Tap chi Cong nghe Sinh hoc 10(3): 407-413, 2012
T A O D O N G R U O l G I A M C H U Y E N G E N M A N G P H U C H O P LAS-DUCH D E N G H I E N c u t ) V A I T R O C U A P R O T E I N U C H - L l D O I VOfI BENH P A R K I N S O N
Phan Nguyen Thuy An', Dang Thj Phuwng Thao', Masamistu YamaguchiS Tran Linh Thir6^c' 'Truong Dgi hgc Khoa hgc lu nhien. Dgi hgc Qudc Gia Thanh pho Hi) Chi Minh
Vien Cong ngh4 Kyoto. Nhat Ban TOM TAT
Benh Pakinson la benh thoai hoa he than kinh van ddng pho bien a nguoi cao tuoi, nguyen nhan la do nao bp nguai b?nh khong san xuat dugc chat dan truyen than kinh dopamine, dan den mat kha nang kiem soat hoai dgng van dgng co. Nhimg nghien cim gan day cung cap nhieu bang chung sinh hoc phan tir lien quan den co che phat sinh benh Parkinson nhu sv lieh ly a-synuclein, sy thuy phan chuoi ubiquitin irong he th6ng ubiquitin-proteasonie bj sat hong, sy mat chCrc nang ciia ty the va hien lugng stress oxy hoa. Trong do, UCH- Ll (ubiquitin carboxyl-terminal hydrolase 1) la mgt protein pho bien o nSo, c6 vai tro quan trgng irong he thong ubiquitin-proieasome nham phan hiiy cac protein gap cugn sai trong te bao. Trong nhirng nam gan day, mac dii nhieu nghien ciiu da dugc th\rc hien de tim ra moi quan h? giua UCH-Ll va benh Parkinson cung nhu cac b^nh thoai hoa than kinh khac, tuy nhien van de nay van con nhieu tranh cai, De hieu ro hon ve vai tro cua protein UCH-Ll, chiing toi tien hanh bieu hien protein dUCH, nuM protein luong dong voi UCH-Ll a ruoi giam. trong ruoi giam chuyen gen \a nghien ciru \a! tro cua no Bang each tao vector pUAST-dUCH, vi tiem vao ruoi giam nham tao ra cac dong ruoi giam chuyen gen mang phirc hop UAS-dUCH \a bieu hien dinh huong dUCH tai cac mo dich thong qua phirc hop GAL4-L AS Khi lai cac dong UAS-dUCH \oi dong GMR- GAL4, dUCH dugc bieu hien dinh huong o mo mat, phan lich cho thay protein dUCH bieu hien manh a viing phan bao thir cap co vai u6 quan trgng trong qua trinh hinh thanh mat va o \iing duoi cua dia tien phan sinh mat chimg to co moi hen he giOa mire dg bieu hien dUCH va kieu hinh mat ruoi Nhu vay, ruoi giam chuyen gen dUCH la mgt mo hinh tot de eg the nghien cim vai tro cija protein nay doi voi benh Parkinson ve sau Tu khoa: gen dUCH. ruoi giam GMR-GAL4. chuyen gen. bieu hien dinh hitaug mo mat
GIOI THIEU
Benh Parkinscm la mot trong nhOng benh ve roi loan than kinh pho bien nhat, dimg sau benh Alzheimer Trong suot the ky 20, yeu to di truyen khong duoc xem la nguyen nhan gay nen benh Parkinson (Corti et al, 2005). Tuy nhien, nhiing nghien ciru gan day da bac bo quan diem nay va quan trgng hem nira la cac nghien ciru nay da cung cap cac bang chimg de hieu duac co che phan tir lien quan den ca che phat sinh benh Parkinson (Gasser.
2007). Nghien ciru chirc nang va hoat dpng ciia nhimg gen nay giiip hieu dugc moi lien quan cua mpt s6 ca che vcn nhau va sir lien he vai co che phat sinh benh Parkinson trong te bao, nhu sir lich tu a- synuclein, su thiiy phan chuoi ubiquitin trong he th6ng ubiquitin-proteasome hu hong, su sai chirc nang ciia ly th6 va hien Urong stress oxy hoa (Dawson, Dawson, 2003).
