proteosoma t cruzi.pdf
TRANSCRIPT
-
5/19/2018 Proteosoma T cruzi.pdf
1/10
P r o t e a s o m e A c t i v i t y I s R e q u i r e d f o r t h e S t a g e - s p e c i f ic
T r a n s f o r m a t i o n o f a P r o t o z o a n P a r a s it e
By Jorge Gonzfilez, E Juarez Ra m alho-Pinto,~ U te Frevertfi
Jorg e G hiso, Step hen Tom linson, Julio Scharfstein,IIE.J. Corey,
andV ictor Nussenzw eig
From the Michael HeidelbergerDivision o f Immunology, Department of Pathology, New York
University Medical Center, Ne w York 10016; the Department of Biochemistry-Immunology,
blstituto de C iendas Biologicas, Universidade Federal de Mina s Gerais, M G, Brazil; the ~Dep artment
of M edical and M olecular Parasitology, Ne w York University Medical Center, Ne w York 10016; the
Iqnstituto de Biofisica, Universidade Federal do Rio deJaneiro, RJ, B razil; and the Dep artment of
Chemistry, Harvard U niversity, Cambridge, Massachusetts 021 38
S u m m a r y
A p ro m in e n t f e a tu re o f th e l i f e c y c le o f i n t r a c e llu l a r p a ra s i te s i s t h e p ro fo u n d mo rp h o lo g ic a l
c h a n ge s t h e y u n d e r g o d u r i n g d e v e l o p m e n t i n t h e v e r t e b r a t e a n d i n v e r t e b r at e h o st s. I n e u k a r y -
o t i c c el ls , mo s t c y to p la s m ic p ro te in s a re d e g ra d e d in p ro te a s o rn e s . H e r e , w e s h o w th a t t h e
t r a n s f o r m a t i o n in a x e n i c m e d i u m o f t r y p o m a s t ig o t e s o f
Trypanosoma cruzi
i n to a ma s t ig o te - l ik e
o rg a ms m s , a n d th e in t ra c e l lu la r d e v e lo p m e n t o f t h e p a ra s it e f ro m a m a s t ig o te s in to t ry p o m a s t i -
g o te s , a re p re v e n te d b y l a c t a c y s t in , o r b y a p e p t id e a ld e h y d e th a t i n h ib i t s p ro te a s o me fu n c t io n .
C la s to - l a c t a c y s tin , a n in a c t iv e a n a lo g u e o f l a c ta c y s tin , a n d c e l l -p e rm e a n t p e p t id e a ld e h y d e in -
h ib i to r s o f
T. cruzi
c y s te in e p ro te in a s e s h a v e n o e f fe c t . W e h a v e a ls o id e n ti f i e d th e 2 0 S p ro te a -
s o m e s f r o m
T. cruzi
a s a t a rg e t o f l a c t a c y s t in in v iv o . O u r r e s u l t s d o c u me n t th e e s s e n t i a l ro l e o f
p ro te a s o me s in th e s t a g e - s p ec i f i c t r a n s fo rm a t io n o f a p ro to z o a n .
n f e c t i o n b y
Trypanosoma cruzi,
t h e c a u s a t iv e a g e n t o f
C h a g a s ' d i s e a s e , i s i n i t i a t e d b y me ta c y c l i c t ry p o ma s t i -
g o te s p re s e n t i n th e f e c es o f t r i a to min e b u g s . T h e t r y p o -
ma s t ig o te s in v a d e h o s t c e l l s a n d e n te r t h e c y to p la s m, w h e re
th e y t r a n s fo rm in to a ma s t ig o te s . T h e a ma s t ig o te s r e p l i c a t e
a n d , a f e w d a y s l a t e r , t r a n s fo rm b a c k in to t ry p o ma s t ig o te s ,
ru p tu re th e h o s t c e l l s , a n d in v a d e th e b lo o d s t re a m (1 ) .
Thus , on two occas ions during i ts in t race l lu la r s tage ,
T. cruzi
u n d e rg o e s s h a p e a n d v o lu me c h a n g e s , r e s t ru c tu re s i t s f l a -
g e l lu m a n d k in e to p la s t , a n d s y n th e si z e s n e w s et s o f su r fa ce
mo le c u le s . T h e s e s t r ik in g mo d i f i c a t io n s a re p re c i s e ly t ime d ,
t a k e p l a c e in a n o rd e r ly f a s h io n , a n d mu s t i n v o lv e s e l e c t iv e
d e g ra d a t io n o f c y to p la s mic p ro te in s .
In e u k a ry o t i c c e l l s , mo s t p ro te in s in th e c y to p la s m a n d
n u c le u s a re d e g ra d e d n o t i n ly s o s o me s , h u t w i th in p ro te a -
s o me s , a f t e r t h e y a re ma rk e d fo r d e s t ru c t io n b y c o v a le n t a t -
t a c h m e n t o f u b i q u i t i n ( U b ) l m o l e c u l e s ( 2 - 5 ) . I n a d d i t i o n
1Abbreviatzons used in this paper:
BSA, bovine serum albumin; CAPS , (3-
[Cyclohexylarmno]-l-Propanesulfonlc acid), Ch-L, Chymotrypsln-hke;
EDT A, ethylene dl-amino tetra acetic acid; E-64, trans-epoxysuccmyl-
L-leucylamldo-3-methyl-butaneethyl ester; FCS , fetal calf serum; F1TC ,
fluoresceln lsothiocyanate; MES, (2-[N-morphohno]-ethanesulfonlcaod);
MG-132, carboxybenzoxyl-leucinyl-leucinyl-leuclnal-H; GPH, pepti-
dylglutamyl peptlde hydrolase; T-L, Trypsin-like; Ub, ub~qultin.
t o t h e i r r o l e i n n o n l y s o s o m a l p r o t e i n t u r n o v e r , p r o t e a -
s o me s a re in v o lv e d in s p e c i f i c c e l lu l a r fu n c t io n s , i n c lu d -
i n g t h e f o l lo w i n g : t h e p r o g r a m m e d i n a c t iv a t i o n o f m i t o t i c
cychns , t ransc r ip t ion fac tors , and t ransc r ip t iona l regu la tors ;
t h e e l i m i n a t i o n o f m u t a t e d o r d a m a g e d p r o te i n s ; a n d a n t i -
g e n p re s e n ta t io n . T h e fu n c t io n o f th e p ro te a s o me s i s a l so
t ig h t ly r e g u la t e d , a n d th e i r s t ru c tu re ma y v a ry to ma tc h
fu n c t io n (6 -7 ) .
T h e e x p e r i m e n t s d e s cr i b e d b e l o w w e r e d e s i g n e d t o d o c -
u m e n t t h e p a r t i c i p a t i o n o f p r o t e a s o m e s i n t h e d e v e l o p m e n -
t a l p a th w a y s o f p ro to z o a n p a ras i te s .
T. cruzi
h a s a n a d v a n -
t a g e a s a n e x p e r ime n ta l mo d e l b e c a u s e i t s t ry p o ma s t ig o te
fo rm c a n b e in d u c e d to c h a n g e ra p id ly in to a ma s t ig o te s in
a x e n ic me d iu m. T h e re s u l t in g a ma s t ig o te -h k e p a ra s i t e s
c a n n o t b e d i s t in g u i s h e d f ro m in t r a c e l lu l a r a ma s t ig o te s b y
l ig h t o r e l e c t ro n mic ro s c o p y , o r b y s t a g e - s p e c i f i c s u r fa c e
ma rk e r s . T h u s , i n th i s mo d e l , t h e e f fe c t s o fp ro te a s e in h ib i -
t o r s o n t r a n s f o r m a t i o n c a n b e s t u d i e d i n d e p e n d e n t l y f r o m
the ir e ffec t on the ce l ls o f the hos t .
a t e r ia l s a n d e t h o d s
Cell Lines .
L L C - M K2 fib rob las ts were ob ta ined from Am en-
can Type Cul tu re Col lec t ion , Rockvi l le , MD (ATCC CCL-7) .
L6E9 myoblasts were a gift of Dr. R. Do cam po (Umversxty of I1-
1909 j. Exp. M ed . The Rockefeller Universxty Press 0022-1007/96/11/1909/10 2.00
Volume 184 Nov em ber 1996 1909-1918
Published November 1, 1996
-
5/19/2018 Proteosoma T cruzi.pdf
2/10
h n o is , U r b a n a - C h a m p a i g n , I L .) . C el ls w e r e g r o w n i n R P M I
1 6 4 0 m e d i u m s u p p l e m e n t e d w i t h 1 0 F C S , 1 0 0 t z g / m l p e n i c i l -
h n , a n d s t r e p t o m y c i n .
Reagents. P r o te a s e i n h lb i to r s E - 6 4 , E - 6 4 d , C b z - P h e - A l a -
F M K , C b z - ( S - B Z ) - C y s - P h e - C H N 2 , a n d f l uo r o ge n ic su bs tr ate s
w e r e p u r c h a s e d f r o m S i g m a C h e m i c a l C o . ( S t. L o re s , M O ) . L a c -
t a c y s t i n a n d c l a s t o - l a c t a c y s t i n we re sy n t h e s i z e d a s p re wo u s l y d e -
s c r i b e d (8 , 9) . M G - 1 3 2 w a s f r o m P r o s c r i p t, I n c . ( C a m b r i d g e ,
M A ) . C h r o m a t o g r a p h y c o l u m n s a n d r e si ns w e r e f r o m P h a r m a c ia
B i o t e c h A B ( U p p s a la , S w e d e n ) .
Inhibi tion of Trypomast igote Transformat ion into Amast igotes . L L C -
M K 2 ce ll s we re i nfe Ct ed wi t h T. cruzi trypomastigotes, Y strata (10).
4 d l a te r , t h e s u p e r n at a n ts c o n t a i n e d m o r e t h a n 9 5 t r y p o m a s t i -
g o t e s a n d s m a l l n u m b e r o f a m a s t ig o t e s o r i n t e r m e d i a t e f o r m s .
