&RPELQDWRULDORPLFVDQDO\VLVUHYHDOVSHUWXUEHGO\VRVRPDOKRPHRVWDVLVLQFROODJHQ9,, GHILFLHQWNHUDWLQRF\WHV
.HUVWLQ7KULHQH%M|UQ$QGUHDV*UQLQJ2OLYLHU%RUQHUW$QLND(U[OHEHQ-XQD/HSSHUW
,RDQQLV $WKDQDVLRX (NNHKDUG :HEHU 'LPLWUD .LULWVL $OH[DQGHU 1\VWU|P 7KRPDV
5HLQKHFNHO 5ROI %DFNRIHQ &ULVWLQD +DV /HHQD %UXFNQHU7XGHUPDQ -|UQ
'HQJMHO
'HSDUWPHQWRI'HUPDWRORJ\0HGLFDO&HQWHU8QLYHUVLW\RI)UHLEXUJ*HUPDQ\ &HQWUHIRU%LRORJLFDO6\VWHPV$QDO\VLV=%6$8QLYHUVLW\RI)UHLEXUJ*HUPDQ\ 'HSDUWPHQWRI&RPSXWHU6FLHQFH8QLYHUVLW\RI)UHLEXUJ*HUPDQ\
,QVWLWXWH RI 3K\VLRORJLFDO &KHPLVWU\ 0HGLFDO )DFXOW\ 0DUWLQ /XWKHU 8QLYHUVLW\ +DOOH
:LWWHQEHUJ*HUPDQ\ ,QVWLWXWHRI0ROHFXODU0HGLFLQHDQG&HOO5HVHDUFK)DFXOW\RI0HGLFLQH8QLYHUVLW\RI)UHLEXUJ *HUPDQ\ &HQWUHIRU%LRORJLFDO6LJQDOOLQJ6WXGLHV%,2668QLYHUVLW\RI)UHLEXUJ*HUPDQ\ 'HSDUWPHQWRI%LRORJ\8QLYHUVLW\RI)ULERXUJ6ZLW]HUODQG 5XQQLQJWLWOHSHUWXUEHGKRPHRVWDVLVLQ&GHILFLHQWNHUDWLQRF\WHV &RUUHVSRQGLQJDXWKRUV -|UQ'HQJMHO/HDG&RQWDFW'HSRI%LRORJ\8QLYHUVLW\RI)ULERXUJ&KHPLQGX0XVpH )ULERXUJ6ZLW]HUODQGHPDLOMRHUQGHQJMHO#XQLIUFKSKRQHID[
/HHQD %UXFNQHU7XGHUPDQ 'HS RI 'HUPDWRORJ\ 0HGLFDO &HQWHU 8QLYHUVLW\ RI )UHLEXUJ +DXSWVWU )UHLEUXJ *HUPDQ\ HPDLOEUXFNQHUWXGHUPDQ#XQLNOLQLNIUHLEXUJGH SKRQHID[
http://doc.rero.ch
Published in "Molecular & Cellular Proteomics doi: 10.1074/mcp.RA117.000437, 2018"
which should be cited to refer to this work.
$EEUHYLDWLRQV %+%HQMDPLQL+RFKEHUJ %0EDVHPHQWPHPEUDQH &FROODJHQ9,, &$)FDQFHUDVVRFLDWHGILEUREODVWV (&0H[WUDFHOOXODUPDWUL[ '(-=GHUPDOHSLGHUPDOMXQFWLRQ]RQH '(%G\VWURSKLFHSLGHUPRO\VLVEXOORVD 06PDVVVSHFWURPHWU\ 1+.QRUPDOKXPDQNHUDWLQRF\WHV 3&$SULQFLSDOFRPSRQHQWDQDO\VLV 6&&VTXDPRXVFHOOFDUFLQRPD 6,/$&VWDEOHLVRWRSHODEHOOLQJE\DPLQRDFLGVLQFHOOFXOWXUH 627$VHOIRUJDQL]LQJWUHHDOJRULWKP
http://doc.rero.ch
6XPPDU\
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
.H\ZRUGV LQIODPPDWLRQ ILEURVLV NHUDWLQRF\WH PDVV VSHFWURPHWU\ FROODJHQ SURWHDVHV O\VRVRPHDXWRSKDJ\7*)EH[WUDFHOOXODUPDWUL[G\VWURSKLFHSLGHUPRO\VLVEXOORVD'(%
ϰ ,QWURGXFWLRQ
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
%LDOOHOLF ORVVRIIXQFWLRQ PXWDWLRQV RI WKH JHQH COL7A1 HQFRGLQJ & FDXVH G\VWURSKLF HSLGHUPRO\VLV EXOORVD '(% DQ LQKHULWHG VNLQ IUDJLOLW\ GLVRUGHU FKDUDFWHUL]HG E\ D EURDG VSHFWUXP RI FOLQLFDO PDQLIHVWDWLRQV VNLQ EOLVWHULQJ DEQRUPDO ZRXQG KHDOLQJ H[FHVVLYH VFDUULQJRIWHQUHVXOWLQJLQDJJUHVVLYHVNLQFDQFHU+RZHYHUWKHYDULHW\DQGPXOWLWXGHRI WKHV\PSWRPVDUHQRWHDVLO\H[SODLQHGE\WKHXQGHUO\LQJCOL7A1 PXWDWLRQV
7RZDUG WKLV HQG µRPLFV¶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
ϱ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in vitroDQGLQKXPDQ'(%VNLQin vivo7KXVZHSURYLGH DGHWDLOHGLPDJHRIG\VUHJXODWHGPROHFXODUSDWKZD\VLQJHQHWLFVNLQIUDJLOLW\DQGVKRZWKDW ORVVRI&OHDGVWRIDUPRUHFRPSOH[FKDQJHVDVDQWLFLSDWHG,PSRUWDQWO\RXUVWXG\UHYHDOV LQWULQVLFHSLGHUPDODOWHUDWLRQVGLUHFWO\FDXVHGE\&GHILFLHQF\DQGLQFUHDVHG7*)EVLJQDOLQJ LQHSLGHUPDONHUDWLQRF\WHVDVLQVWLJDWRUVRIGHUPDOILEURVLV
ϲ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
3DWLHQW'(%KDGDVHYHUHJHQHUDOL]HG'(%ZLWKODFNRI&LQWKHDIIHFWHGVNLQDVVKRZQLQ DVNLQELRSV\WDNHQIURPVFDUUHGQRWDFXWHO\EOLVWHUHGRULQIODPHGVNLQQH[WWRWKHOHIWNQHH 6XSSOHPHQWDO)LJXUH6$VNLQELRSV\WDNHQIURPDFOLQLFDOO\XQDIIHFWHGVNLQSDWFKRQWKH OHIW NQHH UHYHDOHG & UHH[SUHVVLRQ DW DURXQG RI WKH ELRSV\¶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
http://doc.rero.ch
ϳ
$OOVWDWLVWLFDOWHVWVZHUHFRUUHFWHGIRUPXOWLSOHWHVWLQJDVRXWOLQHGLQWKHUHVSHFWLYHSDUDJUDSKV
&HOOO\VDWHVDQG(&0LVRODWLRQRINHUDWLQRF\WHVIRU06DQDO\VLV
$6,/$&DSSURDFKZDVXVHGWRFRPSDUH'(%NHUDWLQRF\WHVWRQRUPDOFRQWUROVE\06EDVHG SURWHRPLFV [ IXOO\ 6,/$&ODEHOHG NHUDWLQRF\WHV ZHUH VHHGHG RQ FP FHOO FXOWXUH
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±+&OS+$OLTXRWVRIWKHGLIIHUHQW(&0VDPSOHV RUFHOOO\VDWHVZHUHXVHGIRU:%DQDO\VLVRUPL[HGDQGSUHSDUHGIRU06DQDO\VLVLQRUGHU WRWHVWDFFXUDWHPL[LQJUDWLRV0L[LQJZDVDGMXVWHGIRUDFWXDO06DQDO\VLVDIWHUZDUGV