Trong do, UCH-Ll la mot protein hien dien rat nhi6u trong nao (khoang 1% - 2%) (Wilkinson et ai, 1989), thupc ho enzyme co vai tro trong viec hinh thanh lien k^t a dku C ciia protein trong quy trinh
gan ubiquiiin cua nhimg protein gap cupn sai, protein gia c6i khong mong muon can phan hiiy bai proteasome (Amerik, Hochstrasser, 2004) UCH-Ll tach chuoi ubiquitin ra khoi protein truoc khi proiein vao he thong phan cat proteasome, ben canh do UCH-Ll con phan tach chuoi ubiquitin thanh cac monomer ubiquitin, lai sir dung ubiquitin cho he thong ubiquilin-proteasome (Larsen el a!., 1998), Mac dii ca che gay benh Parkinson van chua duac bi^t ro, nhung hau het cac nghien cim deu quy \ e mot moi rang su sai hong he thong ubiquitin- proteasome va sir tich tu nhimg protein bi bien doi c6 lien quan den benh Parkinson (Hegde, Upadhya, 2007). Protein UCH-Ll co vai tro trung lam trong boat dpng phan buy protein cua he ihong ubiquUin- proleasome nen cac nha nghien cim cho rang UCH- Ll CO lien quan den benh Parkinson (Leroy et al., 1998; Maraganore et al, 1999; Nishikawa el al, 2003), tuy nhien van de nay van con nhieu tranh cat
Trong nhieu thap ky gan day. benh di truyen a nguiri luon !a de tai thu hiit su quan tam cua cac nha khoa hoc. Ciing vai sir phat men ciia ky thuat smh 407
Phan Nguyen Thuy An el al hpc phan tir, cac cong trinh nghien cim ca che sinh
hpc phan tir ciia benh dupe lien hanh a cic mire dp tir in vilro, le bao dpng vat cho din in vivo, dpng vgt chuyen gen. Trong so cdc mo hinh dpng vgt chuyen gen, vai nhung uu diem vupt irpi, ruoi giam Drosophila melaiiogasler la mo hinh dupe chpn sir dung pho bien trong nghien ciru y - sinh hpc hi?n nay. Trong nghien cina nay, chiing toi xay dyng mo hinh nghien ciru benh tren dpng v^t chuyen gen, mo hinh ruoi giam D metanogasler nham img dung trong nghien cuu vai tro ciia proiein UCH-L1 doi vdn benh Parkinson. CAc kit qua dat dupe bao gom tao dong gen dUCH vao vector pUAST va chuyen vao CO the ruoi giam nham lao ruoi giam chuyen gen mang phirc hpp UAS-dUCH phuc vu cho nghien cuu vai tro ciia protein UCH-Ll doi vai benh Parkinson va xem su bieu hien vupl mire ciia protein dUCH trong ruoi giam chuyen gen.
V A T L I E U VA PHUONG PHAP Chiing vi sinh vat va vector
Chiing E coll DH5a (F-endA 1 hsdRl 7 (rk-/mk-) supE44 thi >.-recAl gyrA96 A lacU169 (4.80 lacZ AM15)) dupe sir dung lam the nhan, nhan va chpn dong.
Vector pUAST (Chromosomal Technology Laboratory, Kyoto Institute Technology, Japan) dupe sir dung chuyen gen dUCH 'vao ruoi giam Vector pUAST bao gom doan UAS la trinh tu gan protein GAL4, doan polylinker giiip viec tao dong duoc de dang va mpt trinh tu ngimg phien ma, tat ca duoc dat trong mot vector co the chuyen vi nho nhan to P, co gen Amp' c6 kha nang khang Ampicillin giiip sang Ipc trong qua trinh tao dong
Chiing ruoi giam Drosophila melanogaster CantonS va W1118 duac su dung nhu dong ruoi hoang dai (Bloomington, My) GMR-Gal4(X) dupe dung de bieu hien vugt mire gen dUCH o tno mat, Thu nhan bo gen ruoi giam
Ruoi truong thanh (10 - 15 con) dupe thu va giu a -20 C qua dem. Sau do, hoa trong 0,1 ml dem ly giai va nghien bang chay, bo sung 0,4 ml dem ly giai. Hon hpp dupe bo sung protease K 0,5 mg/ml va li 1 gio o 37 C, Sau khi ly tam 10 phut vai 10000 vong/phiit, a 4"C, dich noi duac bo sung 250.250 fil
phenol: chloroform, dao ki va lie rung. Sau khi ly tam nhu tren, dich noi dupe hh sung 500 jil chloroform va dao ki. Sau ly tam n h u tren va d(ch n6i dupe b6 sung 1/10 th^ tich 3 M Na acetate pH 5,2 va 500 ml 99% ethanol va li 30 phiit o -20 C, sau do ly lam 30 phiit voi 13000 vong/phiit a 4''C. Tiia dupe rua lai bang 500pl 70% ethanol, ly tam_30 phiit v6i 13000 vong/phiit a 4 ^ . Tiia dugc dS kho hr nhien va gitf trong 50 pi MiliQ.