P a r a si te t r a n s f o r m a t i o n i n t o a m a s t i go t e s w a s i n d u c e d b y l o w e r i n g
t h e p H o f th e i n c u b a t i o n m e d i u m ( 11 , 1 2) . T o a ss ay f o r t h e e f f ec t
o f i n h ib i t o r s i n t h e t r a n s f o r m a t io n , t w o f o l d d i l u n o n s o f e a ch i n -
h i b i t o r w e r e d i s t r i b u t e d m 9 6 - m i c r o w e l l p l a t e s . D i l u t i o n s w e r e
m a d e w i t h D M E M b u f f e re d w i t h 2 0 m M M E S ( p H 5 .0 ) c o n ta i n -
ing 0 .4 BS A. Lactacyst in or c lasto-lactacystin , M G- 132 , E-6 4 ,
C b z - ( S - B Z ) - C y s - P h e q E H N 2 a nd C b z - P h e - A l a - F M K w e r e p re -
p a re d a t 2 0 0 ~ M , a n d 5 0 p ,1 we re a d d e d t o w e l l s t o f i n a l d i l u t i o n s
o f 1 0 0 - 0 .7 8 p ,M . D e p e n d i n g o n t h e i n hi b lt o r s u s e d, D M S O d i -
l u t i o n s o r m e d i u m w e r e u s e d a s c o n t r o l s . T r y p o m a s t i g o t e s w e r e
c e n t n f u g e d ( 3 , 0 0 0 g 1 5 m i n ) a n d r e s u s p e n d e d a t 2 1 0 7 / m l i n
D M E M ( p H 5 . 0) . 5 0 p ~l o f th i s s us p e n s io n w a s a d d e d t o e a c h
w e l l , m i x e d , a n d i n c u b a t e d f o r 4 h a t 3 7 C i n a 5 C O 2 a t m o -
s p h e r e . T h e p l a t e w a s c e n t r i f u g e d a n d t h e s u p e r n a t a n t s w e r e r e -
m o v e d a nd r e p la c e d b y D M E M ( p H 7) c o n t a i ni n g 10 F C S .
T h e p l a te s w e r e r e i n c u b a t e d o v e r n i g h t a t 3 7 C m a C O 2 i n c u b a -
t o r . T h e p e r c e n t a g e o f t r a n s f o r m e d p a r as it e s w a s d e t e r m i n e d b y
mi c ro sc o p i c a l l y sc o r i n g 2 0 0 c e l l s i n e a c h we l l i n a b l i n d e d fa sh -
i o n . A l l e x p e r i m e n t s w e r e c a r r ie d o u t i n d u p h c a t e .
F A C S ~ A n a l y si s . Pa ra sat es (2 .5 1 0 7 ) we re t r a n s fo rme d i n
t h e p re se n c e o r a b se n c e o f p ro t e i n a se i n h l b i t o r s a s d e sc r i b e d . A t
t h e e n d o f t h e i n c u b a t i o n , p a r as i te s w e r e r e s u s p e n d e d i n 2 5 0 } xl
o f D M E M a t 4 C , a n d a n eq u a l v o l u m e o f m o n o c l o n a l a n t ib o d ie s
2 C 2 a n t i - S s p - 4 o r 3 C 9 a n t , - S s p - 3 ( 13 ) w a s a d d e d . T h e i n c u b a -
t i o n p r o c e e d e d f o r 3 0 m m o n i c e . T h e s u s p en s i on w a s t h e n
c e n t r i f u g e d f o r 7 r a i n a t 3 , 5 0 0 r p m i n a r e f r i g e r a t e d c e n t n f u g e
( S o r v al l I < T 6 0 0 0 B ) , u s i n g a h o r i z o n t a l r o t o r . T h e s u p e r n a ta n t
w a s r e m o v e d , a n d t h e pa r as it e s w e r e f i x e d w i t h 4 p a r a f o r m a l d e -
h y d e i n P B S . A f t e r 3 0 m i n a t 4 C , t h e f i x a n v e w a s r e m o v e d a n d
t h e p ar as it es w e r e w a s h e d w i t h 1 m l o f c o l d 0 . 4 B S A - D M E M .
T h e p a r as i te s w e r e t h e n i n c u b a t e d f o r 3 0 r ai n w i t h a n t i - m o u s e
I g G c o n j u g a t e d w i t h F I T C . T h e s u s p e n s i o n s w e r e c e n t r i f u g e d ,
w a s h e d w i t h 0 . 4 B S A - D M E M , r e s u s pe n d e d in 50 [z l o f P B S ,
a n d p o s tf i x e d w i t h 4 p a r a f o r m a l d e h y d e . T h e c e l l s u s p en s i on s
w e r e a n a l y z e d i n a B e c t o n D i c k i n s o n F A C S c a n .
Inhibition o f Developmen t o f lntracellular Parasites. L6 E9 my o b l a s t s
w e r e l r r a & a t e d w i t h 2 , 0 0 0 r a ds ( 1 4) a n d p l a t e d i n 4 - w e l l L a b - T e k
r m c r o c h a m b e r s lid es ( N U N C , N a p e r v l ll e , IL ) . T r y p o m a m g o t e s
we re p re t re a t e d fo r 1 h wi t h 1 0 b t M l a c t a c y s t m o r c l a s t o - l a c t a c y s -
t i n a t 3 7 C . P a r a s it e s w e r e w a s h e d t w i c e , r e s u s p e n d e d i n D M E M
and use d to in fe c t myoblasts a t a parasi te to L6E 9 cel l s rano of 5 :1 .
A f t e r 2 h l n c u b a t m n a t 3 7 C , t r y p o m a s t i g o t e s w e r e r e m o v e d , a n d
t h e L 6 E 9 c e ll s w e r e w a s h e d w i t h D M E M . T o s t ud y t h e e f fe c t o f
inh lb i tors on invasion , one se t o f ce l ls was f ixed wath 4 paraform al-
d e h y d e i n P B S f o r 3 0 m a n . E x t r a c e l lu l a r t ry p o m a s t i g o t e s w e r e d e -
t e c t e d b y l m m u n o f l u o r e s c e u c e w i t h a p o l y c l o n a l a n t i b o d y t o T .
cruzi a n d t h e t o t al n u m b e r o f p ar a si te s w a s d e t e r m i n e d b y s t a i n -
i n g w i t h H o e c h s t d y e ( S i g m a ) a ft e r p e r m e a b i l i z a t i o n o f t h e L 6 E 9
c e ll s w i t h c o l d m e t h a n o l f o r 1 0 m l u . T h e n u m b e r o f i n t r a c e l l u l a r
p a ra s i t e s wa s c a l c u l a t e d b y su b t ra c t i n g t h e e x t ra c e l l u l a r f ro m t h e
t o t a l pa ras it es (1 5 ) . To d e t e rm i n e t h e fa t e o f l a c t a c y sn n - t re a t e d p a ra -
s it es , t h e r e m a i n i n g i n fe c t e d c e l l c u l t u re s we re re i n c u b a t e d a t 3 7 C .
At 2 4 , 4 8 , a n d 7 2 h , t r i p l ic a t e we l l s we re w a sh e d a n d s t a i n e d wi t h
M a y - G r u n w a l d - G i e m s a . T h e s l id e s w e r e e x a m i n e d u n d e r li g ht
m i c r o s c o p y a n d t h e n u m b e r o f ln t r a c e ll u l a r am a s t i g ot e s i n 1 0 0
c e l ls wa s c o u n t e d . R e su l t s a re e x p re s se d a s me a n s + SD.
I n a n o t h e r s e t o f e x p e r i m e n t s , w e s t u d i e d t h e e f f e c t o f m h i b i -
t o r s o n t h e t r a n s fo rm a t i o n o f i n t ra c e l l u l a r a ma s t i g o t e s i n t o t ry p o -
m a s t i g o t e s . C e l l c u l t u r e s w e r e i n f e c t e d w i t h
T . c ruz i
t r y p o m a s n -
g o t e s . 4 8 h a f t e r i n fe c t i o n , t h e c u l t u re s we r e t r e a t e d fo r 2 h wi t h
0 .7 5 , 1 .5 , a n d 3 Iz M o f la c t a c y s t i n o r c l a st o - l a c t a c y s ti n . Th e c u l -
t u r e s w e r e w a s h e d a n d r e i n c u b a t e d a t 3 7 C f o r a n a d d i t i o na l 2 d ,
w h e n t h e f i r st p a ra s i te b u rs t o c c u r re d . T h e c u l t u re su p e rn a t a n t s
w e r e c o l le c t e d an d t h e n u m b e r s o f e x it i n g tr y p o m a m g o t e s w e r e
d e t e r m i n e d i n a N e u b a u e r c h a m b e r . T o d o c u m e n t f u r t h er t h e
i n h i b i t o r y e f f e c t o f l a c ta c y s ti n i n t h e a m a s t i g o t e / t r y p o m a s t i g o t e
t ra n s fo rma t i o n , mfe c t e d c u l t u re s we re l y se d 7 2 , 8 0 , 8 8 , a n d 9 6 h
a f t er i n f e c t i o n w i t h a b u f f e r c o n t a i n i n g 3 n - o c t y l g l u c o p i r a n o -
s id e , 5 0 m M T r i s - H C 1 ( p H 7 . 4 ), 0 .1 m M E D T A , 2 0 b ~M E - 6 4
a n d 5 D g / m l l e u p e p t in , a n t i p a i n , a n d p e p s ta t in . T h e e x t ra c t s w e r e
a n a l y z e d fo r l e v e l s o f t ra n s l a li d a se , a n e n z y m e e x p re s se d i n t ry p o -
m a s t i g o te s , b u t n o t i n a m a s t i g o t es ( 1 6) . M e a s u r e m e n t s w e r e m a d e
i n t r i p l i c a t e sa mp l e s , a n d t r a n si a l id a se a c t i v i t y wa s e x p re s se d a s
c p m + S D .
E n z y m a t i c A s s a y s . Pro t e o l y n c a c t i v i t y wa s a ssa ye d u s i n g a s
s u b s tr a te 1 0 0 p ~M f l u o r o g e n i c p e p t id e s d i l u t e d i n 5 0 m M T n s -
H C l ( p H 7 . 8) . 1 0 I z l o f c h r o m a t o g r a p h i c f r a c ti o n s w a s a d d e d t o
9 0 b ~l o f t h e f l u o ro g e n i c p e p t i d e , a n d t h e mi x t u r e s i n c u b a t e d a t
3 7 C f o r 3 0 m m b e f o r e q u e n c h i n g w i t h 2 0 0 D 1 o f c o l d e t h a n o l .
F l u o r e s c e n c e w a s m e a s u r e d o n a F l u o r o s k a n I I ( L a b s y s t e m s , H e l -
s i n ki , F i n la n d ) u s i n g a n e x c i t a t i o n w a v e l e n g t h o f 3 8 0 n m a n d a n
e m i s s i o n w a v e l e n g t h o f 44 0 n m . F l u o r e s c e n c e v a lu e s w e r e c o m -
p a r e d w i t h a s t a n d a r d c u r v e p r e p a r e d w i t h 7 - a m i n o - 4 - m e t h y l -
c o u ma r i n o r 2 n a p h t h y l a mi d e , a s d e sc r i b e d b y R i v e t t e t a l . (1 7 ) .
