ho plasna nonbvano consisis oI a lipid bilayov

inio which pvoioins and glycopvoioins avo insoviod.

!n gonoval, iho hydvophobic naiuvo oI ihoso lipids
naLos ii inpossiblo Iov iho vasi najoviiy oI popiidos
and pvoioins io cvoss iho nonbvano. A signicani
oxcopiion io ihis vulo havo voconily boon poiniod oui,
wiih iho idoniicaiion oI sovoval pvoioin ivansduciion
donains (!JDs), also Lnown as coll-ponoivaiing pop-
iidos (C!!s), ihai avo capablo oI ivansducing cavgo
acvoss iho nonbvano, allowing pvoioins io accunu-
laio wiihin iho coll. Jho nosi connonly siudiod avo
honoodonain ivanscvipiion Iaciovs such as
Anionnapodia (Anip) [i6], iho hovpos sinplox vivus
(HSV) iypo i pvoioin V!22 [7], and iho hunan
innunodocioncy vivus (H!V-i) ivansaciivaiov JAJ
pvoioin [8i3]. Roconily, poly-avginino (polyR) and
Protein Transduction Technology :
A Novel Therapeutic Perspective
Hirofumi Noguchi

, und Shinichi Mutsumoto

Diole|esBeseorchIns|i|u|eKyo|o,

Dejor|men|ofTronsjlon|o|ionond
Immunoloyy,Kyo|oUniversi|yGroduo|eSchoolofMedicine,

BeseorchIellouof|heJojonSocie|yfor|hePromo|ionofScience(SPD),ond

Tronsjlon|o|ionUni|,Kyo|oUniversi|yIosji|ol,Kyo|o6068507,Jojon
J
Jhc dircct intrucc!!u!ur dc!ivcry of protcins hus, unti! rcccnt!y, hccn di cu!t to uchicvc, duc primur-
i!y to thc hiouvui!uhi!ity hurricr of thc p!usmu mcmhrunc. During thc pust 1 ycurs, u vuricty of
pcptidcs cu!!cd protcin trunsduction domuins (FJDs) or cc!! pcnctruting pcptidcs (CFFs), huvc hccn
chuructcrizcd for thcir uhi!ity to truns!ocutc into !ivc cc!!s. Jhc most common!y studicd urc homc-
odomuin trunscription fuctors such us Antcnnupcdiu, thc hcrpcs simp!cx virus (HSV) typc 1 protcin
VF22, und thc humun immunodccicncy virus (HIV-1) trunsuctivutor JAJ protcin. Rcccnt!y, po!y-
urgininc cxhihits cvcn grcutcr c cicncy in tcrms of dc!ivcry of scvcru! pcptidcs und protcins.
Numcrous cxump!cs of hio!ogicu!!y uctivc fu!!-!cngth protcins und pcptidcs huvc hccn dc!ivcrcd to
cc!!s und tissucs, hoth iuciIro und iucico. Jhcsc studics ocr ncv uvcnucs for trcutmcnt of scvcru!
discuscs. Jhc muin mcchunism of protcin trunsduction is un c!cctrostutic intcruction vith thc p!usmu
mcmhrunc, pcnctrution into cc!!s hy mucropinocytosis, und u rc!cusc to cytop!usm und nuc!ci hy rct-
rogrudc trunsport. Morcovcr, thc intcrcc!!u!ur trunsfcr of cndogcnous trunscription fuctors, such
us JAJ und homcoprotcins, sccms to point to un originu! und importunt modc of signu! trunsduc-
tion. Jhc protcin trunsduction systcms huvc opcncd up scvcru! possihi!itics, not on!y for thc dcvc!op-
mcnt of ncv pcptidc/protcin drugs hut u!so for considcrution of thcir physio!ogicu! und dcvc!opmcn-
tu! imp!icutions.
Kcy vords . pvoioin ivansduciion, pvoioin ivansduciion donain, coll ponoivaiing popiido, nacvopinocyiosis,
iniovcollulav ivansIov
Acta Med. Okayama, 2006
Vol. 60, No. 1, pp. 111
hiip ://www.lib.oLayana-u.ac.jp/www/acia/
Copyvighi 2OO6 by OLayana Univovsiiy Modical School.
Bevieu
Rocoivod Augusi ii, 2OO5 ; accopiod Novonbov 25, 2OO5.

Covvosponding auihov. !hono : 8i7575i4699 ; ax : 8i7575i3896

-nail : noguchihCLuhp.Lyoio-u.ac.jp (H. Noguchi)
poly-lysino havo boon shown io oxhibii ovon gvoaiov
o cioncy in iovns oI dolivovy oI sovoval popiidos and
pvoioins [i4i8]. Signicanily, whon synihosizod as
voconbinani Iusion pvoioins ov covalonily cvoss-
linLod io Iull-longih pvoioins, ihoso !JDs avo capa-
blo oI dolivoving biologically aciivo pvoioins. Jhoso
!JD Iusion pvoioins avo Iound in boih iho cyioplasn
and iho nuclous. Siudios by ouv gvoup, along wiih
ihoso by oihovs, havo dononsivaiod how ihoso pvo-
ioin ivansduciion sysions havo oponod up sovoval
possibiliiios Iov iho dovolopnoni oI now popiido/pvo-
ioin dvugs [i, i2, i4, i5, i9, 2O]. Hovo, wo
voviow sono oI iho nosi voconi advancos in ihis vap-
idly oxpanding avoa oI vosoavch.
Intrucc!!u!ur Dc!ivcry of FJD-Conjugutcd
Mucromo!ccu!cs
Jho nosi inpvossivo aspoci oI !JD-nodiaiod
dolivovy and iis ihovapouiic poioniial is iis sizo indo-
pondonco. Jho abiliiy oI !JDs io dolivov cavgo inio
colls is noi liniiod only io pvoioins ov popiidos.
Sovoval !JDs havo boon shown io nodiaio iho o -
cioni inivacollulav accunulaiion oI non-ovganic nolo-
culos. Jho bvoad uiiliiy oI pvoioin ivansduciion ioch-
nology is illusivaiod schonaiically in ig. i.
IroIciu oud cIidc dcliccry. !voioins
havo boon ovoluiionavily solociod io povIovn spocic
Iunciions. Jhus, iho abiliiy io dolivov a wido vavioiy
oI Iull-longih, Iunciional pvoioins has ivonondous
poioniial as a biological iool Iov siudying collulav
pvocossos as woll as Iov dovoloping novol and poion-
iially vovy spocic ihovapouiic agonis. Jo daio, a
gvowing nunbov oI ivansduciblo pvoioins covoving a
wido vango oI sizos and Iunciional classos havo boon
succossIully usod io siudy inivacollulav nochanisns.
Jhoso includo !DX-i [i3], LJA2/NouvoD [i9],
Ngn3 [2O], onhancod gvoon uovosconi pvoioin
(C!) [i, i9], Cvo voconbinaso [9, iO], and p53
[2i]. !JDs havo boon shown io dolivov pvoioins in
oxcoss oI iOO KDa inio culiuvod colls and nosi, iI
noi all, colls in nannalian nodol sysions [8].