06VDPSOHSUHSDUDWLRQ
In-gel digestion.6DPSOHVZHUHKHDWHGLQ6'6Ǧ3$*(ORDGLQJEXIIHUUHGXFHGZLWKP0'77 IRUPLQDW&DQGDON\ODWHGXVLQJP0LRGRDFHWDPLGHIRUPLQDWURRPWHPSHUDWXUH 3URWHLQPL[WXUHVZHUHVHSDUDWHGRQJUDGLHQWJHOV7KHJHOODQHVZHUHFXWLQWRHTXDO VOLFHVWKHSURWHLQVZHUHLQǦJHOGLJHVWHGZLWKWU\SVLQDQGWKHUHVXOWLQJSHSWLGHPL[WXUHVZHUH SURFHVVHGRQ67$*(WLSVDQGDQDO\]HGE\/&0606 0DVV6SHFWURPHWU\ 06PHDVXUHPHQWVZHUHSHUIRUPHGRQDQ/742UELWUDS;/PDVVVSHFWURPHWHUFRXSOHGWRDQ $JLOHQWQDQRIORZ±+3/&+3/&±FROXPQWLSVIXVHGVLOLFDZLWKPLQQHUGLDPHWHUZHUH VHOISDFNHGZLWK5HSURVLO±3XU2'6±WRDOHQJWKRIFP6DPSOHVZHUHDSSOLHGGLUHFWO\
http://doc.rero.ch
ϴ RQWRWKHFROXPQZLWKRXWDSUH±FROXPQ$JUDGLHQWRI$DFHWLFDFLGLQZDWHUDQG% DFHWLFDFLGLQDFHWRQLWULOHLQZDWHUZLWKLQFUHDVLQJRUJDQLFSURSRUWLRQZDVXVHGIRUSHSWLGH VHSDUDWLRQORDGLQJRIVDPSOHZLWK%VHSDUDWLRQUDPSIURP±%ZLWKLQPLQ7KH IORZUDWHZDVQ/PLQDQGIRUVDPSOHDSSOLFDWLRQQ/PLQ7KHPDVVVSHFWURPHWHUZDV RSHUDWHGLQWKHGDWD±GHSHQGHQWPRGHDQGVZLWFKHGDXWRPDWLFDOO\EHWZHHQ06PD[RI[
LRQV DQG 0606 (DFK 06 VFDQ ZDVIROORZHG E\ DPD[LPXP RIILYH 0606 VFDQV LQWKH OLQHDULRQWUDSXVLQJQRUPDOL]HGFROOLVLRQHQHUJ\RIDQGDWDUJHWYDOXHRI3DUHQWLRQV ZLWKDFKDUJHVWDWHIURP] DQGXQDVVLJQHGFKDUJHVWDWHVZHUHH[FOXGHGIRUIUDJPHQWDWLRQ 7KH PDVV UDQJH IRU 06 ZDV P] ± 7KH UHVROXWLRQ ZDV VHW WR 06 SDUDPHWHUV ZHUH DV IROORZV VSUD\ YROWDJH N9 QR VKHDWK DQG DX[LOLDU\ JDV IORZ LRQ± WUDQVIHUWXEHWHPSHUDWXUH&
,GHQWLILFDWLRQRISURWHLQVDQGSURWHLQUDWLRDVVLJQPHQWXVLQJ0D[4XDQW
7KH 06 UDZ GDWDILOHVZHUH XSORDGHG LQWR WKH0D[4XDQW VRIWZDUH YHUVLRQ IRU SHDN GHWHFWLRQJHQHUDWLRQRISHDNOLVWVRIPDVVHUURUFRUUHFWHGSHSWLGHVDQGIRUGDWDEDVHVHDUFKHV $IXOOOHQJWK8QL3URWKXPDQGDWDEDVHDGGLWLRQDOO\FRQWDLQLQJFRPPRQFRQWDPLQDQWVVXFK DVNHUDWLQVDQGHQ]\PHVXVHGIRULQ±JHOGLJHVWLRQEDVHGRQ8QL3URWKXPDQ)$67$YHUVLRQ 6HSWHPEHUHQWULHVZDVXVHGDVUHIHUHQFH&DUEDPLGRPHWK\OF\VWHLQHZDVVHW DVIL[HGPRGLILFDWLRQDQGSURWHLQDPLQR±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µPDWFKEHWZHHQUXQ¶ RSWLRQ PLQ ZDV
ϵ
XVHG$QQRWDWHGVSHFWUDRIVLQJOHSHSWLGHEDVHG SURWHLQ,'VDUHSURYLGHGDV6XSSOHPHQWDO )LOHV6DQG6
7KH PDVV VSHFWURPHWU\ SURWHRPLFV GDWD KDYH EHHQ GHSRVLWHG WR WKH 3URWHRPH;FKDQJH &RQVRUWLXP YLD WKH 35,'( SDUWQHU UHSRVLWRU\ ZLWK WKH GDWDVHW LGHQWLILHU 3;' 5HYLHZHUDFFRXQWGHWDLOV8VHUQDPHUHYLHZHU#HELDFXN3DVVZRUGP40]-4)Z
ϭϬ 5HVXOWV
'LVHDVHUHOHYDQWWUDQVFULSWLRQDOFKDQJHVLQSULPDU\KXPDQ'(%NHUDWLQRF\WHV
'(% NHUDWLQRF\WHV DUH FKDUDFWHUL]HG E\ D ORVV RI & )LJXUH $ DQG VNLQ RI DIIHFWHG LQGLYLGXDOVH[KLELWVPHFKDQLFDOIUDJLOLW\LQIODPPDWLRQH[FHVVLYHVFDUULQJDQGILEURVLV)LJXUH % :KHUHDV GLVHDVH FRQWULEXWLRQV RI PHVHQFK\PHGHULYHG FHOOV KDYH EHHQ H[WHQVLYHO\ VWXGLHG WKH LQIOXHQFH RI HSLWKHOLDO FHOOV UHPDLQV REVFXUH ZKLFK OHG XV WR LQYHVWLJDWH WKH HSLGHUPDO UHVSRQVHV WR ORVV RI & VHH 7DEOHV DQG ([SHULPHQWDO 3URFHGXUHV IRU GHVLJQDWLRQRIXVHGSULPDU\FHOOV
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Įȕ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
http://doc.rero.ch
ϭϭ
IRXQG WR EH GRZQUHJXODWHG ZKLOH H[SUHVVLRQ RI JHQHV HQFRGLQJ IRU SURWHLQV LQYROYHG LQ UHVSRQVHWRLQMXU\DQG7*)EVLJQDOLQJZHUHXSUHJXODWHGLQ'(%NHUDWLQRF\WHV
$OWHUHG(&0SURWHRPHFRPSRVLWLRQGXHWRORVVRI&
7RFRPSOHPHQWWUDQVFULSWRPHDQDO\VHVDQGGLUHFWO\DVVHVVWKHH[WUDFHOOXODUFRQVHTXHQFHVRI ORVVRI&ZHSHUIRUPHGTXDQWLWDWLYHSURWHRPHDQDO\VHVRIWKHNHUDWLQRF\WH(&0XVLQJ06 &HOOV ZHUH FXOWXUHG LQ WKH SUHVHQFH RI DVFRUEDWH WR HQVXUH FRUUHFW SRVWWUDQVODWLRQDO PRGLILFDWLRQDQGVWDEOHWULSOHKHOL[IRUPDWLRQRIFROODJHQV$IWHUVL[GD\VFHOOVZHUHUHPRYHG FDUHIXOO\ IURP FXOWXUH GLVKHV XVLQJ 7ULWRQ3%6 IROORZHG E\ 1+2+3%6 )LJXUH $
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±3$*(DQGSURWHLQVLQJHOGLJHVWHGZLWKWU\SVLQ *HQHUDWHGSHSWLGHVZHUHLGHQWLILHGDQGTXDQWLILHGE\UHYHUVHSKDVH/&0606 SURWHLQVZHUHLGHQWLILHGLQWZRELRORJLFDOUHSOLFDWHVHDFKRIZKLFKFRXOGEHTXDQWLILHGLQ DW OHDVW RQH VDPSOH DYHUDJH FRUUHODWLRQ FRHIILFLHQW 6XSSOHPHQWDO )LJXUH 6 DQG 6XSSOHPHQWDO7DEOH6VHH6XSSOHPHQWDO)LOH6IRUDQQRWDWHGVSHFWUDRIVLQJOHSHSWLGH,'V 'XH WR WKH KLJK VHQVLWLYLW\ RI PRGHUQ PDVV VSHFWURPHWHUV HYHQ VPDOO DPRXQWV RI FRQWDPLQDWLQJLQWUDFHOOXODUSURWHLQVDUHGHWHFWHGLQWKH(&0IUDFWLRQ7RGLVWLQJXLVKWKHPDQG