Thu nhan plasmid pUAST-dUCH
Plasmid pUAST-dUCH dupe tach chifit tir E coll DH5a theo phuong phap kiem SDS (Bimboim, Doly, 1979),
Khuech dai gen dUCH biing phirong ph^p PCR PCR khuich dai gen bao gom 5 pi dem lOx PCR, 5 pi 8 mM dNTP, 5 pi 25mM MgCl,, 1 ^1 DNA bp gen (Y ng), 1 pi 1 unit Taq polymerase va 1 /il m6i m6i (10 pmol), duac bo sung 31 pi nucrc cat len the tich cu6i ciing la 50 fil, Phan img PCR duoc thuc hien theo che dp nhiet: 95"C/5', 30 chu ky (95''C/1', 55-C/l', 7 2 T / r ) , 7 2 ^ / 1 0 '
Thi^t k^ plasmid va bien nap
San pham PCR gen dUCH duoc cat vai enzyme ban che EcoRl va ,Wiol, chen vao vector pUAST tao thanh plasmid tai to hop pUAST-dUCH va dupe bien nap vao E coli theo phuong phap shock nhiet va CaCL (Sambrook. RusseU, 2001). Khung dpc ciia gen dUCH dupe kiem tra bing PCR, eat han che va dpc trinh tu. Tat ca cac thao tac gen deu thuc hien theo phuong phap co ban trong Sambrook va Russell (2001).
Vi t i e m
Ruoi diing de chuyen gen phai co hai dac diem khong mang gen ma hoa iransposase va khong mang chi thi chpn Ipc (thucmg la ruoi dot bien mat trang), Ruoi duac nuoi trong moi truimg de trung 2 - 3 ngay truac khi vi tiem. Vao ngay vi tiem, cir each 60 - 70 phiit thay moi truong SY ( 5 % duimg, 5% ndm men. 1% agar) mot lan. Truac khi tiem phai de ru6i de trimg trong 20 - 40 phiit trong moi truimg thach SY. Lop mang day bao quanh tning dupe loai bo giiip cho qua trinh vi tiem de dang.
Helper P - element va plasmid mang nhan to P duoc tiem vao trimg cimg mpt liic o phan duoi ciia trimg nai hinh thanh mam te bao.
Tgp chi Cong nghe Sinh hgc 10(3): 407-413, 2012 Tning ruoi sau khi vi tiem dupe cho Ira lai moi truong SY de trimg phat trien. M6i ken ru6i duoc nuoi trong mpt 6ng moi truong, de chiing n a thanh ru6i truong thanh hoac cho cho din khi ken na thanh ru6i truong thanh thi bat trinh nu. Toan bp Ihi he Fu CO mau mat trang. Moi con cai Fo duoc giao phoi vdi ba con dy:c W1118 va m6i con due Fo dupe giao phoi vcri ba con cai W l 118. The he F| co kieu hinh sau- 1 Tat ca the he con F| co mau mat trang. Day la nhirng con ruoi khong dupe chuyen gen, 2. Mot so ruoi F, (khoang 10%) mat co sac to do. Day la dong ruoi dupe chuyen gen 3. The he F| (han 50"o) co nhieu kieu mau mat khac nhau, Dieu nay co nghia la CO nhieu doan gen da dupe chuyen vao bp gen ruoi.
Ruoi CO mau mat nhat duoc diing de lai tiep theo.
Sau khi sang Ipc de thu dupe dong ruoi chuyen gen, cac dong ruoi nay duac xac dmh nhiem sac the mang gen chen bang cac phep lai voi ruoi mang chi th;
nhiem sac the (balancer).
Lai Western
Mau proiein tren gel dien di SDS-PAGE duac chuyen len mang nitrocellulose bang lac nhan dien.
Khoa mang Irong dung dich sua gay 5"o trong PBST (80 mM Na2HP04, 25 mM NaH^POj 100 niM NaCI, 0 , 1 % Tween 20), lac nhe Irong 1 gio cho dung dich tiep xiic deu vai be mat ciia mang lai Sau do, ngam mang lai trong khang thi khang dUCH (1/10000) trong 1 gio, dong thai lac nhe nhang hop. Sau do rira kl mang 5 lan trong dung dich PBST, moi lan 5 phiit.