T h e f o l l o w i n g f l u o r o g en i c p e p n d e s w e r e u s e d: S u c - L e u - L e u -
V a l - T y r - M C A a nd S u c - A I a - A l a - P h e - M C A t o m ea su re c h y -
m o t r y p s i n - h k e ( C h - L ) a c ti v it y , C b z - L e u - L e u - G l u - 2 - n a p h t h y l a -
m i d e t o m e a s u r e p e p t i d y l g l u t a m y l p e p t i d e h y d r o l y z i n g a c t i v i t y
( P G P H ) , a n d B o c - L e u - A r g - A r g - M C A t o m e a su r e tr y p si n -h k e
a c t i v it y (T - L ) . C r u z i p a i n a c t i v i t y w a s m e a s u r e d u s i n g C b z - P h e -
A r g - A M C a s a s u b st r at e .
Puri f icat ion of T . cruzi Proteasomes. F o r p u r i f i c a t i o n o f p r o t e a -
so me s , T . c ruz i e p i ma s t i g o t e s (3 ( s t ra i n ) we re u se d . Pa ra s i t e s we re
h a r v e s t e d f r o m 3 1 o f 6 - d a y c u l tu r e s b y c e n t r f f u g a t io n a t 2 , 0 0 0 g
f o r 2 0 m m a n d w a s h e d t h r e e t i m e s w i t h P B S . P a r a s i t e s w e r e s u s -
p e n d e d m 5 v o f 2 0 m M T n s - H C l , 1 m M E D T A , s o n lc a te d , an d
t h e h o m o g e n a t e c l a ri f ie d b y c e n t r f f u g a t io n . T h e p e l l e t w a s d i s -
c a rd e d a n d t h e su p e rn a t a n t wa s c e n t r i fu g e d a t 1 0 0 ,0 0 0 g fo r 1 h .
T h e 1 0 0 , 0 0 0 g s u p e r n a t an t w a s c o n c e n t r a t e d b y f i l tr a t io n i n a
C e n t r i c o n 1 0 u n i t ( A m i c o n , B e v e r l y , M A ) , a n d f r a c t i o n a t e d b y
f a s t p e r f o m a n c e l i q u i d c h r o m a t o g r a p h y ( F P L C ) u s i n g a S u p e r o s e
6 H I < 1 6 / 5 0 c o l u m n e q u i l ib r a t ed w i t h 25 m M T r i s - H C 1 , 1 m M
E D T A ( p H 7 . 5) . F r a c t i o n s o f 1 .2 m l w e r e c o l l e c t e d a n d a ss ay ed
fo r C h -L a c t i v i t y . Th e a c t i v e f ra c t i o n s we re a g a i n a s sa y e d i n t h e
p r e s e n c e o f 5 0 I x M o f e i t h e r l a c ta c y s t m o r E - 6 4 . T h o s e t h a t w e r e
i n h i b i t e d b y l a c t a c y s t m b u t n o t b y E - 6 4 w e r e p o o l e d a n d l o a d e d
o n t o a M o n o - Q 5 / 5 c o l u m n e q u il i br a t ed w i t h 2 0 m M T r i s - H C 1
( p H 8 . 0 ) . B o u n d p r o t e i n s w e r e e l u t e d u s i n g a 0 - 1 M K C 1 h n e a r
g r a d i e n t in 2 0 t oN I T r i s - H C 1 ( p H 8 . 0) . F r a c t m n s o f 0 .5 m l w e r e
c o l l e c t e d a n d a s sa y e d f o r p r o t e o l y n c a c n v i t y a s a b o v e . T h e a c t i ve
f r a c t i o n s e l u t e d a t a p r o x i m a t e l y 4 0 0 - 5 0 0 m M K C 1 . T h e y w e r e
p o o l e d a n d c o n c e n tr a t e d i n a C e n t r i c o n 1 0 u m t . T h e c o n c e n -
1 9 1 0 P r o te a s o m e C o n t r o l o f M o r p h o l o g y o f T. cruzi
Published November 1, 1996
-
5/19/2018 Proteosoma T cruzi.pdf
3/10
trated sample was loaded onto a Superose 6 HP, 16/30 equili-
brated with 25 mM Tris-HC1, 1 mM EDT A, (pH 7.5) . Fractions
of 0.6 ml we re collected and assayed for Ch-L, T -L and PG PH
activities (17).
Protein Determination.
Protein concentration was determined
by the Bradford method (18), using BSA as a standard.
Electrophoretic Tec hnique s.
Samples were analyzed by SDS-
PAG E electrophoresis according to Laemmli (19) in a 12 sepa-
rating gel and 3 s tacking gel. Two -dim ension al gel SDS -PAG E
electrophoresis was perform ed as in O 'Farrell (20).
Antibodies and Immunoprecipitation Studies. An u- T. cru zi protea-
some antibodies were obtained by injecting rabbits with three
doses of 50 txg of purif ied proteasornes using Titer M ax (Cyt Rx
Corp, Norcross, GA) as adjuvant. The antiserum strongly reacted
with the 25-35 kD proteasorne subunits by Western blotting.
Tw o weaker unidentif ied bands of about 70 kD were also seen on
the blots (data not shown). Fo r immu noprecip itation studies, ali-
quots o f 3 X 10 ? trypornastigotes were incubated for 3 h in trans-
formation medium alone, or in the presence oflactacystin or clasto-
lactacystin. The parasites were washed, resuspended in 20 mM
Tri s-H Cl (pH 7.5), 1 mM EDT A, and sontcated. Sonicates were
centr ifuged for 5 rain at 10,000 g. The supernatants were pre-
treated with preimrnune rabbit serum and protein A-Sepharose
(Pharrnacia Biotech, Uppsala, Sweden), and th en incubated ov er-
n igh t with an t i -T,
cruzi
proteasome antisera diluted 1:250. The
immu nocornplexes were collected by incubation with 100 t~l of a
50 suspension of protein A-Sepharose . The immuno precipitates
were washed and Ch-L activity measured in the presence or ab-
sence of protease inhibltors , as explained in the text and f igure
legends. Experiments were performed in tr iplicate and expressed
as fluorescence units + SD.
Electron Microscopy.
Purified proteasornes (50 I~g/ml) were at-
tached to ca rbon-coa ted and g low-discharged formvar f i lm for
1 nun, and subjected to negative s taining with 1 uranyl acetate
as described (21). Electron micrographs were reco rded with mag -
nif icauon of 80,000 in a Zeiss EM 910 electron m icroscope.
NH2-terminal Sequences.
Samples were separated on SDS-P AGE ,
transferred to polyvinyhdene dif luonde membranes (Immobilon
P, Millipore; Milford, MA) usrng CAPS (Sigma) pH 11, contain-
ing 10 (v/v) methanol, s tamed with Coornassie blue, and the
protein bands were excised and sequenced. Automatic Edrnan
degradation analysts was cam ed out on a 477A protei n sequencer ,
and the resulting phenylt hlohyda ntoin derivatives idenufied using
an online 120A phenyh hioidan toin analyser (Applied Biosystems,
Foster City, CA).
A
100
8O
E
I- 40-
+
2 0 -
0
Clasto-l~tacy~
E~
LactacyWn
I I i
o ,0 ~ o 3 0 20 ~o
Inhibitor Concentnltlon I~M)
B
100 ,
Cbz (S-IBZ)-Cys-Phe-CHN2
90 ~ Cbz-Phe-AJa-FMK
Lactacyl~n
80 q ~
7o M
6 o ~
50 -I
4o -I
3o ~
20 i
i I
0 5 110 115 210 215 30
Inhibitor Conclmtrlltion (I+M)
Figure
1. (A and B) Effect ofprotease inhibltors on the transformauon
o f T. cruzi trypomastlgotes into am astigotes. Parasites were incubated for
4 h at 37C in transformation medtum with the protease mhlbltots, and
then relncubated overnight in DMEM 10 FCS. Transformation was
scored m a double-bhnd fashion by hght microscopy, and results ex-
pressed as mean + SD.
A r g - A M C b y r e c o m b i n a n t c r u za i n ( a g i ft fr o m D r . J+
M c K e r r o w , U n i v e r s i t y o f C a l i fo r n i a, S a n F r a n c i sc o , C A ) ,
o r b y c r u z a in p u r i f i e d f r o m p a r a s i t e e x t r a c t s , wa s n o t a f -
f e c te d b y h ig h c o n c e n t r a t io n s ( 1 00 tx M ) o f l a c ta c y s t in ( d a ta
n o t s h o wn ) . Co n v e r s e ly , p a ra s i t e r e mo d e l in g wa s n o t a f -
f ec te d b y C b z - P h e - A l a - F M K o r C b z - ( S - B z ) C y s - P h e -
CH N 2, ce l l -permea nt inh ib i to r s o f cys te ine pro teases , o r by
E-64 at concentrations as high as 50 IxM (Fig. 1 A and 1 B).
T h e t r y p o ma s t ig o te s t re a te d wi th 1 0 l a M la c ta cy s t in f o r
1 8 h a p p e a r e d n o r ma l o n th e b a s is o f mo t i l i ty a n d mo r -
p h o l o g y , w h e n e x a m i n e d b y l i g h t m i c r o s c o p y ( F i g . 2 C )
a n d e le c t r o n mic r o s c o p y ( d a ta n o t s h o wn ) . Ne v e r th e le s s ,
h ig h e r c o n c e n t r a t io n s o f l ac ta c y s t in we r e to x ic f o r th e p a r -
a s i t e , s imi la r to wh a t h a s b e e n d e s c r ib e d f o r o th e r e u k a r y -
B
o
o H
Na I
~ R ~ s , , , ~ . ~ N . A c O H C0;~1t
H3C , ~ - -
,OH
H- ++~ ~--~s +/ ..
HO .~ -
++
.'A-
HO
~H
lao t~y~n c~sto-lactacv~ln
R e s u l t s
Effect o f Protease Inhibitors on the Transformation of T. cruzi
in Axenic Medium.