ϭϮ
WR REWDLQ D FRPSUHKHQVLYH OLVW RI SRWHQWLDO WUXH (&0 SURWHLQV WKH RULJLQDO SURWHLQ OLVW ZDV ILOWHUHG SURWHLQV GHILQHG LQ WKH ³PDWULVRPH´ DQG WKRVH DQQRWDWHG E\ WKH *2 WHUPV ³H[WUDFHOOXODU´DQGRU³FHOODGKHVLRQ´ZHUHGHILQHGDVWKH³(&0SURWHRPH´7KLVOHGWRD OLVW RI LGHQWLILHG (&0 SURWHLQV RI ZKLFK ZHUH TXDQWLILHG LQ PLQLPDOO\ RQH VDPSOH 6XSSOHPHQWDO7DEOH6
'(%DQGFRQWURO(&0VZHUHFOHDUO\GLVWLQJXLVKHGE\DSULQFLSDOFRPSRQHQWDQDO\VLV3&$ ZLWK'(%(&0EHLQJPRUHKHWHURJHQHRXVWKDQFRQWURO(&06XSSOHPHQWDO)LJXUH67KXV ORVV RI & KDV JOREDO HIIHFWV RQ WKH FRPSRVLWLRQ RI NHUDWLQRF\WHGHULYHG (&0 7R LGHQWLI\ JURXSV RI FRǦ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ĮȕZHUHGRZQUHJXODWHGRQSURWHLQOHYHO 3URWHLQVWKDWDUHLQYROYHGLQWLVVXHUHSDLUVXFKDVILEURQHFWLQDQGWHQDVFLQ&ZHUHXSUHJXODWHG FKDQJHV EHLQJ LQGLFDWLYH RI LQFUHDVHG 7*)E DFWLYLW\ FDXVHG E\ GLVUXSWLRQ RI LQWHJULQ Įȕ VLJQDOLQJDQGLQOLQHZLWKLQFUHDVHGLTBP1 H[SUHVVLRQ)LJXUH&
7DNHQ WRJHWKHU ORVV RI & OHG WR SURQRXQFHG FKDQJHV LQ WKH PLFURHQYLURQPHQW RI '(% NHUDWLQRF\WHV (VSHFLDOO\ EDVHPHQW PHPEUDQH FRPSRQHQWV GLUHFW DQG LQGLUHFW ELQGLQJ SDUWQHUV RI & ZHUH IRXQG WR EH GRZQUHJXODWHG ZKLOH SURWHLQV WKDW DUH LQYROYHG LQ ZRXQG
ϭϯ
KHDOLQJ ZHUH XSUHJXODWHG LQ '(% NHUDWLQRF\WHGHULYHG (&0 ODUJHO\ FRQVLVWHQW ZLWK WUDQVFULSWRPHDQDO\VHV
/RVVRI&OHDGVWRDQLQFUHDVHRILQIODPPDWLRQPDUNHUV
7RGLVWLQJXLVKWUDQVFULSWLRQDOIURPWUDQVODWLRQDORUSRVWWUDQVODWLRQDOUHJXODWLRQPHFKDQLVPVZH GLUHFWO\ DQDO\]HG WKH FRUUHODWLRQ EHWZHHQ P51$ DQG SURWHLQ DEXQGDQFH GLIIHUHQFHV FRPSDULQJ '(% WR FRQWURO NHUDWLQRF\WHV 7KH GLIIHUHQFHV LQ JHQH H[SUHVVLRQ DQG SURWHLQ DEXQGDQFHRI(&0SURWHLQVFRUUHODWHGRQO\ZHDNO\)LJXUH$U 3HDUVRQ7UDQVFULSWV DQGSURWHLQVWKDWZHUHERWKGRZQUHJXODWHGLQ'(%NHUDWLQRF\WHVGLVSOD\*2WHUPVVXFKDV ³H[WUDFHOOXODUUHJLRQ´DQG³FHOODGKHVLRQ´DQGDUHKLJKOLJKWHGLQ\HOORZ)LJXUH$7KLVJURXS FRQWDLQVSURWHLQVDVVRFLDWHGZLWKGHVPRVRPHVOLNHSODNRSKLOLQDQGLQWHJULQDOSKDDVZHOO DVSURWHDVHLQKLELWRUVLHF\VWDWLQ$$GGLWLRQDO(&0SURWHLQVOLNHODPLQLQEHWDDQGJDPPD ZHUHLGHQWLILHGDVGRZQUHJXODWHGZKHUHDVUHVSHFWLYHP51$OHYHOVGLGQRWFRUUHODWHZLWKWKH FKDQJHLQSURWHLQDEXQGDQFHLQGLFDWLQJSHUWXUEHGSRVWWUDQVFULSWLRQDOUHJXODWLRQPHFKDQLVPV 6XSSOHPHQWDO)LJXUH6'RZQUHJXODWHGWUDQVFULSWVRISURWHLQVWKDWZHUHXSUHJXODWHGLQ'(% DUH PDUNHG EOXH DQG DUH DVVRFLDWHG ZLWK *2 WHUPV ³H[WUDFHOOXODU VSDFH´ ³VHFUHWHG´ DQG ³FDOFLXP LRQ ELQGLQJ´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in vivoLQWKHHSLGHUPLVFRPSDULQJLPPXQRIOXRUHVFHQFH VWDLQLQJ RI '(% DQG FRQWURO VNLQ LQGLFDWLQJ WKDW LQIODPPDWLRQ LQ '(% LV SURPRWHG E\ NHUDWLQRF\WHV)LJXUH&
ϭϰ
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
7DNHQWRJHWKHUIROGFKDQJHVRIP51$DQGSURWHLQOHYHOVFRUUHODWHGZHDNO\LQSULPDU\KXPDQ '(%DQGFRQWURONHUDWLQRF\WHVUHJDUGOHVVLIP51$OHYHOVZHUHFRPSDUHGWRLQWUDFHOOXODURU H[WUDFHOOXODU SURWHLQ DEXQGDQFHV 6HYHUDO SURWHLQV WKDW DUH LQYROYHG LQ LQIODPPDWRU\ SURFHVVHVVXFKDV6$DQG6$ZHUHRQO\IRXQGWREHXSUHJXODWHGRQSURWHLQOHYHO LQGLFDWLQJ VR IDU XQNQRZQ NHUDWLQRF\WHLQWULQVLF SRVWWUDQVFULSWLRQDO UHJXODWLRQ PHFKDQLVPV DQGFKDUDFWHUL]LQJNHUDWLQRF\WHVDVDFWLYHO\SURPRWLQJLQIODPPDWLRQLQ'(%
ϭϱ ,QFUHDVHGFDWKHSVLQ%DEXQGDQFHDQGDFWLYLW\LQ&GHILFLHQW'(%VNLQ 7RJHWDJOREDOLPSUHVVLRQRILQWUDFHOOXODUSURWHLQDEXQGDQFHGLIIHUHQFHVLQGXFHGE\WKHORVV RI&ZHDJDLQSHUIRUPHGD627$FOXVWHULQJDQG*2WHUPHQULFKPHQWDQDO\VLV)LJXUH$ IXOOOLVWRI*2WHUPVLQ6XSSOHPHQWDO7DEOH67KH*2WHUPV³O\VRVRPH´DQG³HQGRSHSWLGDVH DFWLYLW\´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et al. FRQGXFWHG D VLPLODU FDWKHSVLQDFWLYLW\DVVD\VDQGFKDUDFWHUL]HGDZHVWHUQEORWVLJQDOMXVWDERYHFDWKHSVLQ%DV FDWKHSVLQ= 7KXVDOVRFDWKHSVLQ=DSSHDUHGWREHPRUHDFWLYHLQ '(% NHUDWLQRF\WHV WKDQLQFRQWUROVDQGPLJKWDVZHOOEHLQYROYHGLQ(&0GHJUDGDWLRQLQ'(%)LJXUH) ,QFUHDVHG O\VRVRPDO SURWHDVH DEXQGDQFH DQG DFWLYLW\ PLJKW LQGLFDWH D JHQHUDO LQFUHDVH LQ O\VRVRPDO SURWHLQ GHJUDGDWLRQ 7KHUHIRUH ZH DVVHVVHG WKH DFWLYLW\ RI DXWRSKDJ\ DQ