TiSp luc ii mang vai khang the thir cap danh dau bang HRP pha loang trong dung dich PBST voi li le 1/20000 trong 1 gia, dong thai lac nhe hop. Sau do rira kT mang bang dung dich PBST 5 lan, moi lan 5 phut Hien phim bang dung dich ECLI va ECL2 (Thermo Fisher Scientific Inc., Wallhain, USA).
Mien dich huynh q u a n g ( I m m u n o Flourcscene) Tach dTa tien phan sinh mat tir au Iriing thir 3 trong PBS (140 pM NaCl, 3 pM KCl, 8 pM N a i H P C , va 15 |iM KH.PO.,, pH 7.4) tren da. Cfl dinh dia Irong 2%. paraformadehyde trong PBS a 4 C qua dem. Sau do rua 6 lan bang PBS o nhiet dp phong, U voi khang Ihe so cap u-dUCH antibody ( 1 / 1 0 0 0 0 ) 6 4 C qua dem, Rira 6 I5n biing PBS va il voi khang the ihir cap a-FITC antibody (1/1500) 4 gia a nhiet do phong hoac 4 C qua dem. Rua 6 Idn biing PBS va ii miu trong 100%
glycerol Irong 30 phiil. Xem tin hieu bang kinh hien vi huynh quang.
KET QUA VA BIEN LUAN
Thiet ki plasmid t^i to hpp pUAST-dUCH San phaiTi gen dUCH vdi khuon DNA bp gen ruoi giam va c^p moi d5c hi^u cd kich thudc 980 bp dung nhu thiet ke (Hinh I A. gieng 2). San pham PCR gen dUCH dupe linh sach. ck vdi EcoR\. Xhal linh sach va niii dau dinh vao vector pUAST cung dupe ck vdi EcoR\ va Xh<i\, bang DNA 8800 bp (Hinh IB, gieng 3) t^o ra plasmid lai to hop pUAST- dUCH, Plasmid duoc bien nap E. coli DH5a lao ra cac d d n g f coh DH5a/pUAST-dUCH.
Kiem tra khuan lac bang PCR vdi cap moi wizl/wiz2, cac khuan lac d gieng 4, 5, 8, 9, 10 cho mpl bang DNA -- 1030 bp niim giiJa 1000 bp va 1500 bp, luong irng vdi san pham PCR plasmid lai to hap pUAST-dUCH vdi cap moi wizl/wiz2. Cac khuan lac mang pUAST-dUCH duac nudi cay va tach plasmid bang phuong phap SDS-kiem dc kiem tra bang moi dac hieu gen
Trong so 5 khuan lac cd PCR khuan lac dirong tinh chi cd 2 plasmid pUAST-dUCH d gieng 5 va 6 liep luc cho san pham PCR 980 bp vdi cap mdi dac hieu cho gen dUCH (Hinh 3, gieng 2) Plasmid (ai to hpp duac cat vdi EcoR\ va A7;f)l (Hinh 4, gieng 4) cho hai bang co kich thudc bang pUAST md vong (Hinh 4, gieng 2) va bang co kich thudc bang vdi gen dUCH (Hinh 4. gieng 3) Plasmid nay cung cho san pham PCR vdi moi dac hieu ciia gen mpl bang ddng kich thudc. Nhu vay gen dUCH da ddng hoa thanh cdng vao plasmid pUAST. Plasmid pUAST- dUCH iai id hop dupe giai trinh tu kiem Ira (Macrogen, Han Qudc) va vi liein vao rudi nham lao rudi mang phirc hop UAS-dUCH,
Bi£u hien dUCH o cac dong ruoi chuyen gen Sau khi chuyen gen bang vi liem, cac ddng ruoi chuyen gen duac luyen chon dua vao gen chi thi w+
cd kieu hinh mau mat dd. Bon dimg rudi dir doan cd mang gen muc iicu (thong qua mau mat) #12-4, /;23- 4, U50-S. va ^59 duac lam Ihuan va xac dinh nhiem s;ic thi mang gen muc tieu (UAS-dUCH).