F ig s. 1 A a n d 1 B s h o w th a t p r o te a -
s o m e i n h i b i t o r s p r e v e n t e d t h e t r a n s f o r m a u o n o f
T. cruzi
t r y p o ma s t ig o te s in to a ma s t ig o te - l ik e p a r as i t es . 5 0 in h ib i -
t io n o f t r a n s f o r ma t io n wa s a c h ie v e d a t 1 - 2 I +M c o n c e n t r a -
t io n s o f la c ta c y s t in a n d M G 1 3 2 , a p e p t i d e a ld e h y d e ( 2 2 )
( F ig . 1 A) . C la s to - l a c ta c y s t in d ih y d r o x y a c id , a n in a c t iv e
ana lo gue of lac tacys t in (Figs . 2 A and 2 B) (23), d id n o t
p r e v e n t t r a n s f o r ma t io n . L a c ta c y s t in h a s n o e f f e ct o n c y s -
t e in e p r o te in a s e s ( 2 4 ), i n c l u d in g c r u z a in ( o r c ru z ip a in ) , t h e
m a j o r l y s o s o m a l c a t h ep s i n L - l i k e e n z y m e o f
T. cruzi
( 2 5 -
2 7 ) th a t h a s b e e n i mp l ic a te d in th e g r o wth a n d d i f f e r e n t i a -
t i o n o f t h e p a r as i te ( 2 8 - 3 0 ). T h e h y d r o ly s i s o f C b z - P h e -
C D
Figure 2. Effe ct of lactacystin and clasto-lactacystm on
T cruzi. A)
Lactacystm. (B) Clasto-lactacystmdlhydroxy acid. (C and D) Morphology
of
T.cruzi
trypomasugotes that were incubated in DMEM (pH 5.0) m the
presence oflactacystln or clasto-lactacystm, espectively.
1911 Gonzfilez et al.
Published November 1, 1996
-
5/19/2018 Proteosoma T cruzi.pdf
4/10
2500
Clasto-Lactacyson
2000 [ ~ Lactacystmn
B ~ t
~ . P - i i * . . . . . i ~ . . . . i P t ~ i ~ 1 ~ = t o m -
m P : e ,
i ~ . . . . . i i * . . . . i - ~ l i P ' t r i ~ - I f * - - - f ~ . . . . i P - l . ~
H
j J -
' ~ , - i l , : ~ - i ~ ~ 0 - - ~ @ - - - t 0 , ~ ~ # .
F l u o r m m o n c o I n t m m d t y
Fi gu re 3 . Ef fec t o f p ro teasom e lnh lb l to rs on the expression o f s tage-
specific epltopes of
T. cmzi
Parasi tes undergo ing t ransformat ion m the
presence or absence o f the p ro teasome lnh lb l to rs lac tacystm (A, B , C , D ) ,
and M G-1 32 (E, F, G , / - / ) , were analyzed by FACS . Trypom asugotes
we r e i n c u b a t e d f o r 4 h i n t h e t r a n s fo r m a t io n m e d m m a lo n e o r m e d iu m
c o n ta in in g i n h ib i t o r , a n d th e n r e ln c u b a te d i n DM E M 1 0 FC S in th e
presence (B , D, F, / - / ) o r absence (A, C , E , G) o f lnh ib l to rs . At the end of
the incubat ion , the parasi tes were washed and sta ined by lmmunof luores-
cence with mAb 2C2 (A, B , E , F) o r 3C9 (C , D, G, H) , and analyzed by
FACS . Th e mA b 2C 2 detec ts Ssp-4 , an amasUgote-specif ic ep l tope , a nd
mA b 3C 9 detects Ssp-3, a trypomasugote-spec~fic epltop e.
O tlC c e ll s . F i g . 2 D s h o w s t h e a m a s t i g o t e - l i k e m o r p h o l o g y
o f t h e p a r a s it e s t h a t h a d t r e a t e d w i t h c l a s t o - l a c t a c y s t in .
T h e p r o t e a s o m e l n h i b i t o r s al so d e la y e d t h e e x p r e s s i o n o f
s t a g e - s p e c i f i c a n t i g e n s , a s s h o w n b y F A C S a n a l ys i s o f p a r -
a s it e sa m p l e s t a k e n a t th e e n d o f t h e t r a n s f o r m a t i o n p r o c e s s .
I n c o n t r o l s a m p l e s , a l a rg e p r o p o r t i o n o f t h e a m a s t i g o t e -
l i k e o r g a n is m s a c q u i r e d t h e a m a s t i g o t e - s p e c i f ic S s p - 4 e p i -
t o p e , a n d l o s t t h e t r y p o m a s t i g o t e - s p e c i f i c S s p - 3 e p i t o p e
( 1 3) , w h i l e m o s t p a r as i te s i n c u b a t e d w i t h l a c t a c y s t i n o r
M G - 1 3 2 r e t a i n e d th e S s p -3 e p i to p e , a n d w e r e S s p - 4 n e g a -
t i v e ( F i g . 3 ) .
Ef fec t o f Pro tease Inh ib i tors on the In tracellu lar Transfor mat ion
o f T c ru z i .
I n o n e s e ri e s o f e x p e r i m e n t s , t r y p o m a s t i g o t e s
w e r e p r e i n c u b a t e d w i t h 1 0 I x M l a c t ac y s t in o r c l a st o - l a ct a -
c y s t in f o r 1 h a t 3 7 C , w a s h e d b y c e n t r i f u g a t i o n , a n d a d d e d
t o c u l t u r e d m y o b l a s t s . T h e m e a n n u m b e r o f i n t r a c e ll u l a r
1500
8
1000
E
5 o o
0 -
_ L i l
24 48
7 2
T i m e h)
Fi gu re 4 . Ef fec t o f lac tacysnn
on cel l invasion by
T. cruzi.
L6E9- ir rad la ted myoblasts were
infe ted with trypomasUgotes
that had bee n preincubated for l h
at 37C with 10 txM lactacystm
or clasto-lactacysnn. After 2 h in-
cubat ion a t 37C, the t rypomas-
t l g o t e s we r e r e m o v e d , a n d t h e
L6E9 cel ls were washed with
DM E M . On e se t o f c el ls wa s
fixed wi th 4 paraformaldehyde
in PBS for 30 ram. The ex tracel-
lu lar t rypomasUgotes w ere detec ted by lmm unof luoresc ence with a po ly-
c]onal an t ibody to
T cruzi
and the to tal n um ber o f parasi tes was deter -
maned by staining wi th Ho ech st dye after permeabflization of the L6E9
cel ls wi th co ld m ethanol fo r 10 ma n . The num ber o f ln t racel lu lar parasi tes
was ca lcu la ted by subtracung th e ex tracellu lar f rom to ta l nu mb er o f para-
si tes . The remoanmg infected ce l l cul tures w ere rem cubated a t 37C. A t
24 , 48 , and 72 h , m phcate wel ls were washed and stmned with M ay- Gm n-
wald-Glemsa. T he sl ides were exarmne d unde r l igh t rmcroscopy and the
num ber o f ln tracel lular am asugotes in 100 ce lls was counted . R esu l ts are
expressed as mean -+ SD.
p a r a s i t e s 2 h a f t e r i n f e c t i o n w a s n o t s i g n i f i c a n t l y d i f f e r e n t
f o r t r y p o m a s t i g o t e s t r e a t e d w i t h l a c t a c y s t i n ( 4 1 . 7 + 5 . 4 ) o r
w i t h c l a s t o - l a c t a c y s t i n (4 1 . 1 + 1 . 2 ), i n d i c a t i n g t h a t p r o t e a -
s o m e a c t i v it y w a s n o t r e q u i r e d f o r c e ll i n v a s i o n . N e v e r t h e -
l e ss , a t 2 4 , 4 8 , a n d 7 2 h a f t e r i n f e c t i o n t h e n u m b e r o f i n t r a -
c e l lu l a r a m a s t i g o t e s w a s m u c h l o w e r i n c e ll s i n f e c t e d w i t h
l a c t a c y s t i n - t r e a t e d t r y p o m a s t i g o t e s ( F i g . 4 ) .
N e x t , w e s t u d i e d t h e e f f ec t o f l a c t a c y s t i n o n t h e i n t r a c e l-
l u l a r t r a n s f o r m a t i o n o f t h e d i v i d i n g a m a s t i g o t e s i n t o t r y p o -
m a s t i g o t e s , a n e v e n t t h a t o c c u rs b e t w e e n 4 0 a n d 4 8 h a f te r
i n f e c t i o n . I n t h e f o l l o w i n g s e t o f e x p e r i m e n t s , t h e m y o -
b l a s ts w e r e t r e a t e d 4 8 h a f t e r i n f e c t i o n w i t h l a c t a c y s t i n o r
c l a s t o - l a c t a c y s t i n . A f t e r 2 h i n c u b a t i o n , t h e d r u g s w e r e r e -
m o v e d , t h e c el ls w e r e t h o r o u g h l y w a s h e d a n d r e i n c u b a t e d
a t 3 7 C . A t v a r i o u s t i m e s t h e r e a f te r , t r y p o m a s t l g o t e s w e r e
c o l l e c t e d in t h e c u l tu r e s u p e m a t a n t s a n d c o u n t e d . I n t h e
A B
800
5000
4000 L a c t a c y s ~ ~
600 A
500 3000
400 /
i 200
~ . ~ 1 o o o
1 o o
o o -
0 0 75 1 5 3 0 80 88 9 6
Concerdrat lon of Lactacy l lt in ~tM) Time h)
Fig u r e 5 . E f fe c t o f l a c ta c y s tm o n a m a su g o te / tr y p o m a sUg o te m t r a c e l l u -
lar transfor rnauon L6E9 i r radia ted myoblasts were in fected with
T. cruzt
trypomastlgotes At 48 h after infect ion, lactacystln or clasto-lactacystm
was added . Af ter 2 h o f inc uba uo n a t 37C, the cu l tu res were w ashed and
re lncubated a t 37C for various per iods o f t ime. The ef fec t o f the d rugs
on parasi te development was evaluated as fo l lows. (A) By count ing ,n a
Ne u b a u e r c h a m b e r t h e n u m b e r o f t r y p o m a su g o te s i n t h e c u l tu r e su p e r n a -
tan ts . Th is was m easured 48 h af ter removal o f the d rugs. (B) By measur -
ing translahdase acuvlty in extracts o f infected cells 72, 80, 88, a nd 96 h
af ter mfecu on , 1 .e . , 24 , 32 , 40 , a nd 48 h af ter removal o f the d rugs. A l l
exper iments were per formed m tnphcate and values expressed as mean
+ SD
1 9 1 2 P r o t e a s o m e C o n t r o l o f M o r p h o l o g y o f
T. cruzi
Published November 1, 1996
-
5/19/2018 Proteosoma T cruzi.pdf
5/10
B
F i g u r e 6 M o rp h o l o g y o f T . c m z i infected cultures treated with lacta-
cystm L6E9-irra&ated myoblasts wer e infected wi th T. cruzi trypomas-
txgotes. At 48 h after infection, lactacysnn or clasto-lactacystin was added.