ϭϲ LQWUDFHOOXODUGHJUDGDWLRQSDWKZD\WDUJHWLQJSURWHLQVDQGRUJDQHOOHVIRUO\VRVRPDOGHJUDGDWLRQ %\DVVD\LQJDFFXPXODWLRQRIWKHOLSLGDWHGDXWRSKDJRVRPDOPDUNHUSURWHLQ0$3/&% /&,,LQWKHSUHVHQFHDQGDEVHQFHRIFRQFDQDP\FLQ$&RQ$DQLQKLELWRURIWKHO\VRVRPDO 9$73DVHZHGHWHFWHGDQLQFUHDVHGDXWRSKDJLFFDSDFLW\LQ'(%NHUDWLQRF\WHV)LJXUH+, ZKLFKLVLQOLQHZLWKLQFUHDVHGFDWKHSVLQDFWLYLW\LQ'(%
)LQDOO\ LQ DJUHHPHQW ZLWK WKH in vitro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in vitroLQPRXVHFHOOVDQGZDVLGHQWLILHGDVLQFUHDVHG LQ'(%VNLQDQGILEUREODVWV7RWHVWLILQFUHDVHGFDWKHSVLQ%DEXQGDQFHLQ'(% NHUDWLQRF\WHVDOVRGHSHQGVRQ7*)EVLJQDOLQJZHDQDO\]HGTGFB1H[SUHVVLRQOHYHOVDQG DFWLYDWLRQRIWKH7*)EWDUJHWSURWHLQV60$',QGHHGTGFB1H[SUHVVLRQZDVVLJQLILFDQWO\ LQFUHDVHG LQ '(% FRPSDUHG WR FRQWURO NHUDWLQRF\WHV )LJXUH $ OHDGLQJ WR LQFUHDVHG 60$'SKRVSKRU\ODWLRQ)LJXUH%7UHDWPHQWRIFRQWURONHUDWLQRF\WHVZLWK7*)EOHGWRD VLJQLILFDQWLQFUHDVHLQFDWKHSVLQ%OHYHOV)LJXUH&'DQGGXDOLQKLELWLRQRI7*)ȕ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
ϭϳ
& OHG WR D SURQRXQFHG UHGXFWLRQ LQ FDWKHSVLQ % OHYHOV LQGLFDWLQJ WKDW & LV VXIILFLHQW WR GHFUHDVHFDWKHSVLQ%OHYHOV)LJXUH*
ϭϴ 'LVFXVVLRQ
1H[W WR LWV LPSRUWDQW VWUXFWXUDO VFDIIROG IXQFWLRQV WKH (&0 LV FULWLFDO IRU FHOO KRPHRVWDVLV SUROLIHUDWLRQDQGGLIIHUHQWLDWLRQ'HILFLHQF\RIWKH(&0SURWHLQ&LQ'(%OHDGVWRVXEVWDQWLDO GHUPDO FKDQJHV LPSDLUHG VNLQ UHJHQHUDWLRQ DQG 7*)EGULYHQ ILEURVLV WKDW IDFLOLWDWHV DJJUHVVLYHVTXDPRXVFHOOFDUFLQRPD6&&+RZHYHU&LVERWKDGHUPDO DQG DQ HSLGHUPDO SURGXFW DQG VR IDU WKH GLUHFW FHOOXODU FKDQJHV WKDW ORVV RI & HYRNHV LQ HSLGHUPDONHUDWLQRF\WHVUHPDLQHGOLPLWHGO\VWXGLHG$SURWHRPLFDQDO\VLVRIWKHVNLQRI '(% PLFH VXJJHVWHG D FHOOLQWULQVLF LQMXU\LQGHSHQGHQW SURJUHVVLRQ RI ILEURVLV LQ '(% WKH HSLGHUPLV EHLQJ D SULPH VXVSHFWIRU SURPRWLQJWKLV DFWLYLW\ ,Q DGGLWLRQ G\VIXQFWLRQLQJ HSLWKHOLDKDYHHPHUJHGDVLQVWLJDWRUVRIILEURVLVLQRWKHUILEURWLFGLVHDVHV7KHDERYHOHG XVWRXVH'(%DVDPRGHOWRVWXG\WKHFRQVHTXHQFHVRIJHQHWLFORVVRIWKHVWUXFWXUDOSURWHLQ &RQHSLGHUPDOFHOOKRPHRVWDVLVE\FRPSUHKHQVLYHµRPLFVDSSURDFKHV¶
,QWULJXLQJO\WKHSUHVHQWVWXG\UHYHDOHGGUDPDWLFFKDQJHVLQNHUDWLQRF\WHGHULYHG(&0DVD FRQVHTXHQFH RI ORVV RI & $EXQGDQFHV RI LQWHJULQ Įȕ FROODJHQ ;9,, DQG ODPLQLQ LQGLUHFWDQGGLUHFWOLJDQGVRI&ZHUHUHGXFHG)LJXUH,QWHJULQĮȕ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in vitro ,PSRUWDQWO\ WKH LQWHJULQ DE UHFHSWRUZDVVKRZQWREHDQHJDWLYHUHJXODWRURI7*)EH[SUHVVLRQDQGLQYHUVHO\LQFUHDVHG 7*)E DFWLYLW\ LQ NHUDWLQRF\WHV PD\ FDXVH UHGXFWLRQ RI ODPLQLQ DQG LQWHJULQ DE DQG XSUHJXODWLRQ RI SURWHLQV DVVRFLDWHG ZLWK WLVVXH GDPDJH 7KXV LQFUHDVHG 7*)E VLJQDOLQJGXHWRWKHORVVRIUHSUHVVLRQPD\OHDGWRPDQ\RIWKHFKDQJHVREVHUYHGLQRXUJOREDO DQDO\VLV
ϭϵ
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±RQFHDQH[WUDFHOOXODUSURWHLQLVV\QWKHVL]HG±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± )FIXVLRQSURWHLQVGLUHFWHGDJDLQVW6$DQG6$KDYHEHHQVKRZQWRLQWHUIHUHZLWK WKHLUDFWLYLW\LQPLFH
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
ϮϬ
DQG GHUPLV DQG FRQWULEXWH WR ILEURVLV LQ '(% ,PSRUWDQWO\ VSRQWDQHRXVO\ JHQHFRUUHFWHG PRVDLF NHUDWLQRF\WHV IURP D '(% SDWLHQW GHPRQVWUDWHG WKDW FDWKHSVLQ % OHYHOV FRUUHODWHG LQYHUVHO\ZLWK&OHYHOV7KHIDFWWKDWFDWKHSVLQ%OHYHOVZHUHUHJXODWHGE\7*)EVXJJHVWVD GHWULPHQWDOSRVLWLYHIHHGEDFNORRSORVVRI&OHDGVWRLQFUHDVHG7*)EVLJQDOLQJLQFUHDVLQJ FDWKHSVLQ%OHYHOVZKLFKLQWXUQPD\SRWHQWLDWH7*)EVLJQDOV)LJXUH7KHFDXVDOLW\NLQHWLF UHJXODWLRQRIHYHQWVDVZHOODVWKHUHJXODWLRQLQKXPDQVNLQin vivoZLOOKDYHWREHDGGUHVVHG LQIXWXUHVWXGLHV
