POdegradesorisstructurallymodified,S1istranslocatedtotllemembranebleb，ａｎｄＳ１９ｉｓｃovalentlydimelizedandsecretedMoreover,tllelnodifiedS19servesasachemoattractantspecificformacrophages、Tllesefindingsindicatethatribosomalproteinsarestructurallymodifiedinapoptoticcellsandlnayacqlliretlleextra-IibosomalfUnctionHowever,thepreviousstudiesdealtwithonlyalilnitednumberofribosomalproteins，andchangesintlleoverallstructureoftllelibosomeinapoptoticcellshavenotbeenintensivelyexalninedHereIanalyzedtlleamount，stmctureandlocalizationofriboSOInalproteinsinaPoptotichumanleukemiccellsusingpolyclonalantibodiesraisedagainstpeptidesandsubunitscorrespondingtopartialaminoacidsequencesof79humanribosomalprOteins．ＲＥＳＵＬｒＳ1)比C"αsejMzM伽肌r〃肋OSC伽ﾉPmre伽伽珈gcWP伽jJ1-1)DetectionofribosomalproteinsbyWesternblottinglfirstdetenninedchangesintheamountoflibosomalproteinsduringapoptosis・Seventy-ninekindsofrabbitantiseraweretestedfortlleirabilitytobindtonativelibosomalproteinｓｂｙＷｅｓｔｅｍｂｌotting・IWenty-eiglltantiseragavesignalsatthepositionscorrespondingtotlleexpectedmolecularmassesofllsmallsubunitproteins：Sa,S3a,Ｓ４Ｘ,Ｓ8,ｓ11,ｓ12,ｓ14,ｓ15,ｓ18,ＳＺ４ａｎｄＳ３０,andl71argesubunitproteins：L5,L7,L7a,L8,L9,L13,L13a,L14,L15,L17,L18,LZ8,L31,L34L35a,L36aandＬ39．Ithenexaminedchangesintheamountoftheseribosomalprotemsinapoptoticcells．1-2)DecreaseofS18,Ｌ5,andL14inapoptoticJuIkatcellsWhole-celllysateswerepreparedfromJurlKatcellsthathadbeenexposedtodoxombicinforvaliouslengtllsoftime,andsubjectedtoWesternblottingwiththeZ8kindsofanti-Iibosolnalproteinantisera・Althoughmostribosomalprotemsdidnotchangeuponapoptosisinduction，threeantiseradetecteddecreaseanddegradation、ThesignalofS18becamelessintenseindoxorubicin-treatedcells、Anti-L5andanti-L14antiseradetectedsignalswitllfastermobilitytllatdisappeaIedintlleprese､ceofthecolrespondingantigenpeptides，indicatingthatthesesignalsweledelivedfromdegradationproductsofeachribosolnalprotein．Thesechangeswitllthreeribosolnalproteinsallbecameapparentaｔ４８ｈｏｒｌａｔｅｒａｆｔｅｒｔｈｅｉｎｄｕｃｔｉｏｎｏｆapoptosis、Iconcludedtllattllleeribosolnalproteins，Ｓ１８，Ｌ５ａｎｄＬ１４,undergodegradationatlatestagesofapoptosis．．-263-1-3)Ribosomalproteindegradationduringcaspase-modiatedapoptosisInextaskediftheribosomalprotemdegradationoccunedduringcaspase-dependentapoptOSis・Julkatcellsweretreatedwithdoxorubicinintllepresenceoftllepan-caspaseinhibitorz-VAD-fmk,andwhole-celllysateswereanalyzedforribosolnalproteinsbyWesternblotting・AppearanceoftllepresumeddegradationproductsofL5andL14waslargelybtlloughnotcolnpletely§abolishedThedecreaseintlleintensityofintactS18wasonlymarginallyinhibitedbyz-VAD-fink，Theseresultsindicatedtllatdegradationofthethreeribosolnalproteinsoccursinamannerpartlydependentontlleactionofcaspases.．1-4)Absenceofthetlllcelibosomalproteinsintheribosolnelnextexalninedwhetllertlleabovechangesareobservedinthelibosomecomplex rｂｄｏｔｈｉｓ，ribosome-enrichfractionsweleplcparedfiDIndoxorubicin-treatedJurkatcellsandanalyzedbyWesternblotting・Decreaseofthethreeproteinswasalsoobservedintlleribosomefraction，However，tllepresunleddegradationproductsOfL5andL14werenotdetectable、TheseresultsindicatedthatthetlⅡeeribosomalproteinsaredegradedanddissociatedfrolntheribosomecomplex．