After 2 h of incubation at 37C, th e cultures we re washed and rem cu-
bated at 37C for another 48 h. The infected cultures were fixed and
stained wi th M ay-Grunw ald-G]em sa and examined by l igh t microscopy
(A) Myoblasts treated with lactacystin showing typical amasugotes. (B)
Myoblasts treated w ath clasto-lactacystm show ing trypomasngotes and in-
termediate forms.
c u l t u re s t r e a t e d w i t h l a c t a c y s ti n a t c o n c e n t r a t i o n s o f 3 a n d
1 . 5 I z M , s i g n if i c a n tl y f e w e r t r y p o m a s t i g o t e s w e r e r e l e a s e d
f r o m t h e c e ll s a s c o m p a r e d w i t h c o n t r o l s t r e a t e d w i t h
c l a s t o- l a c t ac y s t i n o r m e d i u m a l o n e ( F i g . 5 A ) . W e a ls o as -
s a y e d e x t r a c t s o f i n f e c t e d c e l l s f o r t h e p r e s e n c e o f t r a n s ia l i -
d a se , a n e n z y m e e x p r e s s e d o n l y i n t r y p o m a s t i g o t e s . I n c u l -
t u r es t r e a t e d w i t h c l a s t o - la c t a c y s ti n o r m e d i u m a l o n e , t h e
1 9 1 3 Go n z f i l e z e t a l.
B
1 5
1 0
o
_=
e 4
0 0
0 1 5
i
,
i i
b 1 5 3 0
F r a c t i o n N u m b e r
1
0 0
lOOO ~
l
4 5
lOO 1 o
S
a
o
0 1 0
0 0 5
0 O 0
C o~o
o
o
=E 0 0 5
o 0 0
10 20 30
F r a c t i o n N u m b e r
= ~ 0 8
o 6 ~
s o ~ g
6
0 4 ~
.+.
0 O 0
4 0 5 0
1 0 2 0 3 0
F r l c U o n N u m b e r
120
8
i l o
4 O
8 O 0 1 5
0 0 0
~o
- 4 0 0
2 0 0 ~
- 0 0 0
F igure 7. Purification and charactenzation of
T . c m z i
proteasomes. (A)
Ge l fi l trat]on on Superose 6. T he chymotrypsm activity in fractions 17-24
was totally inhibited by lactacystm but unaffected by E-64. (B) Amon-
exchange chromatography o f poole d fractions 17-24 on a M ono Q col-
um n. Bo und proteins wer e eluted using a 0-1 mM KC1 hnear gra&ent.
Fractions that &splayed Ch -L actlv]ty that was m hlbltable b y lactacystin,
bu t no t by E-64 , were e lu ted a t approxamate ly 400-500 mM KC1. (C)
Gel Fdtration on Superose 6. Fracnons eluted from the Mono Q at 400-
500 mM KC1 we re loaded onto Superose 6 16/ 30 . Proteolytlc activit ies
under the major pro te in peak were measured w ath the fo l lowing f luoro-
g e mc p e pt ld e s: Su c -L e u -L e u -V a l -Ty r - M C A fo r C h -L a c ti vi ty
C h - L ) ,
B o c - Le u - Arg -Arg -M C A fo r T -L a c t lv x ty
T - L )
a n d Z - L e u - L e u - G l u -
[JNA f or peptldylglutamyl peptide hydrolase
P G P H ) .
All actlvmes were
strongly inhibited by lactacystm but not by E-64.
e x p r e s s i o n o f t r a n s i a l i d a s e s t ar t s 8 0 h a ft e r i n f e c t i o n , a n d i n -
c r e a s e s u n t i l t h e e n d o f i n t r a c e l l u l a r p a r as i t e d i f f e r e n t i a t i o n .
I n l a c t a c y s t i n - t r e a t e d c u l t u r e s , t h e e x p r e s s i o n o f t r a n s i a l i-
d a s e w a s i n h i b i t e d ( F i g. 5 B ) . F i n a l l y , o n e s e t o f i n f e c t e d
c e ll s w a s s t a i n e d 9 0 h a f te r i n f e c t i o n a n d e x a m i n e d b y l i g h t
m i c r o s c o p y . W h i l e 9 0 p e r c e n t o f c e ll s t r e a t e d w i t h l a c ta -
c y s t in c o n t a i n e d t y p ic a l a m a s t i g o te s , a b o u t 8 0 o f m y o -
b l a s t s t r e a t e d w i t h c l a s t o - l a c t a c y s t i n c o n t a i n e d t r y p o m a s t i -
g o r e - l i k e o r i n t e r m e d i a t e f l a g el l at e f o r m s ( F ig . 6) . A n a l o g o u s
Published November 1, 1996
-
5/19/2018 Proteosoma T cruzi.pdf
6/10
B
O
~ 8o
~ 4 0
~ ~o
~ o
F i g u r e 9
B
1
Control
l a s t o q a c ~ l l c y s t l n
L a c t a c y s t t n
In wvo and m wtro mhibitaonof
T . c m z i
proteasomes by lac-
tacystm. A). Trypomasugotes were incubated for 3 h in transformataon
medium containing 10 ~M lactacystln so h d b a rs ) , or clasto-lactacystln
smp ed b a rs ) or with medium alone o p en b a rs ) . Samples of parasites 3
107) were washed with PBS, resuspended n 200 pJ of 20 mM Trls, sonl-
cated, and centrifuged. Supernatantswere lmmunopreclpltatedwath poly-
clonal antxbodies raised against T . c m z i proteasornes. Irnrnunocomplexes
were collected usmg protean A-Sepharose, and the Ch-L actxvxty associ-
ated with the beads was measured. When parasites were treated with me-
dium and immunoprecipited with preammune serum, no Ch-L activity
was detected. B) As additionalcontrols for the specificityof the lmmuno-
prec~patatlon reaction, untreated parasites were sonlcated, treated with
lactacysm so h d b a rs ) , or c l a s t o - l a c t a c y s t m s t r ip e d b a r s ), or medium o p en
bars) and lrmnunoprecipltated s above. The Ch-L actwlty of the lrnrnu-
nopreclpxtates was then measured. All expenrnents were performed m
tnphcate, and results expressed as mean -+ SD.
Figure 8. A) Composite of SDS-PAGE first track on the left) and
two-dlrnenslonalgel analysisof T. cru z i proteasomes. The arrow points to
an added control protean p l 5 . 2 ) . On the left are the MW markers. Gels
were Sliver-stained. B) Electronnncroscopyof
T . c m z i
proteasomes. Bar,
100 nm
experiments were performed with the cell-permeant cys-
teine proteinase inhibitors E-64d 31) and Cb z- Ph e- Al a-
FM K at concentra tions of 10 /~M. The y had no effect on
the transforma tion of intracellular amastigotes into tr ypo-
mastigotes, or on the expression of transialidase data not
shown).
I d e n t i fi c a t i o n o f t h e L a c t a c y s t in T a r g e t i n T . c r u z i . We used
two approaches to identify the target oflactacyst in in T . c m z i .
First, we isolated the lact acystin-inhibita ble chymotr ypsin
activity from crude extracts of parasite. As show n in Fig. 7 A,
a broad peak o f chymotryps in activity was detected fo llow-
ing filtration of the extracts in a Superose 6 colum n. H ow -
ever, only the activity in the shoulder peak fractions 1 7-24),
containing proteins o f higher molecular mass, was inhib -
itable by lactacystin, but n ot by E-64. In later fractions the
chymotryptic activity was inhibited by E-64 but not by lac-
tacystin. The lactacystin- inhibitable fractions were then
subjected to anion-exchange chromatography in a Mono
Q colu mn. A peak of chymot rypsin activity that was inhi b-
ited by lactacystin eluted at 400-4 50 mM of KC1 Fig. 7 B).
Pooled fractions from this peak were then filtered through
another Superose 6 column. A major symmetrical OD peak
of 670 kD was eluted from the column. It contained the
three characteristic peptidase activities of eukaryotic pro-
teasomes, T-L, Ch -L, an d PG PH Fig. 7 C). All activities
were inhibitable by lactacystin. Using Suc -L eu- Le u-V al-
Tyr-AMC as a substrate, the specific activity of the Ch-L
activity was 1.5 ~M /m g/ h. At concentrations up to 50
p~M, the cruzain inhibi tors Cbz -Ph e-A la- FMK and C bz-
S-B z)C ys- Phe -CH N 2 did not affect the Ch-L activity of
the purifie d proteasomes.
Using SDS-PAG E unde r denaturing conditions the 670 kD
molecules were resolved into subunits with molecular masses
between 25-35 kD. By isoelectrofocusing, their isoelectric
points varied betw een 4.5 and 8.5 Fig. 8 A). The N Hz- ter -
minal protein sequence of the protein from one band TSI-
MAVT FKD ) is identical to that of the [3-subunit of PRE3,
a PG PH activity from yeast proteasomes 32). Electron mi-
croscopy of negatively stained preparations revealed c harac-
teristic images ofproteasomes, i.e., hollow cylinders 18 nm
in length and 12-15 n m in diameter Fig. 8 B).