In situYDOLGDWLRQUHYHDOHGDFOHDULQFUHDVHRIFDWKHSVLQ%LQ'(%VNLQVXSSRUWLQJDQin vivo UROHRIFDWKHSVLQ%LQWKHEOLVWHUSKHQRW\SHLQ'(%$FWLYHFDWKHSVLQ%PLJKWDOVRFRQWULEXWH WR SRVWWUDQVFULSWLRQDOUHJXODWLRQ RI (&0 SURWHLQVLQ '(% VXFK DVWKH GHFUHDVH RI ODPLQLQV EHWD DQG JDPPD )LJXUH ,QFUHDVHG DFWLYLW\ RI PDWUL[ PHWDOORSURWHDVH 003 SURGXFHGE\VNLQILEUREODVWVKDVEHHQVXJJHVWHGWRSOD\DUROHLQWKHEOLVWHUSKHQRW\SHRI'(% +HUHZHVKRZWKDWDOVRNHUDWLQRF\WHVPD\FRQWULEXWHWRH[FHVVLYH(&0GHJUDGDWLRQYLD DQLQFUHDVHGFDWKHSVLQDEXQGDQFHDQGDFWLYLW\
6HFUHWHGFDWKHSVLQVKDYHEHHQLPSOLFDWHGLQNHUDWLQRF\WHPLJUDWLRQDQGZRXQGKHDOLQJ ,QOLQHZLWKWKLVFDWKHSVLQV%DQG=KDYHDOVREHHQVKRZQWRSURPRWHWXPRUSURJUHVVLRQ LQ YDULRXV PRXVH PRGHOV RI KXPDQ FDQFHUV &RQVHTXHQWO\ YDULRXV DSSURDFKHV IRU FDWKHSVLQ LQKLELWLRQ LQ FDQFHUV DUH FXUUHQWO\ LQYHVWLJDWHG DV ³VWDQG DORQH´ RU SDUW RI FRPELQDWRU\WKHUDS\DSSURDFKHVIRUUHYLHZ$V'(%SDWLHQWVRIWHQGHYHORSDJJUHVVLYH 6&&FDWKHSVLQV%DQG=PLJKWDOVREHSURPLVLQJGUXJJDEOHWDUJHWVLQ'(%
7DNHQWRJHWKHUE\DSSO\LQJDFRPSUHKHQVLYHDSSURDFKFRPELQLQJ6,/$&EDVHGTXDQWLWDWLYH 06ZLWKQH[WJHQHUDWLRQ51$VHTXHQFLQJIROORZHGE\ELRLQIRUPDWLFVDQDO\VHVZHJHQHUDWHG DJOREDO\HWGHWDLOHGSLFWXUHRIG\VUHJXODWHGPROHFXODUFRQVHTXHQFHVRI&GHILFLHQF\2XU VWXG\ UHYHDOHG WKDW ORVV RI & IURP HSLGHUPDO NHUDWLQRF\WHV HYRNHV FHOOLQWULQVLF ZRXQG KHDOLQJOLNHUHVSRQVHV7KHVHHSLGHUPDODFWLYLWLHVHVWDEOLVKD7*)EGULYHQSURLQIODPPDWRU\ DQGWLVVXHGHVWDELOL]LQJPLFURPLOLHXHQDEOLQJGHYHORSPHQWRINHUDWLQRF\WHSURPRWHGILEURVLVLQ WKHGHUPLV7KXVE\XQFRYHULQJHSLGHUPDOG\VUHJXODWLRQDVDFDWDO\VWRIGHUPDOILEURVLVRXU VWXG\ LGHQWLILHV SRWHQWLDO QHZ GUXJ WDUJHWV IRU GHVLJQ RI QHZ WUHDWPHQW VWUDWHJLHV IRU '(%
Ϯϭ
,PSRUWDQWO\LWPD\DOVRSURYLGHLQVLJKWVLQWRWKHSDWKRJHQHVLVRIRWKHUFRQGLWLRQVGHSHQGHQW RQFKURQLFXQLQKLELWHG7*)EVLJQDOLQJDVVRFLDWHGZLWKVFDUULQJDQGILEURVLV
ϮϮ 6XSSOHPHQWDO,QIRUPDWLRQ 6XSSOHPHQWDO,QIRUPDWLRQFDQEHIRXQGZLWKWKLVDUWLFOHRQOLQH $XWKRU&RQWULEXWLRQV &RQFHSWXDOL]DWLRQ.7$175/%7DQG-'0HWKRGRORJ\.7%$*2%$(-/,$(:'. $1755%&+/%7DQG-',QYHVWLJDWLRQ.7%$*2%$(-/,$:ULWLQJ±2ULJLQDOGUDIW .775&+/%7DQG-':ULWLQJ±5HYLHZ (GLWLQJ.775$1&+/%7DQG-')XQGLQJ $FTXLVLWLRQ5HVRXUFHV 6XSHUYLVLRQ$1'.755%&+/%7DQG-' $FNQRZOHGJPHQWV
:H WKDQN WKH )UHLEXUJ *DOD[\ 3ODWIRUP IRU ELRLQIRUPDWLFV VXSSRUW KWWSVJDOD[\XQL IUHLEXUJGH7KLVZRUNZDVVXSSRUWHGE\WKH*HUPDQ5HVHDUFK)RXQGDWLRQ')*WKURXJK WKH &5& 5% &5& /%775 -' '( -' 1< 1<$1 .,'.E\WKH*HUPDQ)HGHUDO0LQLVWU\RI(GXFDWLRQDQG5HVHDUFK%0%)JUDQW $$5%XQGHUWKHIUDPHRI(UDUHE\WKH(5$1HWIRU5HVHDUFKRQ5DUH'LVHDVHV (%7KHUD/%7E\WKH([FHOOHQFH,QLWLDWLYHRIWKH*HUPDQ)HGHUDODQG6WDWH*RYHUQPHQWV WKURXJK(;&%,266755%/%7-'DQGE\WKH6ZLVV1DWLRQDO6FLHQFH)RXQGDWLRQ JUDQW$Ǧ-' 5HIHUHQFHV +\QHV527KHH[WUDFHOOXODUPDWUL[QRWMXVWSUHWW\ILEULOVScience
3R]]L $ <XUFKHQFR 3 ' DQG ,R]]R 5 9 7KH QDWXUH DQG ELRORJ\ RI EDVHPHQWPHPEUDQHVMatrix Biol
%RQQDQV & &KRX - DQG:HUE = 5HPRGHOOLQJ WKH H[WUDFHOOXODU PDWUL[ LQ GHYHORSPHQWDQGGLVHDVHNat Rev Mol Cell Biol
+DV & DQG 1\VWURP $ (SLGHUPDO %DVHPHQW 0HPEUDQH LQ +HDOWK DQG 'LVHDVHCurr Top Membr
)LQH-'(DG\5$%DXHU($%DXHU-:%UXFNQHU7XGHUPDQ/+HDJHUW\ $+LQWQHU++RYQDQLDQ$-RQNPDQ0)/HLJK,HWDO7KHFODVVLILFDWLRQ RILQKHULWHGHSLGHUPRO\VLVEXOORVD(%5HSRUWRIWKH7KLUG,QWHUQDWLRQDO&RQVHQVXV 0HHWLQJRQ'LDJQRVLVDQG&ODVVLILFDWLRQRI(%J Am Acad Dermatol 'D\DO-+&ROH&/3RXUUH\URQ&:DWW6$/LP<=6DODV$ODQLV-&
0XUUHOO')0F*UDWK-$6WLHJHU%-DKRGD&HWDO7\SH9,,FROODJHQ UHJXODWHV H[SUHVVLRQ RI 2$73% SURPRWHV IURQWWRUHDU SRODULW\ DQG LQFUHDVHV
Ϯϯ
VWUXFWXUDORUJDQLVDWLRQLQ'VSKHURLGFXOWXUHVRI5'(%WXPRXUNHUDWLQRF\WHV J Cell
Sci
:DWW6$3RXUUH\URQ&3XUGLH.+RJDQ&&ROH&/)RVWHU13UDWW1 %RXUGRQ - & $SSOH\DUG 9 0XUUD\ . HW DO ,QWHJUDWLYH P51$ SURILOLQJ FRPSDULQJFXOWXUHGSULPDU\FHOOVZLWKFOLQLFDOVDPSOHVUHYHDOV3/.DQG&RUIDV WKHUDSHXWLFWDUJHWVLQFXWDQHRXVVTXDPRXVFHOOFDUFLQRPDOncogene .XWWQHU 9 0DFN & 5LJEROW .7 .HUQ - 6 6FKLOOLQJ2 %XVFK + %UXFNQHU 7XGHUPDQ/DQG'HQJMHO-*OREDOUHPRGHOOLQJRIFHOOXODUPLFURHQYLURQPHQW GXHWRORVVRIFROODJHQ9,,Mol Syst Biol