!opiidos can bo usod in a vasi vango oI applica-
iions in phavnacouiical vosoavch. Ly linLing popiido
souoncos io !JDs, pvoviously non-coll-ponoivaiing
popiidos havo boon inivoducod inio colls, ihovoby
alioving oxisiing pvoioin-pvoioin ov pvoioin-oligonu-
clooiido iniovaciions. Sovoval popiidos havo boon
Iusod io !JDs, ihus onabling ihoiv onivy inio iho
cyioplasn. Jho inivoducod popiidos inhibii pvoioin
binding ov pvoioin conplox Iovnaiion [ii, i2, i4
i6]. Subsouonily, sovoval vopovis havo doscvibod
iho dolivovy oI popiidos and pvoioins in vivo [i2,
i4]. Movoovov, LavLa J e|ol. voconily showod iho
advaniagos and vovsaiiliiy oI pvoioin ivansduciion
ovov vival ivansgono dolivovy [22, 23]. Jhoiv siud-
ios using voivovival vociovs oxpvossing -galaciosi-
daso [22] wovo conpavod wiih vosulis Ivon iho injoc-
iion oI JAJ--galaciosidaso Iusion pvoioin in vivo
[23]. !njociion oI JAJ--galaciosidaso by voivo-
gvado duci injociion vosuliod in ivansduciion inio iOO
oI iho vai salivavy gland colls in a conconivaiion-
dopondoni nannov, whovoas vival dolivovy could only
achiovo 3O5O o cioncy.
Dcliccry o] ouIiscusc oligouuclcoIidcs.
Jho uso oI aniisonso iochniuos as a phavnacouiical
iool is iniovosiing bocauso ihis iochnology poioniially
has iho abiliiy io downvogulaio iho oxpvossion oI viv-
iually any dosivod gono. Aniisonso iochniuos avo
basod on souonco-spocic oligonuclooiido analogs
ihai, aIiov inivoduciion io iho cyiosol, can hybvidizo
wiih conplononiavy nRNA sivands. Jhis hybvidiza-
iion causos ivanslaiional avvosi ov vocvuiinoni oI
RNasoH, ihovoby alioving iho gono oxpvossion in iho
coll [24]. Jho dovolopnoni oI aniisonso ihovapy has
Iocusod nainly on inpvoving noihods Iov oligonuclo-
oiido dolivovy inio colls. Alihough iho collulav
upiaLo oI naLod oligonuclooiidos is poov, ii is signi-
canily incvoasod by coupling iho oligonuclooiido io a
ivanspoviov popiido, loading io docvoasod oxpvossion
oI iho gono pvoduci oI iho iavgoiod gono. Movvis e|
ol. doscvibo a poioni now sivaiogy Iov oligonuclooiido
dolivovy basod on iho uso oI a shovi popiido vociov,
iovnod M!C, which coniains a hydvophobic donain,
dovivod Ivon iho Iusion souonco oI H!V gp4i, and a
hydvophilic donain, dovivod Ivon iho nucloav local-
izaiion souonco oI SV4O J-aniigon [25]. Jung e|ol.
voviows iho pvopavaiion and applicaiions oI !JD-
oligonuclooiido conjugaios [26]. Jhoso sivaiogy oI
oligonuclooiido dolivovy inio culiuvod colls basod on
a popiido vociov oovs sovoval advaniagos including
o cioncy, siabiliiy and absonco oI cyioioxiciiy, whon
conpavod io oihov connonly usod appvoachos oI
dolivovy. Jho iniovaciion wiih !JD sivongly
incvoasos boih iho siabiliiy oI iho oligonuclooiido io
iho nucloaso and iho cvossing oI iho plasna non-
2 Acta Med. OkayamaVol. 60, No. 1 Noguchi et al.
3 Protein Transduction Technology February 2006
Cargo Intracellular
Plasma membrane
Liposomes
Protein / Peptide
Plasmid
siRNA
PNA
Antisense
Iron beads
Fe
+PTD
Cargo
delivery
Extracellular
Fig. 1Utility of protein transduction technology. A wide variety of cargo has been covalently linked to PTDs. Currently, most
molecules can be transduced into cells when linked to a PTD.
bvano.
Jho vapid dogvadaiion oI naiuval oligonuclo-
oiidos insido iho coll pvohibiis ihoiv uso in aniisonso
iochnology ; howovov, nany oligonuclooiido analogs
havo boon usod in aniisonso iochniuos, wiih vavying
vosulis. Asiviab-ishov e| ol. showod ihai !JD-
phosphovoihionaio oligonuclooiido conjugaio was
iniovnalizod inio colls undov sovun-voploio condiiions
and inhibiiod oxpvossion oI iho iavgoi noloculo [27].
!JD-!NA could also inhibii iho oxpvossion (soo
Delivery of jej|ide nucleic ocids (PNA) sociion). Jho
dovolopnoni oI nodiod oligonuclooiidos is con-
sianily pvogvossing [24]. !ncvoasod siabiliiy,
onhancod RNA binding a niiy and lowov ioxiciiy avo
jusi sono oI iho aspocis io bo considovod whon choos-
ing a suiiablo oligonuclooiido.
Dcliccry o] cIidc uuclcic ocids (IAAs).
!NAs Iovn siablo conploxos wiih DNA and RNA,
havo low ioxiciiy and, unliLo naiuvally occuvving oli-
gonuclooiidos, avo noi sonsiiivo io nucloasos. Jhoso
Ioaiuvos naLo ihon an idoal iool Iov aniisonso ihov-
apy. As is iho caso Iov iho najoviiy oI lavgo nolo-
culos, iho collulav upiaLo oI ihoso oligonuclooiidos is
poov, bui coupling io !JDs incvoasos ihoiv upiaLo
and ihus ihoiv applicabiliiy as iools Iov iho highly
spocic downvogulaiion oI dosivod gono pvoducis. !n
nany casos iho !JD-!NA consivuci is noi syniho-
sizod as a coniinuous chain bocauso synihosis oI a
coniinuous chain could iniovIovo wiih iho iniovnaliza-
iion oI iho consivuci and iho !NA-nRNA iniovaciion.
JhovoIovo, iho noihod nosi Ivouonily onployod is
coupling oI iho !NA io iho popiido via a disuldo
bond [28, 29]. Jho disuldo bond has no signicani
ooci on iniovnalizaiion and, onco insido iho coll,
iho condiiions oI iho inivacollulav onvivonnoni causo
iho voduciion oI iho disuldo bond, ihovoby voloasing
!NAs, which avo ihon Ivoo io iniovaci wiih iho
dosivod iavgoi [3O]. Movoovov, !JD-!NA has boon
shown io onhanco iho ivansIociion o cacy oI plas-
nids [3i]. !n conbinaiion wiih iho ivansIociion
agoni polyoihylonoinino (!!), iho o cacy oI plas-
nid ivansIociion was inpvovod up io oighiIold (soo
Plosmiddelivery sociion).
siRAA dcliccry. Jho voconi discovovy oI
iho RNA iniovIovonco paihway in a wido vavioiy oI
ouLavyoiic ovganisns has pvovidod a novol noans oI
chavaciovizing gono Iunciion in nannalian colls.
Shovi, iniovIoving RNAs (siRNAs) havo considovablo
poioniial as a powovIul iool in noloculav biology
vosoavch and in iho Iuiuvo as phavnacouiical dvugs.
Howovov, iho najov dvawbacL wiih iho uso oI
siRNA, as wiih nosi oligonuclooiido-basod dvugs, is
iho low yiold oI collulav upiaLo. Ly conivasi, iho uso
oI a nixiuvo oI !JD and siRNA divociod iowavds
glycovaldohydo 3-phosphaio dohydvogonaso (CA!DH)
nRNA incvoasod iho collulav upiaLo oI iho siRNA
sovoval Iold conpavod wiih naLod siRNA, and iho
iavgoiod nRNA was downvogulaiod. !JD and
siRNA avo noi covalonily linLod, bui Iovn a conplox
ihvough olocivosiaiic iniovaciions [32]. Jho popiido
cavviov consiiiuios an oxcolloni iool Iov iho dolivovy
oI siRNA inio colls.