To identify the target of lactacystin in vivo, we incu-
bated samples of trypomastigotes for 2 h in transformation
medium in the presence oflactacystin, clasto-lactacystin, or
medium alone. The parasites were washed, and sonicated
1914 Proteasome Control of Morphology of
T . c r u z i
Published November 1, 1996
-
5/19/2018 Proteosoma T cruzi.pdf
7/10
e x t ra c t s w e r e i m m u n o p r e c i p i t a t e d w i t h a r a b b it a n t i s e ru m
t o pur i f i ed T. cruzi pr o t easomes , o r w i t h nor ma l r abb i t s e -
r u m . I m m u n o p r e c i p i t a t e s w e r e t h e n a s s a y e d f o r c h y m o -
t r yps i n ac t i v i t y . As shown i n F i g . 9 A , t he i mmunopr ec i p i -
t a r ed p r o t easomes f r om pa r as i t e s t ha t had been i ncuba t ed
w i t h l a c t a cy s t in w e r e i n a c t i v e. T h e c o n t r o l i r n m u n o p r e c i p -
i t a t e s f r om pa r as i t e s t r ea t ed w i t h medi um a l one o r c l a s t o -
l ac t acys t i n had Ch - L ac t i v i ty t ha t was i nh i b i t ed by l ac t acys -
t in , b u t n o t b y E - 6 4 . N o e n z y m a t i c a c ti v it y w a s d e t e c t e d in
sampl es i mmunopr ec i p i t a t ed w i t h nor ma l r abb i t s e r um. As
a d d i ti o n a l c o n t r o l s o f t h e s p e c if ic i ty o f t h e i m m u n o p r e c i p i -
t a t ion , t r ypom as t i go t e ex t r ac t s we r e t r ea t ed w i t h l ac tacys t in
or c l a s t o - l ac t acys t i n and t hen i mmunopr ec i p i t a t ed a s de -
s c r i b e d a b o v e . T h e i m m u n o p r e c i p i t a t e s o r i g i n a t i n g f r o m
ext r ac ts t r ea t ed w i t h l ac t acyst i n we r e i nac t i ve F ig . 9 B )
i s c u s s i o n
W e s h o w h e r e t h a t t h e p r o t e a s o m e i n h a b i t o r s M G 1 3 2
a n d l a ct a cy s ti n p r e v e n t e d t h e t r a n sf o r m a t io n o f t r y p o m a s -
t ig o t e s i n to a m a s ti g o te s a n a x e n i c m e d i u m . M G 1 3 2 , a p e p -
t i de a l dehyd e , a l so po t en t l y i nh i b i t s cys t e i ne p r o t eases , bu t
l ac t acyst i n s e l ec ti ve l y inh i b i t s t he pep t i dase ac t i v i ty o f p r o -
t easomes . T h e t r ans i en t i n t e r medi a t e o f lac t acys ti n , c l a st o -
lactacyst in 13 lactone, b inds t ight ly to threon ines in the act ive
s i te o f t he [ 3 subun i t s o f p r o t easo me s 24 , 33). C l as t o - l ac t a -
cys t i n d i hy dr ox y ac id F ig . 2 B ) , t he p r o duc t o f hydr o l ys i s
of the ac t ive 13 lacton e, ha d n o act ivi ty in paras i te t ransfor -
ma t i on . L ac t acys t i n does no t i nh i b i t s e r i ne o r cys t e i ne p r o -
t eases o f mam mal i an ce ll s 24), and d i d no t a f f ec t t he ac t i v -
a ty o f c ruza i n , t he ma j or
T. cruzi
l y s o s o m a l e n z y m e . W e
f ur t he r a sce r t a ined t ha t p r o t easomes a r e t he t a r ge ts o f lac t a -
cys t i n i n t r ypom as t i go t e s by t wo i nde pen den t c r i t e ri a . F ir st ,
p r o t e a so m e s w e r e i s o la t ed t o a p p a r e n t h o m o g e n e i t y f r o m
crud e e xt racts o f paras ites u s ing a lactacy st in-based assay to
f o l l o w p u r i f i c a t i o n . S e c o n d , w h i l e i m m u n o p r e c i p i t a t e s o f
p r o t easom es p r esen t i n ex t r ac t s o f c la s t o - l ac t acys ti n - t r ea t ed
pa r as i t e s had Ch- L ac t i v i t y , t he i mmunopr ec i p i t a t e s f r om
lactacy st in- t reated parasi tes w ere in act ive .
W e a lso s tud i ed t he e f f ec t o f l ac t acys t i n on t he i n f ec t i v i t y
o f T. cmzi t r ypomas t i go t e s t o myobl as t s . I n t hese expe r i -
men t s , we t r i ed t o mi n i m i ze o r ex c l ude poss ib l e e ff ec ts o f
t he d r ug on t he t a r ge t ce l l s . F or exampl e , when s t udy i ng
t h e a t t a c h m e n t a n d p e n e t r a t i o n p h as e s o f in f e c t io n , d r u g -
t r ea t ed pa r as i t e s wer e washed be f or e i ncuba t i on w i t h t he
myobl as t s . W e f oun d t ha t l ac tacys t in had no e f f ec t on i nva -
s ion, an act ive process that requi res paras ite energ y 34) ,
and i s associated wi th c alc ium f luxes in the paras i te 35) .
H o w e v e r , t h e i n t r a ce l lu l a r d e v e l o p m e n t o f th e l a c ta c y s ti n -
t r ea t ed pa r as i t e s was a r r e s t ed . I t canno t be deduced f r om
t hese r e su l t s whe t he r l ac t acys t i n i nh i b i t ed on l y t he t r ypo-
mas t i go t e / amas t i go t e t r ans f o r ma t i on . T he r e i s a d i s t i nc t
poss i b il i ty t ha t l ac t acyst i n i nh i b i t ed amas t i go t e p r o l i f e r a t ion
as we l l , s i nce t he euka r y o t i c ce l l cyc l e i s r egu l a t ed by p r o-
t easomes . I n any case , these exp e r i men t s a lso show t ha t t he
e f f ec t s o f l ac t acy s t i n pe r s i st ed du r i ng t he i n t r acel l u l ar dev e l -
op m en t o f t he pa r as it e . L ac t acys t in is an i r r eve r s ib l e i nh i b i -
t o r o f p r o t e a s o m e s , a n d t h e h a l f- li fe o f T. cruzi p r o t e a s o m e s
m a y b e l o n g . A l t e rn a t iv e l y , d r u g t r e a t m e n t m a y h a v e i r r e -
ve r s i b l y a f fec t ed a p r o t eas om e- de pen den t e s sen ti al pa r as it e
f unc t i on .
L ac t acys t in a lso p r ev en t ed t he t r ans f o r ma t i on o f amas ti -
go t e s i n t o t r ypoma s t i go t e s t ha t occur s a t the e nd o f t he i n -
t r ace l l u l a r phase . I n t hese expe r i ment s , myobl as t s i n f ec t ed
4 8 h p r e v i o u s ly w i t h t r y p o m a s t i g o te s w e r e e x p o s e d f o r 2 h
to 1-3 p~M o f lactacyst in. T he ef fect was s t r iking: as co m -
pared wi th c las to- lactacyst in- t reated cel l s , the lactacyst in-
t r ea t ed ce ll s re l eased f ew er t r ypoma s t i go t e s in t o t he c u l t u r e
medi um, con t a i ned mor e amas t i go t e s i n t he i r cy t op l asm,
and displayed m uc h less t rans ia l idase act ivi ty. In cont ras t ,
h i g h e r c o n c e n t r a t io n s o f c e l l - p e r m e a n t i n h i b i to r s o f c r n z i -
p a i n h a d n o e f f e ct o n t h e a m a s t i g o t e / t ry p o m a s t i g o t e t r a n s-
f o r ma t i on . T h e sma ll concen t r a t i ons o f l ac t acys t i n used , t he
shor t dur a t aon o f d r ug t r ea t me nt , t he spec i f ic i t y o f the ob -
se r ved e f f ec ts , and t he l ack o f e f f ec t o f cys t e i ne p r o t ease i n -
h i b i t o r s a r gue s t r ong l y t ha t t he p r i me t a rge t s o f l act acys ti n
are the t ransfo rmin g paras ites ra ther than th e my oblas t s .
T hese r e su l t s show t ha t p r o t easome ac t i v i t y i s neces sa r y
f or r emod e l i ng , bu t t he subst ra te s t ha t a r e degr aded have
n o t b e e n i d e n t i f i e d . T h e y p r o b a b l y i n c l u d e p r o t e i n s t h a t
ma i n t a i n t he o l d shape , mos t l i ke l y cy t oske l e t a l e l emen t s ,
a se t o f p r o t e i n s a n d e n z y m e s i n v o l v e d i n t h e o l d m e t a -
bol ic pathways, and s tage-speci f ic sur face proteins . In addi -
t i o n t o t h e s e h o u s e k e e p i n g f u n c t i o n s , t h e c l e a v a g e o f k e y
r e g u l a t o r y p r o t e i n s b y p r o t e a s o m e s m a y p r o v i d e t h e c e n -
t r a l sw i t ch i ng mechan i sm t ha t i n i t i a t e s t he s t age - spec i f i c
changes 36) .
In eukaryot ic cel l s , the subst ra tes des t ined for degrada-
t i o n a r e r e c o g n i z e d b y s p e c i f i c E 2 - E 3 U b - p r o t e i n l i g a s e s
3 7) . H o w e v e r , v e r y li tt le is k n o w n a b o u t t h e U b - p r o t e a -
som e sys tem i n p r o t o zoa n pa rasi te s. S ou t he r n and N or t h -
e r n b lo t s o f D N A a n d R N A f r o m v a r io u s str ai ns o f
T. cruzi
r evea l ed l a r ge va r i a t ions in t he nu mb er o f Ub genes 38).
I t s g e n o m e m a y c o n t a i n m o r e t h a n 1 0 0 U b c o d i n g s e -
q u e n c e s , a n u m b e r m u c h l a r g e r t h a n i n o t h e r o r g a n i s m s .
T h e s e a r e e n c o d e d i n f i ve p o l y U b g e n e s a n d f iv e U b f u s io n
genes , whose t r ansc r i p t i on i s a l t e r ed under s t r e s s cond i -
taons . Th er e i s a s igni f icant increase in s teady-s ta te levels of
U b m R N A b e t w e e n t h e m i d lo g p h as e cu l tu r es o f n o n i n -
f ec t i ve ep i mas t i go t e s o f T. cruzi and t he s t a t i ona r y phase
cu l t u r es tha t con t a i n t he mo r pho l og i ca l l y d is t inc t , i n f ec t i ve
met acyc l i c s 39). I t i s no t ew or t h y t ha t hea t - sho ck e l ement s
a r e p re s e n t i n t h e i n t e r g e n i c r e g io n s p r e c e d i n g t h e p o l y U b
genes . P e r haps t he expr es s i on o f t he U b genes in T. cruzi is
r egu l a t ed by t he sh i f t s i n env i r onment a l pH and t emper a -
t u r e , and by o t he r s t r e s s cond i t i ons t ha t l ead t o s t age - spe -
c i f i c r emode l i ng . I n yeas t s t ha t bea r mut a t i ons i n p r o t ea -
somes, sensi twi ty to s t ress i s increased, and under s t ress
cond i t i ons t he mut an t s accumul a t e ub i qmt i na t ed p r o t e i ns .
O t he r p r o t eases have been i den t i f i ed i n
T. cruzi
40- 42) .
O ne o f t hem, c r uza i n , a l ysosomal cat heps i n L - l ike cys t e i ne
pr o t ease , a lso p lays a r o l e i n g r o wt h and d i f f e r en t i a ti on o f
the parasate 28-3 0) . S tudies in di f ferent laborator ies have
s h o w n t h a t s y n th e t i c i n h ib i t o rs o f c ru z a i n, i n c l u d i n g C b z -
1915 Go nzf i lez t a l.
Published November 1, 1996
-
5/19/2018 Proteosoma T cruzi.pdf
8/10
P h e - A l a - F M K a nd C b z - S - B z ) C y s - P h e - C H N 2 , i nh ib it
T. cruzi
i n f e c t i v i ty . H o w e v e r , d i f f e r e n t f r o m l a c t a c y s ti n , th e
c y s t e i n e p r o t e a s e i n h i b i t o r s p r e v e n t p a r a s i t e p e n e t r a t i o n
i n t o t h e h e a r t m u s c l e c el ls 2 8 ) . A s s h o w n h e r e , r e l a t iv e l y
h i g h c o n ce n tr a ti o n s o f C b z - P h e - A l a - F N I K a n d C b z - S -
B z ) - C y s - P h e - C H N 2 d i d n o t a ff ec t t h e r e m o d e l i n g o f T .
cruzi i n a x e n i c m e d i u m o r i n s i d e c e l l s . A l t h o u g h o u r f i n d -
i n g s d o n o t e x c l u d e a r o l e f o r c r u z a i n a n d o t h e r l y s o s o s m a l
e n z y m e s i n t h e e x t e n s i v e p r o t e o l y s i s t h a t m u s t a c c o m p a n y
r e m o d e l i n g , t h e y a r g u e t h a t t h e r o l e o f cr u z a i n is n o t p i v -
o t a l d u r i n g t h e se p h a s e s o f p a ra s it e d e v e l o p m e n t .