1J<=3RXUUH\URQ&6DODV$ODQLV-&'D\DO-+&HSHGD9DOGHV5<DQ ::ULJKW6&KHQ0)LQH-'+RJJ)-HWDO)LEUREODVWGHULYHGGHUPDO PDWUL[ GULYHV GHYHORSPHQW RI DJJUHVVLYH FXWDQHRXV VTXDPRXV FHOO FDUFLQRPD LQ SDWLHQWVZLWKUHFHVVLYHG\VWURSKLFHSLGHUPRO\VLVEXOORVDCancer Res .XWWQHU90DFN&*UHW]PHLHU&%UXFNQHU7XGHUPDQ/DQG'HQJMHO-
/RVV RI FROODJHQ 9,, LV DVVRFLDWHG ZLWK UHGXFHG WUDQVJOXWDPLQDVH DEXQGDQFH DQG DFWLYLW\J Invest Dermatol
1\VWURP$7KULHQH.0LWWDSDOOL9.HUQ-6.LULWVL''HQJMHO-DQG%UXFNQHU 7XGHUPDQ / /RVDUWDQ DPHOLRUDWHV G\VWURSKLF HSLGHUPRO\VLV EXOORVD DQG XQFRYHUVQHZGLVHDVHPHFKDQLVPVEMBO Mol Med
2GRULVLR 7 'L 6DOYLR 0 2UHFFKLD $ 'L =HQ]R * 3LFFLQQL ( &LDQIDUDQL ) 7UDYDJOLRQH$8YD3%HOOHL%&RQWL$HWDO0RQR]\JRWLFWZLQVGLVFRUGDQW IRUUHFHVVLYHG\VWURSKLFHSLGHUPRO\VLVEXOORVDSKHQRW\SHKLJKOLJKWWKHUROHRI7*)EHWD VLJQDOOLQJLQPRGLI\LQJGLVHDVHVHYHULW\Hum Mol Genet
6DNDL1DQG7DJHU$0)LEURVLVRIWZR(SLWKHOLDOFHOOILEUREODVWLQWHUDFWLRQV LQSXOPRQDU\ILEURVLVBiochim Biophys Acta
.HUQ-6*UXQLQJHU*,PVDN50XOOHU0/6FKXPDQQ+.LULWVL'(PPHUW 6%RUR]GLQ:.RKOKDVH-%UXFNQHU7XGHUPDQ/HWDO)RUW\WZRQRYHO &2/$PXWDWLRQVDQGWKHUROHRIDIUHTXHQWVLQJOHQXFOHRWLGHSRO\PRUSKLVPLQWKH 003 SURPRWHU LQ PRGXODWLRQ RI GLVHDVH VHYHULW\ LQ D ODUJH (XURSHDQ G\VWURSKLF HSLGHUPRO\VLVEXOORVDFRKRUWBr J Dermatol
.HUQ-6.RKOKDVH-%UXFNQHU7XGHUPDQ/DQG+DV&([SDQGLQJWKH &2/$ PXWDWLRQ GDWDEDVH QRYHO DQG UHFXUUHQW PXWDWLRQV DQG XQXVXDO JHQRW\SH SKHQRW\SHFRQVWHOODWLRQVLQSDWLHQWVZLWKG\VWURSKLFHSLGHUPRO\VLVEXOORVDJ Invest
Dermatol
0\OO\KDUMX - DQG .LYLULNNR . , &ROODJHQV PRGLI\LQJ HQ]\PHV DQG WKHLU PXWDWLRQVLQKXPDQVIOLHVDQGZRUPVTrends Genet
9ORGDYVN\,3UHSDUDWLRQRIH[WUDFHOOXODUPDWULFHVSURGXFHGE\FXOWXUHGFRUQHDO HQGRWKHOLDODQG3)+5HQGRGHUPDOFHOOVCurr Protoc Cell Biol&KDSWHU8QLW
:LVQLHZVNL-5=RXJPDQ$1DJDUDM1DQG0DQQ08QLYHUVDOVDPSOH SUHSDUDWLRQPHWKRGIRUSURWHRPHDQDO\VLVNat Methods
6KHYFKHQNR $ 7RPDV + +DYOLV - 2OVHQ - 9 DQG 0DQQ 0 ,QJHO GLJHVWLRQ IRU PDVV VSHFWURPHWULF FKDUDFWHUL]DWLRQ RI SURWHLQV DQG SURWHRPHV Nat
Protoc
5DSSVLOEHU - 0DQQ 0 DQG ,VKLKDPD < 3URWRFRO IRU PLFURSXULILFDWLRQ HQULFKPHQWSUHIUDFWLRQDWLRQDQGVWRUDJHRISHSWLGHVIRUSURWHRPLFVXVLQJ6WDJH7LSV
Nat Protoc
&R[ - DQG 0DQQ 0 0D[4XDQW HQDEOHV KLJK SHSWLGH LGHQWLILFDWLRQ UDWHV LQGLYLGXDOL]HGSSEUDQJHPDVVDFFXUDFLHVDQGSURWHRPHZLGHSURWHLQTXDQWLILFDWLRQ
Nat Biotechnol
9L]FDLQR-$&VRUGDV$GHO7RUR1'LDQHV-$*ULVV-/DYLGDV,0D\HU *3HUH]5LYHURO<5HLVLQJHU)7HUQHQW7HWDOXSGDWHRIWKH35,'( GDWDEDVHDQGLWVUHODWHGWRROVNucleic Acids Res'
6DKDULQHQ-DQG.HVNL2MD-6SHFLILFVHTXHQFHPRWLIRI&\VUHSHDWVRI 7*)EHWD ELQGLQJ SURWHLQV /7%3V FUHDWHV D K\GURSKRELF LQWHUDFWLRQ VXUIDFH IRU ELQGLQJRIVPDOOODWHQW7*)EHWDMol Biol Cell
Ϯϰ
*HLJHU7&R[-2VWDVLHZLF]3:LVQLHZVNL-5DQG0DQQ 06XSHU 6,/$&PL[IRUTXDQWLWDWLYHSURWHRPLFVRIKXPDQWXPRUWLVVXHNat Methods 1DED$&ODXVHU.5+RHUVFK6/LX+&DUU6$DQG+\QHV527KH PDWULVRPHLQVLOLFRGHILQLWLRQDQGLQYLYRFKDUDFWHUL]DWLRQE\SURWHRPLFVRIQRUPDODQG WXPRUH[WUDFHOOXODUPDWULFHVMol Cell Proteomics0
+XDQJGD:6KHUPDQ%7DQG/HPSLFNL5$6\VWHPDWLFDQGLQWHJUDWLYH DQDO\VLVRIODUJHJHQHOLVWVXVLQJ'$9,'ELRLQIRUPDWLFVUHVRXUFHVNat Protoc 5RGLXV6,QGUD*7KLEDXOW&3ILVWHU9DQG*HRUJHV/DERXHVVH(/RVV RIDOSKDLQWHJULQVLQNHUDWLQRF\WHVOHDGVWRDQLQFUHDVHLQ7*)EHWDDQG$3VLJQDOLQJ DQGLQH[SUHVVLRQRIGLIIHUHQWLDWLRQJHQHVJ Cell Physiol
7VXUXWD ' .RED\DVKL + ,PDQLVKL + 6XJDZDUD . ,VKLL 0 DQG -RQHV - & /DPLQLQLQWHJULQLQWHUDFWLRQDWDUJHWIRUFDQFHUWKHUDS\"Curr Med Chem
+HUUHUD 2 5 &KULVWHQVHQ 0 / DQG +HOPV 5 $ &DOSURWHFWLQ &OLQLFDO $SSOLFDWLRQVLQ3HGLDWULFVJ Pediatr Pharmacol Ther
%XUUL ( DQG %HJOLQJHU & )DHFDO FDOSURWHFWLQ D XVHIXO WRRO LQ WKH PDQDJHPHQWRILQIODPPDWRU\ERZHOGLVHDVHSwiss Med WklyZ
0ROO5'LYR0DQG/DQJEHLQ/7KHKXPDQNHUDWLQVELRORJ\DQGSDWKRORJ\
Histochem Cell Biol
.QDXS-9HUZDQJHU7*UXEHU&=LHJOHU9%DXHU-:DQG.UDPPHU% (SLGHUPRO\VLV EXOORVD D JURXS RI VNLQ GLVHDVHV ZLWK GLIIHUHQW FDXVHV EXW FRPPRQDOLWLHVLQJHQHH[SUHVVLRQExp Dermatol