Dcliccryo]irouuouoorIiclcs. Woissolod-
ovs gvoup [3335] voconily dolivovod inio colls
4i-nn bioconpaiiblo, doxivan-coaiod supovpavanag-
noiic ivon oxido nanopaviiclos conjugaiod io JAJ-
!JD. Dovivaiizod paviiclos wovo iniovnalizod inio
lynphocyios ovov iOO-Iold novo o cionily ihan non-
nodiod paviiclos. Jhoso paviiclos wovo also iniov-
nalizod inio boih honaiopoioiic and nouval pvogoniiov
colls ai lovols up io iO3O pg pov coll. Movoovov,
iho ivon incovpovaiion did noi aoci coll viabiliiy,
diovoniiaiion, ov pvoliIovaiion oI CD34

hunan
sion colls. ollowing inivavonous injociion inio
innunodocioni nico, 4 oI nagnoiically CD34

colls honod io bono navvow pov gvan oI iissuo, and
singlo colls could bo doiociod by nagnoiic vosonanco
(MR) inaging in iissuo sanplos. Jho noihod has
poioniial applicaiions Iov invivo ivacLing oI nagnoii-
cally labolod colls by MR inaging and Iov vocovoving
inivacollulavly labolod colls Ivon ovgans.
Iiosomc dcliccry. Jho ihovapouiic doliv-
ovy oI polav conpounds is oIion ino cioni owing io
iho di culiy oI cvossing iho lipid nonbvano oI colls.
As a consouonco, dolivovy oI ihoso conpounds
wiihin liposonal cavviovs has boon iho Iocus oI
incvoasing aiioniion, bui has boon hanpovod by ino -
cioni collulav upiaLo and consouoni dogvadaiion
ihvough iho ondocyiic paihway. Roconily, Jovchilin
e| ol. [36] dononsivaiod ihai volaiivoly lavgo dvug
cavviovs, such as 2OO-nn liposonos, can bo dolivovod
inio colls by a JAJ popiido aiiachod io iho liposono
suvIaco. !ncubaiion oI JAJ liposonos wiih sovoval
colls vosulis in inivacollulav localizaiion oI coviain
liposonos. ConIocal analysis showod ihai iho lipo-
sonos vonainod inivacollulavly iniaci i h aIiov ivans-
4 Acta Med. OkayamaVol. 60, No. 1 Noguchi et al.
5 Protein Transduction Technology February 2006
Extracellular Intracellular
Plasma membrane
Macropinocytosis
Retrograde
Transport
Cargo
PTD
Fig. 2Mechanism of protein transduction. The primary mechanism of protein transduction is an electrostatic interaction with the
plasma membrane, penetration into cells by macropinocytosis, and release to the cytoplasm and nuclei by retrograde transport.
duciion. Jho appvoach nay havo inpoviani inplica-
iions Iov dvug dolivovy divocily inio iho coll cyio-
plasn.
Ilosmid dcliccry. Alihough vival noihods
havo sovoval advaniagos, ihoy also havo nany dvaw-
bacLs such as iho innunological vosponso pvovoLod
by oxposuvo io vival inIociion. Oihov cuvvonily
onployod siandavd noihods, including iho applicaiion
oI polycaiionic agonis, olocivopovaiion and nicvoin-
jociion, avo ino cioni Iov uso in vivo. !olycaiionic
agonis havo ioxiciiy and iavgoiing pvoblons, olocivo-
povaiion causos high coll novialiiy and nicvoinjociion
can only bo appliod io ono coll ai a iino. Howovov,
iho uso oI non-vival synihoiic vociovs nininizos iho
visL oI iviggoving an innuno vosponso in iho ivoaiod
individual bocauso ihoso vociovs lacL vival conpo-
nonis. !JDs can also dolivov plasnids. SaLaguchi e|
ol. [37] dosignod a now sysion Iov ivanspoviing oli-
gonuclooiidos inio coll nucloi. Jho vohiclo is con-
posod oI gluiaihiono-S-ivansIovaso, 7 avginino vosi-
duos, iho DNA-binding donain oI CAL4 and a
nucloav localizaiion signal, which avo linLod wiih
oxiblo glycino sivoichos. Jho p53-vosponsivo olo-
noni linLod io iho CAL4 upsivoan aciivaiing
souonco was o cionily ivansIovvod by iho vohiclo
pvoioin inio iho nucloi oI pvinavy culiuvos oI nouvo-
nal colls, onbvyonic sion colls, and vavious hunan
novnal colls. Jho popiido souonco !KKKRKV has
nosily boon usod as a nucloav localizaiion signal
(NLS), bui ii has boon suggosiod ihai ihis popiido
also has coll-ponoivaiing Ioaiuvos, and ii has boon
shown io ivanslocaio pvoioins lavgov ihan 97O LDa
inio iho nuclous [38]. Jho linLing oI iho SV4O NLS
popiido souonco via !NA io a vopoviov gono cavvy-
ing iho vociov, ivansIociing colls and !!, vosuliod
in an up io oighiIold incvoaso in iho nucloav upiaLo oI
C! ov locZ-cavvying plasnids, conpavod wiih
only !! aciing as iho ivansIociing agoni [3i].
JvansIociion by !JD vociovs nighi uso iho
couniov chavgo boiwoon nogaiivoly chavgod plasnid
DNA and iho posiiivoly chavgod popiido. Hyndnan e|
ol. [39] dononsivaiod ihai incovpovaiion wiihoui
covaloni linLago oI a JAJ !JD popiido inio gono
dolivovy lipoploxos inpvovod gono ivansIov.
uvihovnovo, siovilizod R8 (avginino-8) popiidos
havo boon shown io ivansIoci luciIovaso-coniaining
plasnids inio iho nuclous wiih an o cioncy ihai is
conpavablo io LipoIocianino
JM
[4O]. Jo avoid loss
oI ivanslocaiion as a vosuli oI iho !JD iniovaciing
wiih DNA, iho !JD can bo dosignod as a bvanchod
conplox io iniovaci wiih DNA and nodiaio nonbvano
ivanslocaiion. ighi-bvanchod JAJ has boon usod io
ivansIoci colls, wiih vosulis ouivaloni io siandavd
LipoIocianino
JM
sivaiogios [4i]. Jhoso sysions can
bo a convonioni and powovIul iool Iov spocically dis-
vupiing iho Iunciion oI DNA-binding pvoioins.
Dcliccryo]gcuomco]hocIcriolhogcoud
cirus. An oxivono caso is iho inivacollulav
addvossing oI baciovial phagos and vivusos [5, 42,
43]. guchi e| ol. consivuciod voconbinani lanbda
phago paviiclos displaying JAJ-!JD on ihoiv suv-
Iacos and cavvying nannalian navLov gonos as pavi
oI ihoiv gononos [42]. Whon aninal colls avo bvioy
oxposod io iho JAJ-phago, signicani oxpvossion oI
phago navLov gonos is inducod wiih no havnIul
oocis io iho colls. Civon iho sizo oI iho phago
gonono, iho iochnology could ihovoIovo bo usod io
inivoduco up io 2O,OOO bp oI Iovoign coding DNA inio
colls. Sinilavly, incubaiing a conbinaiion oI vival
vociovs and !JDs boIovo vival inIociion can inpvovo
vival gono dolivovy. Jho uso oI adonovivus and Anip-
!JD sinulianoously signicanily inpvovod iho o -
cioncy oI iho gono dolivovy oI gvoon uovosconi pvo-
ioin (C!) and -Cal by adonovivus in vivo and in
vi|ro [43]. Anip-!JD can pvonoio vival upiaLo wiih-
oui gonoiic coupling, povhaps ihvough iho iniovaciion
oI Anip-!JD wiih coai pvoioins. A possiblo nocha-
nisn is ihai iho anphipaihic popiido binds iho vivus
ov phago ihvough iis hydvophobic sido and iho lipids
ihvough iis chavgod sido.
