P r o t e a s o m e Ac t i v i t y Is 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 i c

T r a n s f o r ma t i o n o f a P r o t o z o a n Pa r a s i t e
By Jorge Gonzfilez,* E Juarez Ramalho-Pinto,~ Ute Frevertfi
Jorge Ghiso,* Stephen Tomlinson,* Julio Scharfstein,II E.J. Corey,
andVictor Nussenzweig*
From the *Michael Heidelberger Division of Immunology, Department of Pathology, New York
University Medical Center, New York 10016; the *Department of Biochemistry-Immunology,
blstituto de Ciendas Biologicas, Universidade Federal de Minas Gerais, MG, Brazil; the ~Department
of Medical and Molecular Parasitology, New York University Medical Center, New York 10016; the
Iqnstituto de Biofisica, Universidade Federal do Rio de Janeiro, RJ, Brazil; and the Department of
Chemistry, Harvard University, Cambridge, Massachusetts 02138
S u mma r y
A pr omi nent feature of the life cycle of intracellular parasites is the profound morphol ogi cal
changes t hey undergo during devel opment in the vertebrate and invertebrate hosts. In eukary-
otic cells, most cytoplasmic proteins are degraded in proteasornes. Here, we show that the
transformation in axenic medi um of trypomastigotes of Trypanosoma cruzi into amastigote-like
orgamsms, and the intracellular devel opment of the parasite from amastigotes into t rypomast i -
gotes, are prevent ed by lactacystin, or by a peptide aldehyde that inhibits prot easome function.
Clasto-lactacystin, an inactive analogue of lactacystin, and cel l -permeant peptide aldehyde i n-
hibitors of T. cruzi cysteine proteinases have no effect. We have also identified the 20S prot ea-
somes f r om T. cruzi as a target oflactacystin in vivo. Our results document the essential role of
prot easomes in the stage-specific transformation of a protozoan.
I
nfection by Trypanosoma cruzi, the causative agent of
Chagas' disease, is initiated by metacyclic t rypomast i -
gotes present in the feces of triatomine bugs. The t r ypo-
mastigotes invade host cells and enter the cytoplasm, where
t hey transform into amastigotes. The amastigotes replicate
and, a few days later, transform back into trypomastigotes,
rupture the host cells, and invade the bl oodst ream (1).
Thus, on t wo occasions during its intracellular stage, T. cruzi
undergoes shape and vol ume changes, restructures its fla-
gellum and kinetoplast, and synthesizes new sets of surface
molecules. These striking modifications are precisely timed,
take place in an orderly fashion, and must involve selective
degradation of cytoplasmic proteins.
In eukaryotic cells, most proteins in the cytoplasm and
nucleus are degraded not in lysosomes, hut wi t hi n prot ea-
somes, after t hey are marked for destruction by covalent at-
t achment of ubi qui t i n (Ub) l mol ecul es (2-5). In addition
1Abbreviatzons used in this paper: BSA, bovine serum albumin; CAPS, (3-
[Cyclohexylarmno]-l-Propanesulfonlc acid), Ch-L, Chymotrypsln-hke;
EDTA, ethylene dl-amino tetra acetic acid; E-64, trans-epoxysuccmyl-
L-leucylamldo-3-methyl-butane ethyl ester; FCS, fetal calf serum; F1TC,
fluoresceln lsothiocyanate; MES, (2-[N-morphohno]-ethanesulfonlc aod);
MG-132, carboxybenzoxyl-leucinyl-leucinyl-leuclnal-H; PGPH, pepti-
dylglutamyl peptlde hydrolase; T-L, Trypsin-like; Ub, ub~qultin.
to their role in nonlysosomal prot ei n t urnover, prot ea-
somes are i nvol ved in specific cellular functions, i ncl ud-
ing the following: the pr ogr ammed inactivation of mitotic
cychns, transcription factors, and transcriptional regulators;
the elimination of mut at ed or damaged proteins; and anti-
gen presentation. The function of the proteasomes is also
tightly regulated, and their structure may vary to mat ch
function (6-7).