1-5）DegradationoflibosomalproteinsinJulkatcellsundelgoingFas-mediatedapoptosisInextexaminediftheabovefindingsarerestrictedtocellsthatareundelgoingdoxorubicin-inducedapoptosis、Julkatcells，whichexpresstheapopotosisreceptorFas,wereincubatedwitllCH11，anagonisticanti-humanFasmonoclonalantibodybforvariouslengthsoftiｍｅ,andwhole-celllysatesweleanalyzedfortheribosomalproteinsS18，Ｌ５，ａｎｄＬ１４IfmndtllatallthetllreeribosomalproteinsdegradedinCH11-treatedJulkatcellsinamannersimilartotllatobservedwitlldoxorubicm-treatedcells・TheseresultsindicatedtllatdegradationoflibosolnalproteinsdoesnotdependonthewayofapoptosisinCIuction，ｂｕｔｉｓａｐｈｅｎｏｍｅｎｏｎｃｏｍｍｏｎlyObservedduringapoptosis．2)肋伽陀加伽/１，mario"〃伽80SrjboSo伽ＣＯ叩ﾉどjwmが"g〃叩川iSAbsenceofthetllreeribosomalproteinsmightinfluencethestructuralintegrityoflibosomes・Ｉｔｈｕｓｅｘａｍｉｎｅｄｔｈｅｆｍｎａｔｉｏｎｏｆｔｈｅｒibosomecomplexindoxorubicin-treatedJurkatcells，Whenribosolne-enlichedfractionspreparedfiDmdoxorubicin-treatedJurkatcellswereseparatedinanagarose/PolyacrylamidecompositegelandanalyzedbyWesternblotting・Thesignalfrolntlle80ScomplexdecIeasedastlleculmreperiodwasprolongedwhereastllatfromthe40Sor60Ssubunitwasalmostunchanged・TheseChangesbecameapparentat48hafterinductionofapoptosis，ａｎｄthisiswhentheribosomalproteins,Ｓ18,Ｌ５ａｎｄＬ１４,startedtodegrade・Theseresultsindicatedthatfbnnationoftlle80ScomplexwasinhibitedinapoptoticJulkatcellssimultaneouslywithdegradationofthetlⅡeeribosoInalproteins．3)DecMzsej"伽加陀QﾌﾞＰｍ花加川伽sjs血mzgcWP川jsInordertocorrelatetheabovefindingswithchangesintherateofproteinsynthesis,Jurkatcellsthathadbeentreatedwithdoxorubicinfbrvariouslengtllsoftilnewerepulse-1abeledwitllaradiolabeledaminoacid,andthealnomtoflabeledproteinｓｗａｓdeteminedbySDS-polyacrylamidegelelectrophoresisandautoradiography-264-SignalsderivedfromlabeledproteinsbecameundetectableZ4haftertheinductionofapoptosis・Theseresultsindicatedthattheinhibitionofproteinsynthesisprecedesthestructuralchangesofribosomes,thatis,degradationofthethleeribosomalproteinsanddisappearanceofthe80Sribosoｍｅcomplex．4)Z)wzsjDMiD"〃肋川川ﾉp、花加ZMieM枕ceQﾌﾞ〃〃〃jcceﾉZFSomeIibosomalproteinshavebeenshowntochangetlleirlocalizationduringapoptosis：S1istransferredtotllemembranebleb,andS19issecretedfromcellsaftercovalentmodification・ItllereforeconductedtllreediffelCntexperimentstoexaminewhetheranyribosomalproteinsarepresentonthesulfaceofapoptoticJu1katcells．4-1)FlowcytometrylfirstconductedafIowcytolnetryanalysis・Theseventy-nineantiseraweretestediftlleyboundtoplasmaInembrane-penneabilizedJulkatcellsmoreintenselythantointactcellsinamannerinhibitablebythepreSenceofcorrespondingantigenpeptides．'IWenty-eightantiserawereselectedincludingtlloserecognizinglOsmallsubunitproteins:Ｓａ,Ｓ2,S4X,Ｓ11,ｓ14,ｓ15,ｓ18,Ｓ２４ＳＺ９ａｎｄＳ３０,andl81argesubunitproteins:ＰＯ,Ｐ2,Ｌ3,Ｍ,Ｌ5,Ｌ6,Ｌ7,Ｌ10,Ｌ14,L23a,LZ9,L35a,Ｌ36,L36a,Ｌ37,Ｌ38,L39andLﾑ１１．Julkatcellsthathadbeentreatedwithdoxorubicinwelesuhjectedtoflowcytometrywiththose28antiseratodeteminethesurfaceexpressionofribosomalproteins・Sixantisera，thatrecognizeS15，ＰＯ，Ｌ５，Ｌ６，Ｌ３６ａａｎｄＬ４１，boundtoapoptoticcellsmoreefficientlytllantononnalcells，ThissuggeststhatthoselibosomeplotemsalepresentatthesurfaceofapoptoticJurlKatcells．4-2)IInmunofluorescencel[bfUrtherconfinnthesurfaceexpressionofribosomalproteinsinapoptoticcells，immunofluolescenceanalyseswereadopted・OfthesixantibodiespositiveinflowcytometrybfOurthatrecognizｅＰＯ,Ｌ５，L6andL36ashowedmoreintensebindingtothesulfaceofapoptoticcellsthantononnalcells．