Jhoso daia nay havo signicani inplicaiions Iov
iho Iuiuvo oI oihov noloculo dolivovy sysions invi|ro
and invivo.
Mcchunisms of Frotcin Jrunsduction
Ono sivuciuval collulav nonbvano conpononi is
vouivod Iov !JD iniovnalizaiion. !i is shown ihai
incvoasing conconivaiions oI hopavin could conpoio
wiih JAJ-!JD iniovnalizaiion [44]. Noi suvpvis-
ingly givon ihoiv highly basic chavgo, JAJ popiidos
bind io hopavin [4547]. Jhis wovL lod io iho dis-
covovy ihai coll-suvIaco hopavin sulIaio pvoiooglycans
appoav io bo Loy nodiaiovs oI popiido iniovnalizaiion
[48]. Consisioni wiih ihis obsovvaiion, colls gonoii-
cally inpaivod in iho biosynihosis oI Iully sulIonaiod
6 Acta Med. OkayamaVol. 60, No. 1 Noguchi et al.
hopavin sulIaio pvoiooglycans wovo solociivoly
inpaivod Iov ivansduciion by a JAJ Iusion pvoioin
[48], poly-avginino, and LJA2/NouvoD !JD
(Noguchi e|ol. unpublishod daia). !n addiiion, !JD
upiaLo could bo conpoiiiivoly inhibiiod by iho soloc-
iivo dogvadaiion oI iho hopavin sulIaio sidochains
using glycosaninoglycan lyasos (Noguchi e|ol. unpub-
lishod daia). Jho ubiuiious pvosonco oI hopavin sul-
Iaio nighi oxplain iho obsovvaiion ihai !JDs and
conjugaiod pvoioins avo ablo io oniov a wido vavioiy
oI colls. Jhis iniovaciion ai iho coll suvIaco is liLoly
io nodiaio iho upiaLo oI oihov avginino-vich !JDs.
ov oxanplo, Anip-!JD has also boon Iound io
iniovaci sivongly wiih collulav nonbvanos [49].
avly siudios showod ihai iho iniovnalizaiion oI
JAJ-!JD, Anip-!JD, ov polyR-!JD was noi sig-
nicanily inhibiiod by incubaiion ai 4 `C, by doplo-
iion oI iho collulav AJ! pool, ov by inhibiiovs oI
ondocyiosis ; addiiionally, iniovnalizaiion did noi
dopond on a spocic pvinavy souonco, inplying ihai
vocopiov vocogniiion was noi nocossavy [i8, 5O, 5i].
JhovoIovo, ii had boon connonly accopiod ihai iho
iniovnalizaiion oI !JD-coniaining pvoioins involvos
noiihov ondocyiosis nov spocic pvoioin ivanspoviovs.
Howovov, sono nochanisiic siudios suggosi ihai boih
iho inpovi and nucloav localizaiion oI !JD-coniaining
pvoioins occuv only duving xaiion and noi in living
colls [52, 53]. Lundbovg and Johansson showod ihai
xaiion wiih noihanol inducos an aviicial inux oI
boih V!22 pvoioin Ivon iho IerjesSimjlex vivus and
iho posiiivoly chavgod hisiono Hi [52]. Addiiionally,
boih JAJ-!JD and V!22-!JD wovo shown io nodi-
aio coll binding bui noi ivanslocaiion [53]. Jhoso
daia suggosi ihai iho pvoioin ivansduciion doos noi
occuv in living colls. Howovov, a lavgo nunbov oI
siudios havo voconily vopoviod !JDs in living colls
using boih vopoviov pvoioins and/ov Iunciional pop-
iidos/pvoioins. !ndood, pvoioin ivansduciion MUSJ
wovL in living colls bocauso nany publishod siudios
havo dononsivaiod !JD-nodiaiod dolivovy oI
L!OLOC!CALLY ACJ!V pvoioins/popiidos. Jho
Iollowing ihvoo nodols havo onovgod io oxplain !JD
upiaLo in voconi vopovis.
Ono nodol is iho povpondiculav insoviion oI
anphipaihic popiidos inio iho nonbvano, wiih hydvo-
phobic vosiduos juxiaposing iho lipids and iho asson-
blod hydvophilic Iacos Iovning a channol ihvough
oligonovizaiion. Jhis would allow o cioni passago
oI hydvophilic cavgoos, bui would also bo a ihvoai io
coll suvvival. Sovoval popiidos can Iovn povos ai iho
coll suvIaco and, indood, induco coll doaih. Ono
oxanplo oI ihis is pvovidod by doIonsins [54], a
class oI snall popiidos ihai Iovn holos in nicvobial
nonbvanos and avo pvoducod by iho invoviobvaio
innuno sysion and by sono voviobvaio colls.
A socond nodol pvoposos ihai popiidos bind io
iho polav hoads oI lipids in iho nonbvano ihvough
olocivosiaiic iniovaciion, vosuliing in ivansioni Iov-
naiion oI invoviod nicollos ihai ongulI iho popiido
and iis cavgo, ovon ai 4 `C. Jho vo-oponing oI iho
nicollo insido iho coll would ihon vosuli in voloaso oI
iho !JD cavgo consivuci inio iho cyioplasn. Sono
oxpovinonis suggosi ihai Anip-!JD nighi uso ihis
paihway. Jho addiiion oI Anip-!JD io a ln oI
bvain lipids inducos iho Iovnaiion oI invoviod
nicollos. [55] A popiido oI iho sano longih, chavgo
and hydvophobiciiy in which a ivypiophan has boon
changod inio a phonylanino (W6 vaviani) siill binds
iho lipids [56, 57] bui is noi iniovnalizod [58] and
doos noi induco invoviod nicollo Iovnaiion. Howovov,
invoviod nicollo Iovnaiion would linii cavgo sizo and
vouivo iho pvosonco oI hydvophobic anino acids,
which avo absoni in JAJ-!JD and poly-avginino-
!JDs ; ihus, such a nochanisn is unliLoly io bo a
connon !JD dolivovy nochanisn.
A ihivd nodol is ondocyiosis Iollowod by pas-
sago Ivon iho vosiclo inio iho cyioplasn ; alihough
ihis inplios vosiclo disvupiion, iho plasna nonbvano
and coll viabiliiy avo noi aociod. Sono siudios havo
shown ihai JAJ-!JD, polyR-!JD, Anip-!JD, and
avginino-vich popiido SynL oniov colls by ondocyiosis
vaihov ihan by ionpovaiuvo- ov onovgy-indopondoni
ivanslocaiion [59, 6O]. Wo obsovvod, Iov iho vsi
iino, pvoioin ivansduciion in voal iino. JAJ-!JD,
iiR-!JD, and LJA2/NouvoD ivanscvipiion Iac-
iov, which has an avginino- and lysino-vich !JD,
ponoivaios inio colls by nacvopinocyiosis, a iypo oI
ondocyiosis, and is voloasod Ivon iho ondosono
honogonoously inio iho cyioplasn and nucloi by voi-
vogvado ivanspovi [2, i9]. Anip-!JD and !DX-i
pvoioin, which has Anip-liLo !JD, is also iniovnal-
izod inio colls by iho sano nochanisn [2, 3].