Phirc hop gen UAS-dUCIl Irong ca the chuyen gen dupe khuech dai PCR \ a i cap moi dac hieu bao quanh vimg gen UAS-dUCH (Hinh 5A). Theo ly lluiyet, dudi lac dong ctia yeu Id chuyen vi gen I', khi plasmid pUAST-dUCII duoc dira v;io plidi riiiii, vdi boat dpng cua transposase, phirc hpp UAS-dUCH nam giira cac trinh lu IR cua yeu Id P dupe chen vao nhiem sac the cua rudi giam. Theo thiet ke thi nghiem chuyen gen, Iran.sposase chi hien dien duy
Phan Nguyen Thyy An el al nhal Irong the he P (phoi ruiji) khi chuyen gen v:)
ho:in loan khong hi^n dii;n d ciic the hi^ sau dd. Do dd, gen chen vao nhiem siic the ton tai on djnh,
Nhimg dong rudi chii\cii gen \;i rudi hoang dyt dupe lai Western dc kiem Ira si,r bieu hi?n d l K l l protein Ircii rudi chii>cii gen (Hinh 5B). Budng irirng cua cac ddng rudi chu>cii gen \a ddng hoang dai duac thu nhan vi protein dUCII duac bieu hiC'n in;iiili a budng irinig nidi. Kliinig the dliCII nhan dicn dupe dllCII piolein ( 30 kDa) d rudi chuyen gen \ii rudi huangdi,ii.
Bii-ii hien djnh hudng liii nu'i imll d tidiig ruoi (;Mi*-(;Ai.4
Su dung ddng iiidi (iMR-(iAl,4 mang promolei Lhuvcii hii'l elm md iiial cho plii-p \cni \cl sir bii-ii hicn \irpl niirc piolcin iinie lieu udi.i phan smh iiial Hidi. hinh lluii iniil o rudi trirong Ihanh. cimg nhu phiin lich sM\ luni \ i - i;ic ddng cua prolcin muc lieu (UCI l-LI) Icn chu tiinh le b;io \a su bicI hoa ciic le biio o niirc dd md phan sinh.
Di' Ihu nhiin c mdi chuyen gen mang
ddng Ihdi hai yeu Id UAS-dUCH va GMR-GAL4 Irong ea I h i , ma l6 hpp gen mang promoter GMR va GAL4 nam Ircn nhilm sSc the X, cho nen rudi due mang phirc hpp gen UAS-dUCH dupe lai vdi ruii ciii GMR-(iAL4. Do ca t h i rudi cai mang GMR- ( i A L 4 ddng hpp l u Iren nhiem sac the X nen lai ca rudi eon o t h i h ^ Fl deu mang GMR-(JA[.4.
Ban diiu, mire dp bieu hien proiein dUC'H d rudi chuyen gen dirpc kiem tra bang lai Western giira prolcin L\\.fi}c Itieh ra lir t;ic dTa lien phan sinh mai ciia iiu iriing ihir ba va khiing the khang dUCTI, cac ddng mdi chiiycn gen cd sir bieu hi^'n VUITI mi'rc proiein dUCll (ilinh 6A). Ngoiii ra, hinh lliai m,il cua runi Irucmg lliiinh lii kicii hinh mill nhiiin (rough eye) a ciie ddng rudi chuyen gen dUCll so vdi kieu hinh mill hinh linriTug <r rudi hoang dai (Hinh CiB).