0DLHU . +H < :ROIOH 8 (VVHU 3 5 %UXPPHU 7 6FKHPSS & %UXFNQHU 7XGHUPDQ / DQG +DV & 89%LQGXFHG FXWDQHRXV LQIODPPDWLRQ DQG SURVSHFWV IRU DQWLR[LGDQW WUHDWPHQW LQ .LQGOHU V\QGURPH Hum Mol Genet
*UHHQEDXP'0HG]LKUDGV]N\.)%XUOLQJDPH$DQG%RJ\R0(SR[LGH HOHFWURSKLOHV DV DFWLYLW\GHSHQGHQW F\VWHLQH SURWHDVH SURILOLQJ DQG GLVFRYHU\ WRROV
Chem Biol
2UORZVNL*0&ROEHUW-'6KDUPD6%RJ\R05REHUWVRQ6$DQG5RFN. /0XOWLSOH&DWKHSVLQV3URPRWH3UR,/EHWD6\QWKHVLVDQG1/530HGLDWHG ,/EHWD$FWLYDWLRQJ Immunol
*UHW]PHLHU&(LVHOHLQ6-RKQVRQ*5(QJHONH51RZDJ+=DUHL0.XWWQHU 9 %HFNHU $ & 5LJEROW .7 * +R\HU+DQVHQ 0 HW DO 'HJUDGDWLRQ RI SURWHLQ WUDQVODWLRQ PDFKLQHU\ E\ DPLQR DFLG VWDUYDWLRQLQGXFHG PDFURDXWRSKDJ\
Autophagy
-LDQJ < :RRVOH\ $ 1 6LYDOLQJDP 1 1DWDUDMDQ 6 DQG +RZH 3 + &DWKHSVLQ%PHGLDWHG FOHDYDJH RI 'LVDEOHG UHJXODWHV 7*)EHWDLQGXFHG DXWRSKDJ\Nat Cell Biol
1\VWURP $ 9HODWL ' 0LWWDSDOOL 9 5 )ULWVFK $ .HUQ - 6 DQG %UXFNQHU 7XGHUPDQ/&ROODJHQ9,,SOD\VDGXDOUROHLQZRXQGKHDOLQJJ Clin Invest .LULWVL'9DODUL00LFKRV$.DUDNRVWD9DQG+DV&7KHP\VWHULHVRI PRVDLFLVPSKHQRW\SLFYDULDELOLW\LQDIDPLO\ZLWKLQFRQWLQHQWLDSLJPHQWLEur J Dermatol :DWW6$'D\DO-+:ULJKW65LGGOH03RXUUH\URQ&0F0LOODQ-5.LPEOH 503ULVFR0*DUWQHU8:DUEULFN(HWDO/\V\O+\GUR[\ODVH/RFDOL]HV WR (SLGHUPDO %DVHPHQW 0HPEUDQH DQG ,V 5HGXFHG LQ 3DWLHQWV ZLWK 5HFHVVLYH '\VWURSKLF(SLGHUPRO\VLV%XOORVDPLoS OneH
GH 3HUHGD - 0 2UWHJD ( $ORQVR*DUFLD 1 *RPH]+HUQDQGH] 0 DQG 6RQQHQEHUJ $ $GYDQFHV DQG SHUVSHFWLYHV RI WKH DUFKLWHFWXUH RI KHPLGHVPRVRPHVOHVVRQVIURPVWUXFWXUDOELRORJ\Cell Adh Migr
:DONR*&DVWDQRQ0-DQG:LFKH*0ROHFXODUDUFKLWHFWXUHDQGIXQFWLRQ RIWKHKHPLGHVPRVRPHCell Tissue Res
Ϯϱ
9LOORQH')ULWVFK$.RFK0%UXFNQHU7XGHUPDQ/+DQVHQ8DQG%UXFNQHU 36XSUDPROHFXODULQWHUDFWLRQVLQWKHGHUPRHSLGHUPDOMXQFWLRQ]RQHDQFKRULQJ ILEULOFROODJHQ9,,WLJKWO\ELQGVWREDQGHGFROODJHQILEULOVJ Biol Chem &KXQJ+-DQG8LWWR-7\SH9,,FROODJHQWKHDQFKRULQJILEULOSURWHLQDWIDXOW LQG\VWURSKLFHSLGHUPRO\VLVEXOORVDDermatol Clin 1\VWURP$%RUQHUW2DQG.XKO7&HOOWKHUDS\IRUEDVHPHQWPHPEUDQH OLQNHGGLVHDVHVMatrix Biol %UHPHU-%RUQHUW21\VWURP$*RVW\QVNL$-RQNPDQ0)$DUWVPD5XV$ YDQGHQ$NNHU3&DQG3DVPRRLM$0$QWLVHQVH2OLJRQXFOHRWLGHPHGLDWHG ([RQ 6NLSSLQJ DV D 6\VWHPLF 7KHUDSHXWLF $SSURDFK IRU 5HFHVVLYH '\VWURSKLF (SLGHUPRO\VLV%XOORVDMol Ther Nucleic AcidsH
)ULWVFK$/RHFNHUPDQQ6.HUQ-6%UDXQ$%RVO05%OH\7$6FKXPDQQ +YRQ(OYHUIHOGW'3DXO'(UODFKHU0HWDO$K\SRPRUSKLFPRXVHPRGHO RIG\VWURSKLFHSLGHUPRO\VLVEXOORVDUHYHDOVPHFKDQLVPVRIGLVHDVHDQGUHVSRQVHWR ILEUREODVWWKHUDS\J Clin Invest
9RJHO&DQG0DUFRWWH(0,QVLJKWVLQWRWKHUHJXODWLRQRISURWHLQDEXQGDQFH IURPSURWHRPLFDQGWUDQVFULSWRPLFDQDO\VHVNat Rev Genet
6FKZDQKDXVVHU%%XVVH'/L1'LWWPDU*6FKXFKKDUGW-:ROI-&KHQ: DQG6HOEDFK0*OREDOTXDQWLILFDWLRQRIPDPPDOLDQJHQHH[SUHVVLRQFRQWURO
Nature
(PLQJ6$.RFK0.ULHJHU$%UDFKYRJHO%.UHIW6%UXFNQHU7XGHUPDQ/ .ULHJ 7 6KDQQRQ - ' DQG )R[ - : 'LIIHUHQWLDO SURWHRPLF DQDO\VLV GLVWLQJXLVKHV WLVVXH UHSDLU ELRPDUNHU VLJQDWXUHV LQ ZRXQG H[XGDWHV REWDLQHG IURP QRUPDOKHDOLQJDQGFKURQLFZRXQGVJ Proteome Res
7KRUH\,65RWK-5HJHQERJHQ-+DOOH-3%LWWQHU09RJO7.DHVOHU6 %XJQRQ35HLWPDLHU%'XUND6HWDO7KH&DELQGLQJSURWHLQV6$ DQG6$DUHHQFRGHGE\QRYHOLQMXU\UHJXODWHGJHQHVJ Biol Chem
<XL 6 1DNDWDQL < DQG 0LNDPL 0 &DOSURWHFWLQ 6$6$ DQ LQIODPPDWRU\SURWHLQFRPSOH[IURPQHXWURSKLOVZLWKDEURDGDSRSWRVLVLQGXFLQJDFWLYLW\
Biol Pharm Bull
4LQ + /HUPDQ % 6DNDPDNL , :HL * &KD 6 & 5DR 6 6 4LDQ - +DLOHPLFKDHO < 1XULHYD 5 'Z\HU . & HW DO *HQHUDWLRQ RI D QHZ WKHUDSHXWLF SHSWLGH WKDW GHSOHWHV P\HORLGGHULYHG VXSSUHVVRU FHOOV LQ WXPRUEHDULQJ PLFHNat Med
$NNDUL/*RFKHYD9.HVWHU-&+XQWHU.(4XLFN0/6HYHQLFK/:DQJ + : 3HWHUV & 7DQJ / + .OLPVWUD ' 6 HW DO 'LVWLQFW IXQFWLRQV RI PDFURSKDJHGHULYHGDQGFDQFHUFHOOGHULYHGFDWKHSVLQ=FRPELQHWRSURPRWHWXPRU PDOLJQDQF\YLDLQWHUDFWLRQVZLWKWKHH[WUDFHOOXODUPDWUL[Genes Dev %HQJVFK)%XFN$*XQWKHU6&6HL]-57DFNH03IHLIHU'YRQ(OYHUIHOGW
' 6HYHQLFK / +LOOHEUDQG / ( .HUQ 8 HW DO &HOO W\SHGHSHQGHQW SDWKRJHQLF IXQFWLRQV RI RYHUH[SUHVVHG KXPDQ FDWKHSVLQ % LQ PXULQH EUHDVW FDQFHU SURJUHVVLRQOncogene