Roconily, iho volo oI ondosonal acidicaiion and voi-
vogvado ivanspovi Iov iho upiaLo oI Anip-!JD,
JAJ-!JD, and 9R-!JD was vopoviod [6i]. A nun-
bov oI woll-chavaciovizod ioxins voach iho cyiosol oI
7 Protein Transduction Technology February 2006
ouLavyoiic colls aIiov binding io iho coll suvIaco,
undovgoing ondocyiosis and voivogvado ivanspovi io
iho Colgi appavaius and ondoplasnic voiiculun [62,
63, 64]. Jho avginino-vich noiiI oI 8iO anino acids
in iho A subuniis oI ihoso ioxins, vopoviod io bo
ivanspoviod by noans oI voivogvado ivanspovi, is
sinilav io ihoso oI JAJ-!JD, Anip-!JD [6i] and
!DX-i-!JD. !JDs, ovon Anip-!JD, ivanspovi by
a sinilav nochanisn io ihai oI ihoso pvoioin ioxins.
!n conclusion, iho pvinavy nochanisn oI pvoioin
ivansduciion is an olocivosiaiic iniovaciion wiih iho
plasna nonbvano, ponoivaiion inio colls by nacvopi-
nocyiosis, and voloaso io iho cyioplasn and nucloi by
voivogvado ivanspovi. Howovov, ii is noi possiblo io
conploioly olininaio iho possibiliiy oI paviial upiaLo
acvoss iho plasna nonbvano via oihov nochanisns.
Iuturc Ohjcctivcs
!voioin ivansduciion iochnology has sono disad-
vaniagos. Ono is ihai ivansducod pvoioins only havo
a liniiod aciivo halI-liIo, so ihai ihoiv oocis avo
only ivansioni. Michiuo e| ol. showod ihai nuiani
pvoioins oI p53 in which nuliiplo lysino vosiduos in
iho C-iovninal wovo voplacod by avgininos wovo ooc-
iivoly dolivovod inio gliona colls and wovo vosisiani
io Mdn2-nodiaiod ubiuiiinaiion [65]. Jho nuiani
pvoioins displayod highov ivanscvipiion vogulaiovy
aciiviiy and inducod powovIul inhibiiion oI gliona coll
pvoliIovaiion. Jhoso ndings suggosi ihai ubiuiiina-
iion-vosisiani pvoioins nay bocono a novo oociivo
sivaiogy Iov pvoioin ivansduciion iochnology.
Anoihov disadvaniago is accunulaiion oI !JDs
ov !JD-Iusod pvoioins in iho ondocyiic conpavinoni.
Jho nain nochanisn oI pvoioin ivansduciion is pono-
ivaiion inio colls by nacvopinocyiosis and voloaso io
iho cyioplasn and nucloi by voivogvado ivanspovi.
Alihough !JDs ov !JD-Iusod pvoioins can bo ivans-
ducod inio colls by nacvopinocyiosis, such pvoioins
avo oIion onivappod insido nacvopinosonos in iho
cyioplasn. JhovoIovo, high doso conconivaiions oI
!JDs ov !JD-Iusod pvoioins avo noodod Iov iho
iochnology io Iunciion oociivoly. Jo ovovcono ihis
pvoblon, iho pvoioins wovo linLod wiih iho NH
2
-
iovninal donain oI inuonza vivus honaggluiinin-2
subunii (HA2), which is a pH-dopondoni Iusogonic
popiido ihai inducos iho lysis oI nonbvanos ai low
pH lovols [2i, 66]. Jho pH-sonsiiivo HA2 popiido
navLodly and spocically onhancod nacvopinosono
oscapo [66]. HA2- and polyavginino-Iusod p53
inducod p2i(WAi) ivanscvipiional aciiviiy and
inhibiiod iho gvowih oI cancov colls novo oociivoly
ihan a polyavginino-Iusod p53 pvoioin [2i].
Movoovov, wo dovolopod noihods oI onhancing ondo-
sono oscapo using phoio-sonsiiivo !JD [6]. Jhoso
iochniuos can onhanco iho o cioncy oI iho pvoioin
ivansduciion sysion.
!n sono pvoioin ivansduciion oxpovinonis, iho
aciiviiy oI iniovnalizod pvoioins was noi voiainod Iol-
lowing insoviion inio iho coll. Jo vosolvo ihis issuo,
pvoioins and conpounds could bo couplod io !JDs
via a linLov dosignod io voloaso iho aciivo pvoioins
and dvugs wiihin colls. Ovgan-spocic dolivovy involv-
ing !JDs also posos a conplox challongo wiih
vospoci io iho uiiliiy oI ihis iochnology as a clinical
iool. Jho iochnology cannoi bo appliod io insolublo
pvoioins duo io di culiios associaiod wiih puvica-
iion oI ihoso noloculos. Ovovconing ihoso obsiaclos
will vouivo advancos involving vonod !JD
souoncos and inpvovod applicaiion.
Fhysio!ogicu! und Dcvc!opmcntu! Imp!icutions
!JDs avo donod by ihoiv abiliiy io voach iho
cyioplasnic and/ov nucloav conpavinonis in livo
colls aIiov iniovnalizaiion. Jho iniiial discovovy oI
!JDs oviginaiod Ivon iho unoxpociod obsovvaiion
ihai coviain Iull-longih pvoioins ov pvoioin donains
can ivanslocaio acvoss iho plasna nonbvano. Jhis
was vsi shown Iov iho H!V JAJ ivansaciivaiov [67,
68] and Iov iho honoodonain oI iho Drosojhilomelo-
noyos|er ivanscvipiion Iaciov Anionnapodia [69], and
has sinco oxpandod io includo non-naiuval popiidos
ihai shavo ihis pvopoviy. !n iho caso oI honoodo-
nain- and JAJ-dovivod popiidos, and in conivasi
wiih nany oihov !JDs, upiaLo soons io vooci an in
vivo biological pvocoss. !n suppovi oI ihis, iho ivans-
locaiing aciiviiy oI ihoso popiidos covvolaios wiih
ihai oI ihoiv pavonial Iull-longih pvoioins [7O],
sivongly suggosiing ihai, in sono civcunsiancos,
ihoso pvoioins nay havo pavacvino aciiviiios as pavi
oI a physiological pvocoss, in ihai ihoy avo voloasod
by ono Lind oI coll and ihon iniovnalizod by oihov
colls. Accovdingly, iho Iull-longih pvoioins avo also
socvoiod, pvobably by unconvoniional socvoiion paih-
ways ihai do noi involvo a socvoiion signal souonco
8 Acta Med. OkayamaVol. 60, No. 1 Noguchi et al.
[7i, 72]. Roconi vopovis oI honoopvoioins show
ihai HOXL4 pvoioin siinulaios iho non-oncogonic
pvoliIovaiion oI CD34 haonaiopoioiic sion colls [73],
and ihai !DX-i pvoioin can ivansIovn duci colls inio
insulin pvoducing colls [i3]. Movoovov, wo
vopoviod ihai LJA2/NouvoD pvoioin, ono oI iho
bHLH ivanscvipiion Iaciovs, can also bo ivansducod
inio colls duo io iis own avginino- and lysino-vich
souonco [i9]. LJA2/NouvoD and !DX-i pvo-
ioins avo inpoviani in pancvoaiic dovolopnoni [74,
75]. Jho jdx-1 gono has A-boxos, and iho !DX-i
pvoioin can posiiivoly auiovogulaio iis oxpvossion
[76] ; iho BITA:/NeuroD gono also has -boxos,
and iho LJA2/NouvoD pvoioin can siinulaio iis
own ivanscvipiion [77]. Jho posiiivo auiovogulaiion
oI ihoso pvoioins loads us io considov a poioniial
pavacvino volo in noogonosis. Onco LJA2/NouvoD
and !DX-i pvoioins avo ivansducod inio pvogoniiov
colls, ondogonous BITA:/NeuroD and jdx-1 gono
ivanscvipiion avo anpliod by ihoso pvoioins, siinu-
laio insulin ivanscvipiion, and nay Iaciliiaio ihoiv
diovoniiaiion io insulin-pvoducing colls.