S o m e p u b l i c a t i o n s r e p o r t t h e p r e s e n c e o f p r o t e a s o m e s i n
Trypanosoma 4 3 , 4 4 ) a n d Entamoeba 4 5 ), b u t t h e i r f u n c t i o n
h a s n o t b e e n s t u d i e d . W e f o u n d t h a t t h e st r u c t u r a l f ea t u r e s
a n d a r c h i t e c t u r e o f t h e T. cruzi p r o t e a s o m e s w e r e s i m i l a r
t o t h o s e o f o t h e r s p e ci e s. B y S D S - P A G E t h e c y l i n d ri c al
2 0 S s t r u c t u r e w a s r e s o l v e d i n t o t h e t y p i c a l 6 - 8 b a n d s o f
2 5 - 3 5 k D . H o w e v e r , m o r e t h a n 2 0 p r o te i n s, w i t h w i d e ly
d i v e r s e p l s , w e r e s e e n i n T. cruzi p r o t e a s o r n e s a n a l y z e d b y
t w o - d i m e n s i o n a l P A G E . I t as g e n e r a ll y a c c e p t e d t h a t t h e 2 0 S
p r o t e a s o r n e i s a d i m e r o f 1 4 s u b u n i t s a r r a n g e d otT[37137oL . I n
t h e y e a s t
Saccharomyces cerevisiae
h e r e a r e f o u r t e e n g e n e s e n -
c o d i n g 7 o t a n d 7 [3 s u b u n i t s , a n d t h e d e n d r o g r a m r e p r e -
s e n t i n g t h e a l i g n m e n t s o f al l e u k a r y o t i c p r o t e a s o m e s e -
q u e n c e s y i e l d s o n l y 1 4 s u b g r o u p s c o n t a i n i n g a s i n g l e y e a s t
m e m b e r . T h e e x p l an a t io n f o r th e l ar g e n u m b e r o f T. cruzi
p r o t e a s o m e - a s s o c i a t e d p r o t e i n s m a y b e t r iv i al : s o m e e x t r a
s p o t s c o u l d r e p r e s e n t p o s t t r a n s l a ti o n a l m o d i f i c a t i o n s o f a
p o l y p e p t i d e , o r s i m p l y c o n t a m i n a n t s . A l t e r n at i v e ly , a n u n -
u s u a l f e a t u r e o f T. cruzi i s t h a t i t s p r o t e i n s a r e f r e q u e n t l y
e n c o d e d b y s e v e r a l t a n d e m l y a r r a n g e d g e n e s t h a t a r e p o l y -
c l s t r o n i c a l l y t r a n s c r i b e d f r o m a s i n g l e p r o m o t e r a n d a r e
c o n c u r r e n t l y e x p r e ss e d . S e q u e n c e v a r i a ti o n o f g e n es f o u n d
i n o n e s u c h t r a n s c r i p t i o n u n i t c o u l d a d d t o t h e a p p a r e n t
s u b u n i t h e t e r o g e n e i t y . F u r t h e r s t u d i e s a r e n e c e s s a r y t o c l a r -
i fy th i s i s sue .
T h e p r e s e n t p a p e r d e m o n s t r a t e s t h a t p r o t e a s o m e a c t i v i t y
i s e s s e n t i a l f o r
T. cruzi
r e m o d e l i n g . V e r y s i m i l a r r e s u l t s
w e r e r e c e n t l y o b t a i n e d w i t h o t h e r p r o t o z o a n p a ra s it es . I n a
r o d e n t m a l a r i a m o d e l S i n n i s , P . , B . G u t i e r r e z , M . B r i o n e s ,
a n d V . N u s s e n z w e r g m a n u s c r i p t i n p r e p a r a ti o n ) s h o w e d t h a t
l a c ta c y s ti n d i d n o t p r e v e n t t h e p e n e t r a t i o n o f th e Plasmo-
dium berghei
c r e s c e n t - s h a p e d s p o r o z o i t e s i n t o h e p a t o c y t e s ,
b u t s t r o n g l y i n h i b i t e d t h e i r t r a n s f o r m a t i o n i n t o t h e r o u n d
h e p a t o c y t e s t a g es a n d s u b s e q u e n t d e v e l o p m e n t . E i c h i n g e r,
D . , V . N u s s e n z w e i g , a n d J . G o n z a l e z m a n u s c r i p t in p r e p a -
r a ti o n ) d e m o n s t r a t e d t h a t la c ta c y s ti n p r e v e n t e d t h e e n c y s t a -
t i o n o f Entamoeba invadens . Trypanosoma Entamoeba a n d
Plasmodium b e l o n g t o p h y l a w i d e l y s e p a r a t e d i n e v o l u t i o n .
T h e r e f o r e , i t is l i k e l y t h a t t h e m e c h a n i s m s g o v e r n i n g s t a g e -
s p e c if i c m o r p h o l o g i c a l c h a n g e s i n p r o t o z o a a r e c o n s e r v e d ,
a n d p r o t e a s o m e - d e p e n d e n t , a n d th a t p r o t e a s o m e in h i b l to r s
m a y h a v e a b r o a d r a n g e o f t ar g e ts . E n c o u r a g i n g f e a tu r e s fo r
a t t e m p t i n g t o d e v e l o p t h is c la ss o f c h e m o t h e r a p i c a g e n ts
a r e t h a t s o m e p a r a s i t e s , s u c h a s
Plasmodium
u n d e r g o c o n -
s t a n t a n d r a p i d r e m o d e l i n g i n t h e m a m m a l i a n h o s t . T h u s ,
e f f ec t iv e d ru g s n e e d n o t b e a d m i n i s t e re d f o r p r o l o n g e d p e -
r i o ds o f t i m e t o a r re s t p a r as i te d e v e l o p m e n t . F u r t h e r m o r e ,
t h e a c c u r a t e d i s c r i m i n a t i o n b e t w e e n t h e o l d a n d n e w p r o -
t e in s t h a t c o e x is t w i t h i n t h e s a m e c e ll d u r i n g r e m o d e l i n g o f
p r o t o z o a m a y r e q u i r e s p e c ia l iz e d f e a tu r e s o f th e p r o t e a -
s o m e / U b s y st em . P r o t e a s o m e s f r o m i n tr a ce l lu l ar p r o t o z o a n
p a r a s it e s m a y a l so d i f f e r s i g n i f i c an t l y i n s t r u c t u r e f r o m t h o s e
o f t h e h o s t c e l l, r e n d e r i n g t h e i n f e c t e d c e ll s s u s c e p t i b l e t o
d e s t r u c t io n b y c e ll s o f t h e i m m u n e s y s te m . H o p e f u l l y ,
s o m e o f t h e s e a p p r o a c h e s t o t h e r a p y w i l l y i e l d t o e x p e r i -
m e n t a l a t t a c k .
W e a re g ra te ful to D rs . Ch r i s tophe r C a rdoz o a nd M a r t in Re c hs te ine r fo r he lpfu l d i s c uss ions a nd prov id ing
reagents . W e th ank D r. Da nie l E~chmger for he lping w ith the t ransia l idase assays and fo r revtew ing the
manuscr ipt . W e than k Mrs . Bessy Gu tierrez for the prep ara t ion o f f igures .
Thi s w o rk w a s suppor te d by gra n t s f rom the N a t iona l Ins ti tu t e s o f H e a l th to V . N usse nz w e ig , a nd E . J . Co -
re y. J . G onz f il e z ha s be e n supp or te d by a pos tdoc tora l f el low ship f rom the P e w La t in A me r ic a n Fe llow s Pro -
gra m. F .J . Ra m a lho-P in to w a s suppor te d by CN Pq Bra zi l) .
A ddres s c or r e sponde nc e to D r . Jorge G onz f il e z, D e pa r tm e nt o f Pa tho logy , Mic ha e l H e l lde lbe rge r D iv i s ion
o f I m m u n o l o g y , 5 5 0 F i rs t A v e . , N e w Y o r k , N Y 1 0 0 16 .
Received or publication 19 August 199 6 and in revised orm 5 September 199 6.
e f e r e n c e s
1. Bre ne r , Z . 1973 . B io log y of Trypanosoma cruzi. Annu. Rev.
Microbiol. 2 7 : 3 4 7 - 3 6 2 .
2 . O r low skl , M. 1990 . The mul t i c a ta ly t i c p ro te ma se c omple x , a
m a j o r e x t r a l y s o s o m a l p r o t e o l y n c s y s t e m . Biochemistry 29:
1 0 2 8 9 - 1 0 2 9 7 .
3 . Coux , O . , K . Ta na ka , a nd A . L . G oldbe rg . 1996 . S t ruc tura l
func t ions o f the 20S a nd 26S pro te a som e s .
Annu. Rev. Bio-
chem.
6 5 : 8 0 1 - 8 4 7 .
4 . 1K e c hste me r, M. , L . H of fm a n, a nd W. D ubie l . 199 3 . Th e
mul t i c a ta lync a nd 26S pro te a se s .
J. B io l . Chem .
2 6 8 : 6 0 6 5 -
6068.
5 . H i l t , W. , a nd D . H . Wo l f . 1996 . Pro te a some s : de s t ruc t ion a s a
1 9 1 6 P r o te a so m e C o n t r o l o f M o r p h o l o g y o f T. cn~zi
Published November 1, 1996
-
5/19/2018 Proteosoma T cruzi.pdf
9/10
program.
Trends B iochem. Sc i .
21:96-102.
6. Cardozo, C., A.M. Eleuteri, and M. Orlowska, M. 1995. Dif-
ferences in catalytic activities and su bunit pattern o fmul tica t-
alytic protemases (proteasomes) isolated from bovin e pitu -
itary, lung and live r.J.
B i o l . Che m .
270:22645-22651.
7. Nandi, D., H. Jiang, and J.J. Monaco. 1996. Identification of
MECL- I(LMP -10) as the third IFN-~/-inducible proteasome
subunit.J. I m m u n o l . 156:2361-2364.