4-3)WesternblottingFinallyゥproteinspresentonthesurfaceofJurkatcellswereisolatedandanalyzedbyWesternblottingwiththreeanti-ribosomalproteinantiserathatwereusablefbrtllispurpose・JulkatcellswelCsulfacelabeledwitllbiotin,andtllelabeledproteinswereaffinity・purifiedusingavidin-conjugatedlnagneticbeadsWhentheisolatedsulfaceproteinswereanalyzedwitllanti-S1５，－L５，and-L36aantisera，signalscolTespondingtoallthetllreeribosomalproteinsweredetectableonlyinthesulfaceproteinspreparedfromapoptoticcells・TheseresultsindicatedthattllethreeribosomalproteinsexistatthesmfaceofapoptoticJurkatcells、TheresultsfromtheabovethreeexpelinJentsaccordedwitheachotherandindicatedthatfivelargesubunitproteins，ＰＯ，Ｌ５，Ｌ６，Ｌ３６ａａｎｄＭ１，andonesmallsubunitprotein，S15arerelocatedandexpressedatthesulfaceofJurkatcellsunde埴oingdoxombicin-treatedapoptosis．ＤＩＳＣＵＳＳＩＯＮlhereanalyzedchangesintheamountandlocalizationofeukaryoticribosomal-265-proteinsduringapoptosis・TheanalysisbyWesternblottingof281ibosomalproteinsrevealedtllattllreeoftllem，Ｓ18,Ｌ５ａｎｄＬ１４，underwentdegradationandwelcdissociatedfromribosomesduringapoptosisinducedbytheanticancerdmgdoxorubicinoranagonisticanti-FasmonoclonalantibodybTheseresultssuggestedthatribosolneslackingparticularlibosolnalproteinsexistinapoptoticce11s・Moreover，ＩｆＯｕｎｄｔｈａｔｔｈｅａｍｏｕｎｔｏｆｔｈｅ８０Ｓｒｉｂosomecomplexdecreasedinapoptoticcells、Thesechangesshouldlnakeribosomesunfimctionalinproteinsynthesis・However，tｈｅｙｄｏｎｏｔｓｅｅｍｔｏｂｅｔｈｅｍａ】orcauseoftlledecreasedrateofproteinsynthesisinapoptoticcells，becauseproteinsynthesisdeclineｄｗｅｌｌｂｅｆｏｒｅｔｈｅｃｈａｎｇｅｓｉｎｔｌｌｅlibosomalproteinsbecalnedetectable・Ispeculatetllatthedegradationofribosolnalproteinsoccurstocompletelyinactivategeneexpressio、attllefinalstepintlleprocessofapoptosis，Anotllerpossibleroleofthestructuralchangesofribosomesisregulationoftlleextra-ribosomalfimctionoflibosolnalproteins，Ifbundthatsixlibosomalproteinsmovedtothesulfaceofapoptoticcells、Ihypothesizethattheseproteinscaｎｂｅｍarker(s)forrecognitionofapoptoticcellsbyphagocyticcells、Apoptoticcellsarerapidlyandselectivelyeliminatedfromtheorganismbyphagocytosis，ａｎｄthisisdefinedbyspecificrecognitionoftalgetcellsbyphagosytes、Phagocyticcellscouldrecognizetheexternalizedribosomalproteinsandengulftheapoptoticcells、HoweveLtllisisonlyahypotllesisandneedstobevelifiedbyfUrthersmdies．学位論文審査結果の要旨西田純氏から提出された学位論文について,上記５名の審査委員による査読の後に平成１４年２月４日に口頭発表会が行われた。同日に最終の審査委員会が開かれ，審議の結果，以下のとおり判定した。本論文は，アポトーシス過程におけるリボソームの変化について解析したものである。タンパク質合成で中心的な役割を果たすリポソームタンパク質は，アポトーシスに伴って分解や局在性の変化を受ける例が報告されている。しかし，これまでの研究ではごく一部のリボソームタンパク質でしか調べられておらず，また細胞の種類に依存するケースも知られている｡本研究では７９種類のヒトリボソームタンパク質すべてについて網羅的に解析することを試みた。作製された７９種類のウサギ抗血清のうち，実際の解析に使用できたのは２８種類であったが，このうち３種類でアポトーシスに伴う抗原リボソームタンパク質の分解を認めた。また，これらの分解はリボソームからの遊離を引き起こし，８０Ｓリボソームの形成阻害に導くことが強く示唆された。一方，フローサイトメトリー，および蛍光抗体法を用いて，アポトーシス細胞表層におけるリボソームタンパク質の存在を調べ，６種類のリボソームタンパク質がアポトーシスの初期に細胞表面へ移行することを見出した。本研究は，アポトーシスの過程で複数のリボソームタンパク質が構造と局在性の変化を受けることを明らかにしたものであり，リボソームタンパク質の新しいリボソーム外機能を示唆したものと高く評価され，博士（薬学）の学位に値すると判定した。-266-

アポトーシス過程におけるリボソームの構造変化 : ドキソルビシンで処理したJurkat細胞でのリボソームタンパク質の分解と局在変化

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アポトーシス過程におけるリボソームの構造変化 : ドキソルビシンで処理したJurkat細胞でのリボソームタンパク質の分解と局在変化

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