The experi ment s described bel ow were designed to doc-
ument the participation of pr ot easomes in the devel opmen-
tal pathways of prot ozoan parasites. T. cruzi has an advan-
tage as an experimental model because its t rypomast i got e
form can be induced to change rapidly into amastigotes in
axenic medi um. The resulting amast i got e-hke parasites
cannot be distinguished from intracellular amastigotes by
light or electron microscopy, or by stage-specific surface
markers. Thus, in this model , the effects of pr ot ease inhibi-
tors on transformation can be studied i ndependent l y f r om
their effect on the cells of the host.
Ma t e r i a l s a n d Me t h o d s
Cell Lines. LLC-MK 2 fibroblasts were obtained from Amen-
can Type Culture Collection, Rockville, MD (ATCC CCL-7).
L6E9 myoblasts were a gift of Dr. R. Docampo (Umversxty of I1-
1909 j. Exp. Med. The Rockefeller Universxty Press 0022-1007/96/11/1909/10 $2.00
Volume 184 November 1996 1909-1918

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Published November 1, 1996
hnois, Ur bana- Champai gn, IL.). Cells wer e gr own in RP MI
1640 medi um suppl ement ed wi t h 10% FCS, 100 t zg/ ml peni ci l -
hn, and st rept omyci n.
Reagents. Protease inhlbitors E-64, E-64d, Cb z - P h e - Al a -
FMK, Cb z - ( S - BZ) - Cy s - P h e - CHN2 , and fl uorogeni c substrates
wer e purchased f r om Sigma Chemi cal Co. (St. Lores, MO) . Lac-
tacystin and clasto-lactacystin wer e synthesized as prewousl y de-
scribed (8, 9). MG- 132 was f r om Proscript, Inc. (Cambri dge,
MA). Chr omat ogr aphy col umns and resins wer e f r om Pharmacia
Bi ot ech AB (Uppsala, Sweden).
Inhibition of Trypomastigote Transformation into Amastigotes. LLC-
MK 2 cells were infeCted wi t h T. cruzi trypomastigotes, Y strata (10).
4 d later, t he supernatants cont ai ned mor e than 95% t rypomast i -
gotes and small number o f amastigotes or i nt ermedi at e forms.
Parasite transformation i nt o amastigotes was i nduced by l ower i ng
the p H o f t he i ncubat i on medi um (11, 12). To assay for t he effect
o f inhibitors in t he transformation, t wof ol d di l unons o f each i n-
hi bi t or wer e distributed m 96- mi cr owel l plates. Di l ut i ons wer e
made wi t h DME M buffered wi t h 20 mM MES (pH 5.0) cont ai n-
i ng 0.4% BSA. Lactacystin or clasto-lactacystin, MG- 132, E-64,
Cb z - ( S- BZ) - Cy s - Ph e q EHN2 and Cb z - Ph e - Al a - FMK were pre-
pared at 200 ~M, and 50 p,1 wer e added to wells to final dilutions
o f 100-0. 78 p,M. Dependi ng on t he inhibltors used, DMS O di -
lutions or medi um wer e used as controls. Trypomast i got es wer e
cent nf uged (3, 000g 15 min) and resuspended at 2 107/ml in
DME M (pH 5.0). 50 p~l o f this suspension was added to each
well, mi xed, and i ncubat ed for 4 h at 37C in a 5% CO2 at mo-
sphere. The plate was cent ri fuged and the supernatants wer e r e-
move d and replaced by DME M (pH 7) cont ai ni ng 10% FCS.
The plates wer e rei ncubat ed over ni ght at 37C m a CO2 i ncuba-
tor. The percent age of transformed parasites was det er mi ned by
mi croscopi cal l y scoring 200 cells in each wel l in a bl i nded fash-
ion. All experi ment s wer e carried out in duphcate.