8. Corey, E.J., and G.A.R .eichard. 1992. Total synthesis oflac-
tacystin.J.
A m . C h e m . S o c .
114:10677-10678.
9. Corey, E.J., and S. Choi. 1993. An enantioselec tive synthesis
of (6R.)-lactacystin.
Tetrahedrom Letters.
34:6969-6972.
10. Silva, L.H.P., and V. Nussenzweig. 1953. Sobre urna cepa de
Trypanosoma cruzi
altamente virulenta para o camundongo
branco. Fol . Cl in. B iol . 20: 191-207.
11. Kanbara, H., H. Uemura, S. Nakazawa and T. Fukama.
1990. Effect of low pH on transformation of
Trypanosoma
c ruz i t r y pom as t i g o t e . J ap . J . P aras i to l .
39:226-228.
12. Tomhnso n, S., F. Vandekerckhove, U. Frevert, and V. Nus-
senzweig. 1995. The i nduct ion of
Trypanosoma cruzi
trypo-
mastigote to amastigote trans formation by low pH.
Parasitol-
ogy . 110:547-554.
13. Andrews, N.W., K.S. Hong, E.S.R.obbins, and V. Nussen-
zwexg. 1987. Stage-specific surface antigens expressed dunng
the morphogenesis of vertebrate forms of
T ry panosom a c m z i .
Exp. Parasi tol .
64:474-484.
14. Schmatz, D. M., and P.K. Murray.1982. Culti vation of T ry p-
anosom a c ruz i in irradiated muscle cells: improved synchron i-
zation and enhanced trypomastlgote production. Parasitology.
85:115-125.
15. Schenkman, S., C. Diaz, and V. Nussenzwesg. 1991. Attach-
ment of
T ry panosom a c ruz i
trypomastigotes to receptors at re-
stricted cell surface domains.
Exp, Parasi tol .
72:76-86.
16. Schenkman, S., J. Man-Shiow, G.W. Hart, and V. Nussen-
zweig. 1991. A nove l cell surface transialidase of
Trypanosoma
c ruz i generates a stage-specific epitope required for invasion
of mammalian cells.
Cel l .
65:1117-1125.
17. R.ivett, A.J., P .J. Savory, and H. Djaballah. 1994. Multicata -
lytlc endopeptidase complex: proteasome.
M e t h . E n z y m o l .
244:330-350.
18. Bradford, M. 1976. A rapid and sensitive method for the
quantltatxon of microgram quantities of protein uuhzi ng the
principle of protein-dye binding.
A nal . B i oc he m .
72:248-254.
19. Laemmli, U.K. 1970. Cleavage of structural protein during
the assembly of the head of bacteriophage T4.
Nat ure L ond . ) .
227:680-685.
20. O'Farrell, P. H. 1975. High resolution two-dimension al elec-
trophoresis.J.
B i o l . Che m .
250:4007-4021.
21. Baumeister, W., B. Dahlman n, R . Hegerl, F. Koop, L.
Kuehn, and G. Pfeifer. 1988. Elec tron microscopy and xmage
analysis of the multicatalytic proteinase.
Fed. Exp. B iol . Soc .
Le t t .
241:239-245.
22. R.ock, K.L., C. Gramm, L. R.othsteln, K. Clark, R.. Stewm,
L. Dick, D. Hwang, and A.L. Goldberg. 1994. Inhibitors of
the proteasome block the degradation of most cell proteins
and the gene ration of peptides presented o n MH C class I
molecules.
Cel l .
78:761-771.
23. Fenteany, G., R..F. Standaert, G.A. R'eichard, E.J . Corey, and
S.L. Schreiber. 1994. A [3-1actone related to lactacystin in -
duces neurite outgrowth in a neuroblastoma cell line and in-
hibits cell cycle progression in an osteosarcoma cell line.
Proc.
Na t l . A c ad . Sc i . U SA .
91:3358-3362. -
24. Fenteany, G., IL.F. Standaert, W.S. Lane, S. Choi, E.J. Co-
rey, and S.L. Schreiber. 1995. Inh ibiti on ofproteasome activ-
sties and subunit-specific amino-t erminal hreonin e modifica-
tion by lactacystin.
Sc i e nc e W ash . D C) .
268:726-731.
25. Cazzulo, J.J., R.. Couso, A. R.axmondi, C. Wernstedt, and U.
Hellman. 1989. Further characterization and partial amin o
acid sequence of a cysteine proteinase from
Trypanosoma
cruzi . Mol . B iochem. Parasi tol .
33:33-42.
26. Murta, A.C.M., P.M. Persechini, T. Souto -Padr6n, W. D e
Souza, J.A. Guimaraes andJ. Scharfsteln. 1990 . Structural and
functional identification of GP 57/51 antigen of
Trypanosoma
cruzi
as a cysteino proteinase.
Mol. B iochem. Parasi tol .
43:27-38.
27. Eakin, A.E., J. Bouvler, J.A. Sakanari, C.S. Craik, and J.H.
McKerrow. 1990. Amplification and sequencing of genomac
DNA fragment encoding cysteine proteinase from protozoan
parasites. Mol. B iochem. Parasi tol . 39:1-8.
28. Meirelles, M.N.L., L. Juhano, E. Carmona , S.G. Silva, E.M.
Costa, A.C. Murta, andJ. Scharfstein. 1992. Inhibitors of the
major cysteinyl proteinase (GP57/51) impair host cell inva-
sion and arrest the intracellular development of
Trypanosoma
cruzi in v i t ro. Mol . B iochem. Parasi tol .
52:175-184.
29. Harth, G., N. Andrews, A.A. Mills, J.C. Engel, R.. Smith,
andJ .H. McKerrow. 1993. Peptide-fluoromethyl ketones ar-
rest lntracellu lar replicat ion and interce llular transmission of
Trypanosoma cruzi . Mol . B iochem. Parasi tol .
58:17-24.
30. Franke de Cazzulo, B.M., J. Martinez, M.J. North, G.H.,
Coombs, G.H. and J.J. Cazzulo. 1992. Effects of proteinase
inhlbitors on the growth and differentiation of Trypanosoma
cruzi . FE M S M icrobiol. Le t ters . 124:81-86.
31. Wang, K.K.W., and P.W. Yuen. 1994. Calpain inhibition:
an overview of its therapeutic potential.
Trends. Biochem. Sci.
15:412-419.
32. Hilt, W., and D.H. Wolf. 1995. Proteasomes o f the yeast S.
cerevisiae:
genes, structure and functaons.
Mol. B iol . Rep.
21:3--10.
33. Dick, R..L., L.G. Cruikshank, F.D. Melandri, S.L. Nunes,
and R. Stein. 1996. Mechanistic studies on the inactivation of
the proteasome by lactacystln.J.
Biol . Chem.
271:7273-7276.
34. Schenkman, S., E.S.R.obbins and V. Nussenzweig. 1991. At-
tachment of
Trypanosoma cruzi
to mammalian cell s requires
parasite energy, and invasion can be indepe ndent of the target
cell cytoskeleton.
I n fe c t. hn m u n .
59:645-654.
35. Moreno, S.N.J., J. Silva, A.E. Vercesi, and P,. Docampo.
1994. Cytosohc-free calcium elevation m
Trypanosoma cruzi
is
requi red for cell invasion._]. E x p . M e d . 180:1535-1540.
36. Palombella, V.J., O.J.R.ando, A.L., Goldberg and T. Mania-
tlS. 1994. The ubxquitin-proteasome pathway as requi red for
processing the NF-KB1 precursor protein and the activation
of NF-KB.
Cel l .
78:773-785.
37. Ciechan over, A. 1994. The Ub -prote asome proteolytlc path-
way.
Cel l .
79:13-21.
38. Kirchhoff, L. V., K.S. Klrn, D.M. Engman, and J.E. D one l-
son. 1988. Ublquitm genes in Trypanosomatidae. J . B iol .
C h e m . 263:12698-12704.
39.-Swindle, J., J. Afioka, H. Eisen, B. Sanwal, C. Jacquemot, z .
Browder, and G. Buck. 1988. The genomic organization and
the transcription of the ubi quitm genes of
Trypanosoma cruzi .
E M B O E u ro p . M o l . B i ol . O r g . ) J .
7:1121-1127.
40. Ashall, F. 1990. Cha ract enzation of an alkaline peptidase o f
Trypanosoma cruzi
and other trypanosomatxds.
Mol. B iochem.
Parasitol.
38:77-88.
41. Santana, J.M., P. Grelher, M.-H. R'odier, J. Schrevel, and A.
Telxeira. 1992. Purification and characterization of a new
120 kD alkaline proteinase of
Trypanosoma cmzi . B iochem. B io-
p h y s . R e s . C o m m .
187:1466-1473.
1917 Gonzfilez et al.
Published November 1, 1996
-
5/19/2018 Proteosoma T cruzi.pdf
10/10
42 . Bur le igh , B .A. , and N.W. Andrews . 1995 . A 120-Da a lka l ine
p e p t i d a se f r o m Trypanosoma cruzi i s i n v o l v e d i n t h e g e n e r a -
t i o n o f a n o v e l C a 2 - s l g n a l i n g f a c t o r f o r m a m m a l i a n -c e l l s.J .
Biol . Chem. 2 7 0 : 1 - 9 .
4 3 . H u a , S . , W . T o , T . T . N g u y e n , M . L . W o n g , a n d C . C .
W a n g . 1 9 96 . P u r i f i c a t i o n a n d c h a r a c t e n z a t i o n o f p r o t e a so m e s
f r o m
Trypanosoma bruce i . M ol . B iochem. Parasi tol .
78:33--46.
44 . L ima , B .D. , M.H.G. Va ins te in , and C . Mar t ins de Sa . 1993 .
M u l t i c a t a l y ti c p r o te i n a s e c o m p l e x p u r i f i e d f r o m d i f fe r e n t
stains of T r yp a n os o m a c r u z i A c o m p a r a t i v e s t u d y . M e m . I ns t .
O s w a ld o C r u z R i o J . 88 :BQ29 , 143 .
4 5 . S c h o l z e , H . , S . F r e y , Z . C e j k a , a n d T . B a k k e r - G r u n w a l d .
1 9 96 . E v i d e n c e f o r t h e e x i s t e n ce o f b o t h p r o t e a s o m e s a n d a
n o v e l h i g h m o l e c u l a r w e i g h t p e p t i d a s e i n
Entamoeba his toly t
ica. J . B iol . Ch em. 2 7 1 : 6 2 1 2 - 6 2 1 6 .
1 9 1 8 P r o t e as o m e C o n t r o l o f M o r p h o l o g y o f T. cruzi
Published November 1, 1996