Alihough iho oxaci Iunciion oI ihis iniovcollulav
ivansIov is noi yoi cloav, voconi daia on ivanscvipiion
Iaciovs, in paviiculav, honoopvoioins, suggosi ihai,
aIiov ivansIov, iho ivanscvipiion Iaciov can vogulaio
ivanscvipiion and ivanslaiion in iho vocipioni coll,
ihus aciing as a nossongov pvoioin [4, 78]. As
honoopvoioin oxpvossion is vogionally spociod, ii
has boon spoculaiod ihai ihis covvosponds io iho pas-
sago oI posiiional inIovnaiion boiwoon colls [4, 78].
Conc!usions
Jho pvosoni pvoioin ivansduciion sysion has low
ioxiciiy and a high yiold oI dolivovy. Jho uso oI ihis
sysion opons up iniovosiing ihovapouiic povspociivos.
Jho iniovosi in pvoioin ivanslocaiion acvoss non-
bvanos is noi liniiod io !JDs and ihoiv uso as a iool
wiih which io invosiigaio coll biology, ov convovsoly
io ihoiv bioiochnological applicaiions. Jho iniovcollu-
lav ivansIov oI ondogonous ivanscvipiion Iaciovs,
such as JAJ and honoopvoioins, soons io poini io
an oviginal and inpoviani nodo oI signal ivansduc-
iion. Jho physiological signicanco oI ihoso paih-
ways vouivos Iuvihov oxplovaiion.
AcLnowlodgononis.Wo ihanL Dv. Koichi JanaLa, Shivoh uiaLi,
MichiLo Uoda, YusuLo NaLai, Hidoo Nagaia, Yasuhivo !wanaga, Jovu
OLiisu, YuLihido YonoLawa, and ALoni !shii (Kyoio Univovsiiy),
MasayuLi Maisushiia, HidoLi Maisui, Naoya Kobayashi, and NoviaLi
JanaLa (OLayana Univovsiiy), and Covdon C Woiv, and Susan Lonnov-
Woiv (Havvavd Modical School) Iov ihoiv valuablo suggosiions.
Rcfcrcnccs
1. Noguchi H, Kaneto H, Weir GC and Bonner-Weir S : PDX-1 pro-
tein containing its own antennapedia-like protein transduction
domain can transduce pancreatic duct and islet cells. Diabetes
(2003) 52 : 17321737.
2. Noguchi H, Matsushita M, Matsumoto S, Lu YF, Matsui H and
Bonner-Weir S : Mechanism of PDX-1 protein transduction.
Biochem Biophys Res Commun (2005) 332 : 6874.
3. Noguchi H, Matsumoto S, Okitsu T, Iwanaga Y, Yonekawa Y,
Nagata H, Matsushita M, Wei FY, Matsui H, Minami K, Masui Y,
Futaki S and Tanaka K : PDX-1 protein is internalized by lipid raft-
dependent macropinocytosis. Cell Transplant (2005) 14: 637-645.
4. Prochiantz A : Messenger proteins : homeoproteins, TAT and oth-
ers. Curr Opin Cell Biol (2000) 12 : 400406.
5. Joliot A and Prochiantz A : Transduction peptides : from technol-
ogy to physiology. Nat Cell Biol (2004) 6 : 189196.
6. Matsushita M, Noguchi H, Lu YF, Tomizawa K, Michiue H, Li
ST, Hirose K, Bonner-Weir S and Matsui H : Photo-acceleration
of protein release from endosome in the protein transduction sys-
tem. FEBS Lett (2004) 572 : 221226.
7. Elliott G and OHare P : Intercellular tra cking and protein delivery
by a herpesvirus structural protein. Cell (1997) 88 : 223233.
8. Schwarze SR, Ho A, Vocero-Akbani A and Dowdy SF : In vivo
protein transduction : delivery of a biologically active protein into
the mouse. Science (1999) 285 : 15691572.
9. Watanabe T, Shibata N, Westerman KA, Okitsu T, Allain JE,
Sakaguchi M, Totsugawa T, Maruyama M, Matsumura T,
Noguchi H, Yamamoto S, Hikida M, Ohmori A, Reth M, Weber A,
Tanaka N, Leboulch P and Kobayashi N : Establishment of immor-
talized human hepatic sPhonelate scavenger cells to develop bio-
articial livers. Transplantation (2003) 75 : 18731880.
10. Matsumura T, Takesue M, Westerman KA, Okitsu T, Sakaguchi M,
Fukazawa T, Totsugawa T, Noguchi H, Yamamoto S, Stolz DB,
Tanaka N, Leboulch P and Kobayashi N : Establishment of an
immortalized human-liver endothelial cell line with SV40T and
hTERT. Transplantation (2004) 77 : 13571365.
11. Bonny C, Oberson A, Negri S, Sauser C and Schorderet
DF : Cell-permeable peptide inhibitors of JNK : novel blockers of
beta-cell death. Diabetes (2001) 50 : 7782.
12. Kaneto H, Nakatani Y, Miyatsuka T, Kawamori D, Matsuoka TA,
Matsuhisa M, Kajimoto Y, Ichijo H, Yamasaki Y and Hori
M : Possible novel therapy for diabetes with cell-permeable JNK-
inhibitory peptide. Nat Med (2004) 10 : 11281132.
13. Embury J, Klein D, Pileggi A, Ribeiro M, Jayaraman S, Molano
RD, Fraker C, Kenyon N, Ricordi C, Inverardi L and Pastori
RL : Proteins linked to a protein transduction domain e ciently
transduce pancreatic islets. Diabetes (2001) 50 : 17061713.
14. Noguchi H, Matsushita M, Okitsu T, Moriwaki A, Tomizawa K,
Kang S, Li ST, Kobayashi N, Matsumoto S, Tanaka K, Tanaka
N and Matsui H : A new cell-permeable peptide allows successful
allogeneic islet transplantation in mice. Nat Med (2004) 10 : 305
309.
15. Noguchi H, Nakai Y, Matsumoto S, Kawaguchi M, Ueda M,
9 Protein Transduction Technology February 2006
Okitsu T, Iwanaga Y, Yonekawa Y, Nagata H, Minami K, Masui Y,
Futaki S and Tanaka K : Cell Permeable Peptide of JNK Inhibitor
Prevents Islet Apoptosis Immediately After Isolation and Improves
Islet Graft Function. Am J Transplant (2005) 5 : 18481855.
16. Matsushita M, Tomizawa K, Moriwaki A, Li ST, Terada H and
Matsui H : A high-e ciency protein transduction system demon-
strating the role of PKA in long-lasting long-term potentiation. J
Neurosci (2001) 21 : 60006007.
17. Mai JC, Shen H, Watkins SC, Cheng T and Robbins
PD : E ciency of protein transduction is cell type-dependent and
is enhanced by dextran sulfate. J Biol Chem (2002) 277 : 30208
30218.
18. Futaki S, Suzuki T, Ohashi W, Yagami T, Tanaka S, Ueda K
and Sugiura Y : Arginine-rich peptides. An abundant source of
membrane-permeable peptides having potential as carriers for
intracellular protein delivery. J Biol Chem (2001) 276 : 58365840.