F A C S ~ Analysis. Parasates (2.5 107) wer e transformed in
t he presence or absence of proteinase inhlbitors as described. At
t he end o f t he i ncubat i on, parasites wer e resuspended in 250 }xl
of DME M at 4C, and an equal vol ume of monoc l ona l antibodies
2C2 anti-Ssp-4 or 3C9 ant, -Ssp-3 (13) was added. The i ncuba-
t i on pr oceeded for 30 mm on ice. The suspension was t hen
cent ri fuged for 7 rai n at 3,500 r pm in a refrigerated cent nf uge
(Sorvall I<T6000B), using a hori zont al rotor. The supernatant
was r emoved, and t he parasites wer e fixed wi t h 4% paraformalde-
hyde in PBS. Aft er 30 mi n at 4C, t he fi xanve was r emoved and
t he parasites wer e washed wi t h 1 ml of col d 0.4% BSA- DMEM.
The parasites wer e t hen i ncubat ed for 30 rain wi t h ant i - mouse
IgG conj ugat ed wi t h FI TC. The suspensions wer e centrifuged,
washed wi t h 0.4% BS A- DMEM, resuspended in 50 [zl of PBS,
and postfixed wi t h 4% paraformaldehyde. The cell suspensions
wer e analyzed in a Bect on Di cki nson FACScan .
Inhibition of Development of lntracellular Parasites. L6E9 myoblasts
wer e lrra&ated wi t h 2,000 rads (14) and plated in 4- wel l Lab- Tek
r mcr ochamber slides ( NUNC, Napervl l l e, IL). Tr ypomamgot es
wer e pret reat ed for 1 h wi t h 10 btM lactacystm or clasto-lactacys-
tin at 37C. Parasites wer e washed twice, resuspended in D M E M ,
and used to infect myoblasts at a parasite to L6E9 cells rano of 5:1.
Aft er 2 h l ncubat mn at 37C, trypomastigotes wer e r emoved, and
t he L6E9 cells wer e washed wi t h DMEM. To study the effect of
inhlbitors on invasion, one set of cells was fixed wath 4% paraformal-
dehyde in PBS for 30 man. Extracellular trypomastigotes wer e de-
t ect ed by l mmunof l uor esceuce wi t h a pol ycl onal ant i body to T.
cruzi, and t he total number of parasites was det er mi ned by stain-
i ng wi t h Hoechst dye (Sigma) after permeabi l i zat i on o f t he L6E9
cells wi t h col d met hanol for 10 ml u. The number ofi nt racel l ul ar
parasites was calculated by subtracting t he extracellular f r om t he
total parasites (15). To determine the fate oflactacysnn-treated para-
sites, t he remai ni ng infected cell cultures were reincubated at 37C.
At 24, 48, and 72 h, triplicate wells wer e washed and stained wi t h
May- Gr unwal d- Gi ems a. The slides wer e exami ned under light
mi cr oscopy and the number of lntracellular amastigotes in 100
cells was count ed. Resul t s are expressed as means + SD.
In anot her set o f experi ment s, we studied t he effect o f mhi bi -
tors on t he transformation of intracellular amastigotes i nt o t rypo-
mastigotes. Cel l cultures wer e i nfect ed wi t h T. cruzi t r ypomasn-
gotes. 48 h after i nfect i on, t he cultures wer e treated for 2 h wi t h
0.75, 1.5, and 3 IzM of lactacystin or clasto-lactacystin. The cul -
tures wer e washed and rei ncubat ed at 37C for an additional 2 d,
when t he first parasite burst occurred. The culture supernatants
wer e col l ect ed and t he numbers of exi t i ng t r ypomamgot es wer e
det er mi ned in a Neubauer chamber. To document furt her the
i nhi bi t ory effect o f lactacystin in t he amast i got e/ t rypomast i got e
transformation, mfect ed cultures wer e lysed 72, 80, 88, and 96 h
after i nfect i on wi t h a buffer cont ai ni ng 3% n- oct yl gl ucopi r ano-
side, 50 mM Tr i s- HC1 (pH 7.4), 0.1 mM EDTA, 20 b~M E-64
and 5 Dg/ ml l eupept i n, antipain, and pepstatin. The extracts wer e
analyzed for levels of translalidase, an enzyme expressed in t rypo-
mastigotes, but not in amastigotes (16). Measurement s wer e made
in triplicate samples, and transialidase activity was expressed as
cpm + SD.