*RJLQHQL95*XSWD51DOOD$.9HOSXOD..DQG5DR-6X3$5 DQG FDWKHSVLQ % VK51$ LPSHGHV 7*)EHWDGULYHQ SUROLIHUDWLRQ DQG LQYDVLRQ RI PHQLQJLRPDFHOOVLQD;,$3GHSHQGHQWSDWKZD\Cell Death DisH
0ROHV $ 7DUUDWV 1 )HUQDQGH]&KHFD - & DQG 0DUL 0 &DWKHSVLQ % RYHUH[SUHVVLRQ GXH WR DFLG VSKLQJRP\HOLQDVH DEODWLRQ SURPRWHV OLYHU ILEURVLV LQ 1LHPDQQ3LFNGLVHDVHJ Biol Chem
&RORPEL0*DUGHOOD5=RSSL10RUR/0DULQL'6SXUU1.DQG%DUODWL6 ([FOXVLRQRIVWURPHO\VLQVWURPHO\VLQLQWHUVWLWLDOFROODJHQDVHDQGILEURQHFWLQ JHQHVDVWKHPXWDQWORFLLQDIDPLO\ZLWKUHFHVVLYHHSLGHUPRO\VLVEXOORVDG\VWURSKLFD DQGDIRUPRIFHUHEHOODUDWD[LDHum Genet
%XWK + :ROWHUV % +DUWZLJ % 0HLHU%RUQKHLP 5 9HLWK + +DQVHQ 0 6RPPHUKRII&36FKDVFKNH10DFKOHLGW:)XVHQLJ1(HWDO+D&D7
Ϯϲ
NHUDWLQRF\WHVVHFUHWHO\VRVRPDOF\VWHLQHSURWHLQDVHVGXULQJPLJUDWLRQEur J Cell Biol
%XWK+/XLJL%XWWLJLHJ32VWDIH55HKGHUV0'DQQHQPDQQ656FKDVFKNH 1 6WDUN + - %RXNDPS 3 DQG %UL[ . &DWKHSVLQ % LV HVVHQWLDO IRU UHJHQHUDWLRQ RIVFUDWFKZRXQGHG QRUPDO KXPDQ HSLGHUPDONHUDWLQRF\WHV Eur J Cell
Biol
5HLQKHFNHO73HWHUV&.UXJHU$7XUN%DQG9DVLOMHYD2'LIIHUHQWLDO ,PSDFWRI&\VWHLQH&DWKHSVLQVRQ*HQHWLF0RXVH0RGHOVRI'HQRYR&DUFLQRJHQHVLV &DWKHSVLQ%DV(PHUJLQJ7KHUDSHXWLF7DUJHWFront Pharmacol
2OVRQ 2 & DQG -R\FH - $ &\VWHLQH FDWKHSVLQ SURWHDVHV UHJXODWRUV RI FDQFHUSURJUHVVLRQDQGWKHUDSHXWLFUHVSRQVHNat Rev Cancer
Ϯϳ )LJXUH/HJHQGV
)LJXUH/RVVRI&LQ'(%LVDVVRFLDWHGZLWKVNLQIUDJLOLW\LQIODPPDWLRQDQGVFDUULQJ $ $QWL& ZHVWHUQ EORW & LV GHILFLHQW LQ WKH (&0 LVRODWHG IURP FXOWXUHG SULPDU\ '(% NHUDWLQRF\WHVGHULYHGIURPGHSLFWHGSDWLHQWV%&OLQLFDOSKHQRW\SHVRI'(%/RVVRI&LV DVVRFLDWHGZLWKVNLQIUDJLOLW\LQIODPPDWLRQDQGVFDUULQJ7KHDJHRISDWLHQWLVLQGLFDWHG
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
ITGA6/ITGB4 ZHUH IRXQG WR EH GRZQUHJXODWHG LQ '(% NHUDWLQRF\WHV ZKLOH WKH WUDQVFULSW HQFRGLQJ/7%3ZDVPRUHDEXQGDQWLQ'(%NHUDWLQRF\WHV
)LJXUH$QDO\VLVRIWKHH[WUDFHOOXODUSURWHRPHRI'(%NHUDWLQRF\WHV$&HOOFXOWXUH VFKHPH .HUDWLQRF\WHV ZHUH NHSW LQ FXOWXUH IRU VL[ GD\V )UHVK FXOWXUH PHGLXP FRQWDLQLQJ DVFRUEDWHZDVDGGHGGDLO\7KH(&0ZDVKDUYHVWHGDWGD\XVLQJ6'6%$QDO\VLVRI (&0LVRODWLRQSURFHGXUH:HVWHUQEORWDQDO\VHVZHUHSHUIRUPHGWRDGGUHVVWKHTXDOLW\RI (&0LVRODWLRQ,QWUDFHOOXODUSURWHLQVUHSUHVHQWLQJWKHFHOOFRPSDUWPHQWVQXFOHXV7%3*ROJL DSSDUDWXV *2/*$ HQGRSODVPLF UHWLFXOXP +63$ DQG F\WRVRO *$3'+ ZHUH RQO\
Ϯϴ
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Įȕ DQG ODPLQLQ ZHUH VLJQLILFDQWO\ GRZQUHJXODWHG LQ (&0 LVRODWHGIURP '(% NHUDWLQRF\WHV ZKHUHDVSURWHLQVLQYROYHGLQILEURVLVDQGLQIODPPDWLRQZHUHVLJQLILFDQWO\XSUHJXODWHG:HOFKV WWHVW3HUPXWDWLRQEDVHG)'57KHILJXUHZDVSURGXFHGXVLQJ6HUYLHU0HGLFDO$UW
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n vivo DFFXPXODWLRQ RI 6$ ,PPXQRIOXRUHVFHQFH VWDLQLQJ RI '(% VNLQ UHYHDOV D VWURQJ LQFUHDVHRI6$LQWKHHSLGHUPLVJUHHQVLJQDO%OXH '$3,VWDLQLQJ'&RUUHODWLRQRI JHQH WUDQVFULSWLRQ DQG LQWUDFHOOXODU SURWHLQ DEXQGDQFH *HQH H[SUHVVLRQ DQG SURWHLQ
Ϯϵ
DEXQGDQFH FRUUHODWHG ZHDNO\ U *HQHV LQ HDFK TXDGUDQW ZLWK D IROG FKDQJH ! RQ P51$ DQG SURWHLQ OHYHO ZHUH FRORUHG DQG WHVWHG E\ '$9,' IRU *2 HQULFKPHQW ELRORJLFDO SURFHVV%3FHOOXODUFRPSDUWPHQW&&PROHFXODUIXQFWLRQ0)DQG63B3,5B.(<:25'6 .:6HOHFWHGWHUPVDUHVKRZQ
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
ϯϬ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
ϯϭ
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
ϯϮ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
http://doc.rero.ch
ϯϯ 7DEOH&KDUDFWHULVWLFVRISULPDU\FRQWUROFHOOVXVHGLQWKLVVWXG\ ,' $JHDW ELRSV\ JHQGHU &WUO \HDUVP &WUO \HDUVP &WUO \HDUVP &WUO \HDUVI &WUO \HDUVI &WUO \HDUP &WUO \HDUVI &WUO \HDUVP &WUO \HDUVP &WUO \HDUVI &WUO \HDUVI &WUO \HDUVP
http://doc.rero.ch
ϯϰ )LJXUH
ϯϱ )LJXUH
http://doc.rero.ch
ϯϲ )LJXUH
http://doc.rero.ch
ϯϳ )LJXUH
http://doc.rero.ch
ϯϴ )LJXUH
http://doc.rero.ch
ϯϵ )LJXUH
ϰϬ )LJXUH