19. Noguchi H, Bonner-Weir S, Wei FY, Matsushita M and
Matsumoto S : BETA2/NeuroD protein can be transduced into
cells due to an arginine- and lysine-rich sequence. Diabetes (2005)
54 : 28592866
20. Dominguez-Bendala J, Klein D, Ribeiro M, Ricordi C, Inverardi L,
Pastori R and Edlund H : TAT-mediated neurogenin 3 protein trans-
duction stimulates pancreatic endocrine dierentiation in vitro.
Diabetes (2005) 54 : 720726.
21. Michiue H, Tomizawa K, Wei FY, Matsushita M, Lu YF,
Ichikawa T, Tamiya T, Date I and Matsui H : The NH2 terminus of
inuenza virus hemagglutinin-2 subunit peptides enhances the anti-
tumor potency of polyarginine-mediated p53 protein transduction. J
Biol Chem (2005) 280 : 82858289.
22. Barka T and Van der Noen HM : Retrovirus-mediated gene transfer
into salivary glands in vivo. Hum Gene Ther (1996) 7 : 613618.
23. Barka T, Gresik EW and van Der Noen H : Transduction of TAT-
HA-beta-galactosidase fusion protein into salivary gland-derived
cells and organ cultures of the developing gland, and into rat sub-
mandibular gland in vivo. J Histochem Cytochem (2000) 48 : 1453
1460.
24. Kurreck J : Antisense technologies. Improvement through novel
chemical modications. Eur J Biochem (2003) 270 : 162844.
25. Morris MC, Vidal P, Chaloin L, Heitz F and Divita G : A new pep-
tide vector for e cient delivery of oligonucleotides into mammalian
cells. Nucleic Acids Res (1997) 25 : 27302736.
26. Tung CH and Stein S : Preparation and applications of peptide-oli-
gonucleotide conjugates. Bioconjug Chem (2000) 11 : 605618.
27. Astriab-Fisher A, Sergueev DS, Fisher M, Shaw BR and Juliano
RL : Antisense inhibition of P-glycoprotein expression using pep-
tide-oligonucleotide conjugates. Biochem Pharmacol (2000)
60 : 8390.
28. Pooga M, Soomets U, Hallbrink M, Valkna A, Saar K, Rezaei K,
Kahl U, Hao JX, Xu XJ, Wiesenfeld-Hallin Z, Hokfelt T, Bartfai
T and Langel U : Cell penetrating PNA constructs regulate galanin
receptor levels and modify pain transmission in vivo. Nat
Biotechnol (1998) 16 : 857861.
29. Ostenson CG, Sandberg-Nordqvist AC, Chen J, Hallbrink M,
Rotin D, Langel U and Efendic S : Overexpression of protein-tyro-
sine phosphatase PTP sigma is linked to impaired glucose-induced
insulin secretion in hereditary diabetic Goto-Kakizaki rats. Biochem
Biophys Res Commun (2002) 291 : 945950.
30. Hallbrink M, Floren A, Elmquist A, Pooga M, Bartfai T and
Langel U : Cargo delivery kinetics of cell-penetrating peptides.
Biochim Biophys Acta (2001) 1515 : 101109.
31. Branden LJ, Mohamed AJ and Smith CI : A peptide nucleic acid-
nuclear localization signal fusion that mediates nuclear transport of
DNA. Nat Biotechnol (1999) 17 : 784787.
32. Simeoni F, Morris MC, Heitz F and Divita G : Insight into the
mechanism of the peptide-based gene delivery system
MPG : implications for delivery of siRNA into mammalian cells.
Nucleic Acids Res (2003) 31 : 27172724.
33. Josephson L, Tung CH, Moore A and Weissleder R : High-
e ciency intracellular magnetic labeling with novel superparamag-
netic-Tat peptide conjugates. Bioconjug Chem (1999) 10 : 186
191.
34. Lewin M, Carlesso N, Tung CH, Tang XW, Cory D, Scadden DT
and Weissleder R : Tat peptide-derivatized magnetic nanoparticles
allow in vivo tracking and recovery of progenitor cells. Nat
Biotechnol (2000) 18 : 410414.
35. Dodd CH, Hsu HC, Chu WJ, Yang P, Zhang HG, Mountz JD Jr,
Zinn K, Forder J, Josephson L, Weissleder R, Mountz JM and
Mountz JD : Normal T-cell response and in vivo magnetic reso-
nance imaging of T cells loaded with HIV transactivator-peptide-
derived superparamagnetic nanoparticles. J Immunol Methods
(2001) 256 : 89105.
36. Torchilin VP, Rammohan R, Weissig V and Levchenko TS : TAT
peptide on the surface of liposomes aords their e cient intracel-
lular delivery even at low temperature and in the presence of meta-
bolic inhibitors. Proc Natl Acad Sci USA (2001) 98 : 87868791.
37. Sakaguchi M, Nukui T, Sonegawa H, Murata H, Futami J,
Yamada H and Huh NH : Targeted disruption of transcriptional reg-
ulatory function of p53 by a novel e cient method for introducing a
decoy oligonucleotide into nuclei. Nucleic Acids Res (2005)
33 : e88.
38. Yoneda Y, Semba T, Kaneda Y, Noble RL, Matsuoka Y,
Kurihara T, Okada Y and Imamoto N : A long synthetic peptide
containing a nuclear localization signal and its anking sequences
of SV40 T-antigen directs the transport of IgM into the nucleus e -
ciently. Exp Cell Res (1992) 201 : 313320.
39. Hyndman L, Lemoine JL, Huang L, Porteous DJ, Boyd AC and
Nan X : HIV-1 Tat protein transduction domain peptide facilitates
gene transfer in combination with cationic liposomes. J Control
Release (2004) 99 : 435444.
40. Futaki S, Ohashi W, Suzuki T, Niwa M, Tanaka S, Ueda K,
Harashima H and Sugiura Y : Stearylated arginine-rich peptides : a
new class of transfection systems. Bioconjug Chem (2001)
12 : 10051011.
41. Tung CH, Mueller S and Weissleder R : Novel branching mem-
brane translocational peptide as gene delivery vector. Bioorg Med
Chem (2002) 10 : 36093614.
42. Eguchi A, Akuta T, Okuyama H, Senda T, Yokoi H, Inokuchi H,
Fujita S, Hayakawa T, Takeda K, Hasegawa M and Nakanishi
M : Protein transduction domain of HIV-1 Tat protein promotes e -
cient delivery of DNA into mammalian cells. J Biol Chem (2001)
276 : 2620426210.
43. Gratton JP, Yu J, Gri th JW, Babbitt RW, Scotland RS, Hickey R,
Giordano FJ and Sessa WC : Cell-permeable peptides improve cel-
lular uptake and therapeutic gene delivery of replication-decient
viruses in cells and in vivo. Nat Med (2003) 9 : 357362.
44. Mann DA and Frankel AD : Endocytosis and targeting of exoge-
nous HIV-1 Tat protein. EMBO J (1991) 10 : 17331739.
45. Rusnati M, Coltrini D, Oreste P, Zoppetti G, Albini A, Noonan D,
dAdda di Fagagna F, Giacca M and Presta M : Interaction of
HIV-1 Tat protein with heparin. Role of the backbone structure,
sulfation, and size. J Biol Chem (1997) 272 : 1131311320.
46. Rusnati M, Tulipano G, Urbinati C, Tanghetti E, Giuliani R,
10 Acta Med. OkayamaVol. 60, No. 1 Noguchi et al.
Giacca M, Ciomei M, Corallini A and Presta M : The basic
domain in HIV-1 Tat protein as a target for polysulfonated heparin-
mimicking extracellular Tat antagonists. J Biol Chem (1998)
273 : 1602716037.