Enz ymat i c Assays. Pr ot eol ync activity was assayed using as
substrate 100 p~M fl uorogeni c peptides di l ut ed in 50 mM Tn s -
HCl (pH 7.8). 10 Izl of chromat ographi c fractions was added to
90 b~l of the fl uorogeni c peptide, and t he mi xt ures i ncubat ed at
37C for 30 mm before quenchi ng wi t h 200 D1 of col d ethanol.
Fl uorescence was measured on a Fl uoroskan II (Labsystems, Hel -
sinki, Finland) using an exci t at i on wavel engt h of 380 nm and an
emission wavel engt h o f 440 nm. Fl uorescence values wer e com-
pared wi t h a standard curve prepared wi t h 7- ami no- 4- met hyl -
coumar i n or 2 napht hyl ami de, as described by Ri ve t t et al. (17).
The f ol l owi ng fl uorogeni c pepndes wer e used: Su c - Le u - Le u -
Va l - Ty r - MCA and Su c - AI a - Al a - Ph e - MCA to measure chy-
mot r ypsi n- hke (Ch-L) activity, Cb z - Le u - Le u - Gl u - 2 - n a p h t h y l a -
mi de to measure pept i dyl gl ut amyl pept i de hydrol yzi ng activity
(PGPH), and Bo c - Le u - Ar g - Ar g - MCA to measure t rypsi n-hke
activity (T-L). Cruzi pai n activity was measured using Cb z - P h e -
Ar g - AMC as a substrate.
Purification of T. cruzi Proteasomes. For purification of prot ea-
somes, T. cruzi epimastigotes (3(strain) wer e used. Parasites wer e
harvested f r om 3 1 o f 6-day cultures by centrffugation at 2,000 g
for 20 mm and washed three times wi t h PBS. Parasites wer e sus-
pended m 5 v of 20 mM Tn s - HCl , 1 mM EDTA, sonlcated, and
t he homogenat e clarified by centrffugation. The pellet was dis-
carded and t he supernatant was cent ri fuged at 100,000 g for 1 h.
The 100,000 g supernatant was concent r at ed by filtration in a
Cent r i con 10 uni t (Ami con, Beverl y, MA), and fractionated by
fast per f omance l i qui d chr omat ogr aphy (FPLC) using a Superose
6 HI< 16/ 50 col umn equi l i brat ed wi t h 25 mM Tri s-HC1, 1 mM
EDTA (pH 7.5). Fractions of 1.2 ml wer e col l ect ed and assayed
for Ch- L activity. The active fractions wer e again assayed in the
presence of 50 IxM of ei t her lactacystm or E-64. Those that wer e
i nhi bi t ed by lactacystm but not by E- 64 wer e pool ed and l oaded
ont o a Mo n o - Q 5/ 5 col umn equi l i brat ed wi t h 20 mM Tr i s- HC1
(pH 8.0). Bound proteins wer e el ut ed using a 0 - 1 M KC1 hnear
gradient in 20 toNI Tr i s- HC1 (pH 8.0). Fractmns of 0.5 ml wer e
col l ect ed and assayed for pr ot eol ync acnvi t y as above. The active
fractions el ut ed at aproxi mat el y 400- 500 mM KC1. They wer e
pool ed and concent rat ed in a Cent r i con 10 umt . The concen-
1910 Proteasome Cont r ol of Mor phol ogy of T. cruzi

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Published November 1, 1996
trated sample was loaded onto a Superose 6 HP, 16/30 equili-
brated with 25 mM Tris-HC1, 1 mM EDTA, (pH 7.5). Fractions
of 0.6 ml were collected and assayed for Ch-L, T-L and PGPH
activities (17).
Protein Determination. Protein concentration was determined
by the Bradford method (18), using BSA as a standard.
Electrophoretic Techniques. Samples were analyzed by SDS-
PAGE electrophoresis according to Laemmli (19) in a 12% sepa-
rating gel and 3% stacking gel. Two-dimensional gel SDS-PAGE
electrophoresis was performed as in O' Farrell (20).