PI oleiii dl K 'I I cd the dirae bicii hicn vmrl mire, giiy dde li- biio va ga> nen sir bien ddi Irong qua Irinh hinh ihiinli nial <i c.ic ddng rudi eluiyen gen, priHeiti dUCH biC'ii hicn manh IT vimg phan hao ihu ciip. noi ddng \ai Ird quan Irong Irong qua Irinh hinh Ihanh infil Ml a Ming dudi (posterior) cua dTa lien phim smh mill (liinhdD)
bp 1 2 1 2 3
Hinh 1 San pham PCR gen dUCH (1A), pUAST/EcoRI, Xho\ (3B), pUAST {2B), thang DNA chuan 1 kb(l)
1000 bp
Hinh 3. San pham PCR plasmid vdi moi dac hieu cua gen dUCH VCTI khuon gen dUCH (2, chung duo'ng), hhuon pUAST (chung am, 3), khuon plasmid tach tu cac khu^n lac (4-8), thang DNA chu^n 100 bp C*
Hinh 2. San pham PCR khuSn lac voi mfii wiz1/wiz2 100 bp va khuon pUAST (chung am, 2), khuon nuo'c (3), khuon cac khuSn Igc sSng loc (4-10), thang DNA chuin (1),
Hinh 4. San pham c^l b§ng EcoRI vA Xhol cOa pUAST (2), gen dUCH (3), pUAST-dUCH (4), SSn ph^m PCR cCia pUAST-dUCH b^ng c$p mei (5?c hi$u gen (5), thang DNA chu^n 1 kb (1) 4 1 0
Tap chi Cong nghe Smh hgc 10(3): 407-413, 2012
Cs #12-4 1123-4 #50-5 #59
Hinh 5. A Di0n di 66 san phAm PCR gen UAS-dUCH 6 cac dong ru6i chuyen gen s u dung khuon pUAST-dUCH (2, 3, Chung duong), khuon nuoc (4, 5, chung am), khuon bo gen CanlonS (6, 7, chung npi), khuon bO gen #12-4 (8, 9), khuon bp gen #23-4 (10, 11), khuon bp gen #50-5 (12. 13), khuon bo gen S59 (14,15) w6i cap moi dac hieu plasmid (gi4ng ch^n) va moi dac hieu gen ( g i l n g le trir 1), thang DNA chuan 100 bp (1) B Lai Western dUCH protein bieu hien o moi chuyen gen vol khang Ih^ khang dUCH
(-) #12-4 #23-4 #50-5 #59
Hinh 6 Bi^u hiSn dUCH dinh h u d n g tai mo mat d d6ng ruoi GMR-Gal4 A, Muc do bieu hien dUCH o dTa lien phan smh mk d rufli hoang dai (-) va rudi chuyen gen dUCH B-C, Hi6n tupng mat nhSm d ru6i hoang dai CanlonS va ru6i chuyen gen dUCH khi protein dUCH d u a c bi6u hiSn v i r o l muc a mat ru6i (B) Hang tren va hSng duo'i tuong ung voi dp phong dai 200x va 700x. (C) Nhuflm ^ a ti^n phan sinh mat cua ru6i hoang d^i va ru6i chuyen gen bang khang th^ khang dUCH (a) dia dup'c chup a vjrt kinh lOx; (a')' dTa duwc chup cr vat kinh 40x Mui tfeniprotein dUCH duo'c bieu hien manh cr second mitotic wave va o duoi mat
Phan Nguyen Thuy An el al
KET LUAN
Gen dUCH da diroc lao ddng Ihanh cdng vao plasmid pUAST va chuyen van i.a ihc rudi giam, Uio rudi giam chuyen gen md hinh mang gen dUCI I. Kel L]ua budc dau da cho Ihiiy indi lien L|uan gifra mire di) bieu hicn prolcin U C I M . l vii kieu hinh ihiii mfil rudi Han nira. md hinh ii;i\ chinh hi nen tang cho cac Ihi nghiem phim lich \ai trdcii,i proiein UCH-Ll ddi MTi bi'iih Parkinson \ c s a u
l.i)i c;iin o'li: /)<• k'li dteoc tliiiv hicn voi sw ho lio kinh phi lir (/i/r Nafosled vi'i su lii'i lur ky lliiial run phong ihi iigliiciii kC ihiidl iiliiciii \('ir ilic. i'lrn Kv ihiu'it. Cong nghe Kyoto. S'hdl Han
I A I i II U I I I A M K H A O
M (2im-l) Mcch.ini^ni ;IIKI
•; c n / y n i t ' s liici iniii Huii'liv^
( urti <). ll,iiii|x-1 , \y, iHWf^) I'aikinson'v dis RBio/}2m) I.1I-I4.
inLvl". KubcigM.Hrii
I VL (2003) Molcculai palliWLiys II Parkinson's di.seasc Si n-i.
(iiisscr T ( 2 0 0 7 ) U p d a t e o n t h e g e n e t i c s o f Parkinson's disc.isc. A / r ' r / > w < j r J 2 2 S u p p l 17: S.343-350- l l c g i l c A N . U p a d h y a S C ( 2 0 0 7 ) I h c u b i q u i l i n - p r o t e a s o m e p a t h w a y m h e a l t h a n d disea.sc ol" i h c n e r v o u s system.
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L a r s e n C N , Kiiinlz H A , W i l k i n s o n K l ) ( I 9 9 S ) Subslrale s p c c i l i c i t y o l ' d c u b i q u i l i n a l i n g c n / y m c s ubiquitin C- I c r m i n a l h y d i o k i s c s Bioiliauriti v 37( 10)- 3358-33fiX.
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