47. Hakansson S, Jacobs A and Carey M : Heparin binding by the
HIV-1 tat protein transduction domain. Protein Sci (2001) 10 : 2138
2139.
48. Tyagi M, Rusnati M, Presta M and Giacca M : Internalization of
HIV-1 tat requires cell surface heparan sulfate proteoglycans. J
Biol Chem (2001) 276 : 32543261.
49. Thoren PE, Persson D, Karlsson M and Norden B : The antenna-
pedia peptide penetratin translocates across lipid bilayers - the rst
direct observation. FEBS Lett (2000) 482 : 265268.
50. Vives E, Brodin P and Lebleu B : A truncated HIV-1 Tat protein
basic domain rapidly translocates through the plasma membrane
and accumulates in the cell nucleus. J Biol Chem (1997)
272 : 1601016017.
51. Derossi D, Calvet S, Trembleau A, Brunissen A, Chassaing G
and Prochiantz A : Cell internalization of the third helix of the
Antennapedia homeodomain is receptor-independent. J Biol Chem
(1996) 271 : 1818818193.
52. Lundberg M and Johansson M : Positively charged DNA-binding
proteins cause apparent cell membrane translocation. Biochem
Biophys Res Commun (2002) 291 : 367371.
53. Falnes PO, Wesche J and Olsnes S : Ability of the Tat basic
domain and VP22 to mediate cell binding, but not membrane
translocation of the diphtheria toxin A-fragment. Biochemistry
(2001) 40 : 43494358.
54. Ganz T : Defensins : antimicrobial peptides of innate immunity.
Nat Rev Immunol (2003) 3 : 710720.
55. Berlose JP, Convert O, Derossi D, Brunissen A and Chassaing
G : Conformational and associative behaviours of the third helix of
antennapedia homeodomain in membrane-mimetic environments.
Eur J Biochem (1996) 242 : 372386.
56. Christiaens B, Symoens S, Verheyden S, Engelborghs Y, Joliot A,
Prochiantz A, Vandekerckhove J, Rosseneu M and Vanloo
B : Tryptophan uorescence study of the interaction of penetratin
peptides with model membranes. Eur J Biochem (2002) 269 : 2918
2926.
57. Magzoub M, Eriksson LE and Graslund A : Comparison of the
interaction, positioning, structure induction and membrane pertur-
bation of cell-penetrating peptides and non-translocating variants
with phospholipid vesicles. Biophys Chem (2003) 103 : 271288.
58. Dom G, Shaw-Jackson C, Matis C, Bou oux O, Picard JJ,
Prochiantz A, Mingeot-Leclercq MP, Brasseur R and Rezsohazy
R : Cellular uptake of Antennapedia Penetratin peptides is a two-
step process in which phase transfer precedes a tryptophan-depen-
dent translocation. Nucleic Acids Res (2003) 31 : 556561.
59. Richard JP, Melikov K, Vives E, Ramos C, Verbeure B, Gait MJ,
Chernomordik LV and Lebleu B : Cell-penetrating peptides. A
reevaluation of the mechanism of cellular uptake. J Biol Chem
(2003) 278 : 585590.
60. Drin G, Cottin S, Blanc E, Rees AR and Temsamani J : Studies
on the internalization mechanism of cationic cell-penetrating pep-
tides. J Biol Chem (2003) 278 : 3119231201.
61. Fischer R, Kohler K, Fotin-Mleczek M and Brock R : A stepwise
dissection of the intracellular fate of cationic cell-penetrating pep-
tides. J Biol Chem (2004) 279 : 1262512635.
62. Sandvig K and van Deurs B : Entry of ricin and Shiga toxin into
cells : molecular mechanisms and medical perspectives. EMBO J
(2000) 19 : 59435950.
63. Lord JM and Roberts LM : Toxin entry : retrograde transport
through the secretory pathway. J Cell Biol (1998) 140 : 733736.
64. Noguchi H and Matsumoto S : Protein transduction technology
oers a novel therapeutic approach for diabetes. J Hepatobiliary
Pancreat Surg (2006) (in press).
65. Michiue H, Tomizawa K, Matsushita M, Tamiya T, Lu YF,
Ichikawa T, Date I and Matsui H : Ubiquitination-resistant p53 pro-
tein transduction therapy facilitates anti-cancer eect on the
growth of human malignant glioma cells. FEBS Lett (2005)
579 : 39653969.
66. Wadia JS, Stan RV and Dowdy SF : Transducible TAT-HA fuso-
genic peptide enhances escape of TAT-fusion proteins after lipid
raft macropinocytosis. Nat Med (2004) 10 : 310315.
67. Frankel AD and Pabo CO : Cellular uptake of the tat protein from
human immunodeciency virus. Cell (1988) 55 : 11891193.
68. Green M and Loewenstein PM : Autonomous functional domains of
chemically synthesized human immunodeciency virus tat trans-
activator protein. Cell (1988) 55 : 11791188.
69. Joliot A, Pernelle C, Deagostini-Bazin H and Prochiantz
A : Antennapedia homeobox peptide regulates neural morphogene-
sis. Proc Natl Acad Sci U S A (1991) 88 : 18641868.
70. ChaPhonein L, Volovitch M, Joliot AH, Perez F and Prochiantz
A : Transcription factor hoxa-5 is taken up by cells in culture and
conveyed to their nuclei. Mech Dev (1996) 55 : 111117.
71. Hel l and DE, Wel l es JL, Caputo A and Hasel ti ne
WA : Transcellular transactivation by the human immunodeciency
virus type 1 tat protein. J Virol (1991) 65 : 45474549.
72. Joliot A, Maizel A, Rosenberg D, Trembleau A, Dupas S,
Volovitch M and Prochiantz A : Identication of a signal sequence
necessary for the unconventional secretion of Engrailed homeopro-
tein. Curr Biol (1998) 8 : 856863.
73. Amsellem S, Pumio F, Bardinet D, Izac B, Charneau P, Romeo
PH, Dubart-Kupperschmitt A and Fichelson S : Ex vivo expansion
of human hematopoietic stem cells by direct delivery of the HOXB4
homeoprotein. Nat Med (2003) 9 : 14231427.
74. Naya FJ, Huang HP, Qiu Y, Mutoh H, DeMayo FJ, Leiter AB
and Tsai MJ : Diabetes, defective pancreatic morphogenesis, and
abnormal enteroendocrine dierentiation in BETA2/neuroD-de-
cient mice. Genes Dev (1997) 11 : 23232334.
75. Jonsson J, Carlsson L, Edlund T and Edlund H : Insulin-promoter-
factor 1 is required for pancreas development in mice. Nature
(1994) 371 : 606609.
76. Marshak S, Benshushan E, Shoshkes M, Havin L, Cerasi E and
Melloul D : Functional conservation of regulatory elements in the
pdx-1 gene : PDX-1 and hepatocyte nuclear factor 3 beta transcrip-
tion factors mediate beta-cell-specic expression. Mol Cell Biol
(2000) 20 : 75837590.
77. Miyachi T, Maruyama H, Kitamura T, Nakamura S and Kawakami
H : Structure and regulation of the human NeuroD (BETA2/BHF1)
gene. Brain Res Mol Brain Res (1999) 69 : 223231.
78. Prochiantz A and Joliot A : Can transcription factors function as
cell-cell signalling molecules? Nat Rev Mol Cell Biol (2003) 4 : 814
819.
11 Protein Transduction Technology February 2006