Antibodies and Immunoprecipitation Studies. Anu-T. cruzi protea-
some antibodies were obtained by injecting rabbits with three
doses of 50 txg of purified proteasornes using Titer Max (CytRx
Corp, Norcross, GA) as adjuvant. The antiserum strongly reacted
with the 25-35 kD proteasorne subunits by Western blotting.
Two weaker unidentified bands of about 70 kD were also seen on
the blots (data not shown). For immunoprecipitation studies, ali-
quots of 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-HCl (pH 7.5), 1 mM EDTA, and sontcated. Sonicates were
centrifuged 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 then incubated over-
night with anti-T, cruzi proteasome antisera diluted 1:250. The
immunocornplexes were collected by incubation with 100 t~l of a
50% suspension of protein A-Sepharose. The immunoprecipitates
were washed and Ch-L activity measured in the presence or ab-
sence of protease inhibltors, as explained in the text and figure
legends. Experiments were performed in triplicate and expressed
as fluorescence units + SD.
Electron Microscopy. Purified proteasornes (50 I~g/ml) were at-
tached to carbon-coated and glow-discharged formvar film for
1 nun, and subjected to negative staining with 1% uranyl acetate
as described (21). Electron micrographs were recorded with mag-
nificauon of 80,000 in a Zeiss EM 910 electron microscope.
NH2-terminal Sequences. Samples were separated on SDS-PAGE,
transferred to polyvinyhdene difluonde membranes (Immobilon
P, Millipore; Milford, MA) usrng CAPS (Sigma) pH 11, contain-
ing 10% (v/v) methanol, stamed with Coornassie blue, and the
protein bands were excised and sequenced. Automatic Edrnan
degradation analysts was camed out on a 477A protein sequencer,
and the resulting phenylthlohydantoin derivatives idenufied using
an online 120A phenyhhioidantoin analyser (Applied Biosystems,
Foster City, CA).
A
100
8O
E
I- 40-
+
20-
0
Clasto-l~tacy~
E~4
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
6o~
50 -I
4o -I
3o ~
20 i
i I
0 5 110 115 210 215 30
In hibitor Conclmtrlltion (I+M)
Figure 1. (A and B) Effect ofprotease inhibltors on the transformauon
of T. cruzi trypomastlgotes into amastigotes. 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.
Ar g- AMC by r ecombi nant cruzain (a gift from Dr. J+
McKer r ow, Uni versi t y of California, San Francisco, CA),
or by cruzain puri fi ed from parasite extracts, was not af-
fected by hi gh concent rat i ons (100 txM) ofl act acyst i n (data
not shown). Conversel y, parasite r emodel i ng was not af-
fected by Cbz - Phe - Al a - FMK or Cbz- ( S- Bz) Cys - Phe-
CHN2, cell-permeant inhibitors of cysteine proteases, or by
E-64 at concentrations as high as 50 IxM (Fig. 1 A and 1 B).
The t rypomast i got es t reat ed wi t h 10 laM lactacystin for
18 h appeared normal on the basis of mot i l i t y and mor -
phol ogy, when exami ned by light mi croscopy (Fig. 2 C)
and el ect ron mi croscopy (data not shown). Nevertheless,
hi gher concent rat i ons of lactacystin were t oxi c for the par-
asite, similar to what has been descri bed for ot her eukary-
A B
o
o H
N a I
~ . ~ R ~ s , , , ~ . ~ N . A c O' H C0;~1t
H3C : , ~ - - ,OH
H- ++~'~--~s +/ ..+
HO .~ - "
++" .'A-"
HO" ~H
l ao' t ~y~n c~sto-lactacv~ln
Resul t s
Effect of Protease Inhibitors on the Transformation of T. cruzi
in Axenic Medium. Figs. 1 A and 1 B show that pr ot ea-
some i nhi bi t ors pr event ed the t ransformauon of T. cruzi
t rypomast i got es i nt o amast i got e-l i ke parasites. 50% i nhi bi -
t i on of t ransformat i on was achi eved at 1- 2 I+M concent ra-
tions of lactacystin and MG132, a pept i de al dehyde (22)
(Fig. 1 A). Clasto-lactacystin di hydr oxy acid, an inactive
anal ogue of lactacystin (Figs. 2 A and 2 B) (23), di d not
prevent transformation. Lactacystin has no effect on cys-
teine proteinases (24), i ncl udi ng cruzain (or cruzipain), the
maj or lysosomal cathepsin L- l i ke enzyme of T. cruzi ( 25-
27) that has been i mpl i cat ed in the gr owt h and differentia-
t i on of the parasite (28-30). The hydrolysis of Cbz - Phe -
C D
Figure 2. Effect of lactacystin and clasto-lactacystm on T cruzi. (A)
Lactacystm. (B) Clasto-lactacystm dlhydroxy acid. (C and D) Morphology
of T.cruzi trypomasugotes that were incubated in DMEM (pH 5.0) m the
presence oflactacystln or clasto-lactacystm, respectively.
1911 Gonzfilez et al.

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Published November 1, 1996
2500
Clasto-Lactacyson
2000 [ ~ Lactacystmn
" B ~t
" ~.P - i i * . . . . . i ~ . . . . i P t~ i ~ " 1~= " tom- mP : 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 gur e 3. Effect of proteasome lnhlbltors on the expression of stage-
specific epltopes of T. cmzi Parasites undergoing transformation m the
presence or absence of the proteasome lnhlbltors lactacystm (A, B, C, D),
and MG-132 (E, F, G, /-/), were analyzed by FACS . Trypomasugotes
were incubated for 4 h in the transformation medmm alone or medium
containing inhibitor, and then relncubated in DMEM 10% FCS in the
presence (B, D, F,/-/) or absence (A, C, E, G) oflnhibltors. At the end of
the incubation, the parasites were washed and stained by lmmunofluores-
cence with mAb 2C2 (A, B, E, F) or 3C9 (C, D, G, H), and analyzed by
FACS . The mAb 2C2 detects Ssp-4, an amasUgote-specific epltope, and
mAb 3C9 detects Ssp-3, a trypomasugote-spec~fic epltope.
OtlC cells. Fi g. 2 D s h o ws t h e a ma s t i g o t e - l i k e mo r p h o l o g y
o f t h e par as i t es t h a t h a d t r e a t e d wi t h c l a s t o- l a c t a c ys t i n.
T h e p r o t e a s o me l n h i b i t o r s al so d e l a y e d t h e e x p r e s s i o n o f
s t a ge - s pe c i f i c a nt i ge ns , as s h o wn b y F ACS anal ysi s o f p a r -
asi t e s a mpl e s t a k e n at t h e e n d o f t h e t r a n s f o r ma t i o n pr oces s .
I n c o n t r o l s ampl es , a l ar ge p r o p o r t i o n o f t h e a ma s t i g o t e -
l i ke o r g a n i s ms a c q u i r e d t h e a ma s t i g o t e - s p e c i f i c Ss p- 4 e p i -
t o p e , a n d l os t t h e t r y p o ma s t i g o t e - s p e c i f i c Ss p- 3 e p i t o p e
( 13) , wh i l e mo s t par as i t es i n c u b a t e d wi t h l a c t a c ys t i n o r
MG- 1 3 2 r e t a i n e d t h e Ss p- 3 e p i t o p e , a n d we r e Ss p- 4 n e g a -
t i ve (Fi g. 3).
Effect of Protease Inhibitors on the Intracellular Transformation
of T, cruzi. I n o n e ser i es o f e x p e r i me n t s , t r y p o ma s t i g o t e s
we r e p r e i n c u b a t e d wi t h 10 I xM l a c t a c ys t i n o r c l a s t o- l a c t a -
c ys t i n f or 1 h at 3 7 C, wa 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 myobl a s t s . T h e me a n n u mb e r o f i n t r a c e l l u l a r
1500
8
1000
E
5 o o
0 -
_ L , i l
2 24 48 7 2
T i m e (h)
Fi gur e 4. Effect of lactacysnn
on cell invasion by T. cruzi.
L6E9-irradlated myoblasts were
infected with trypomasUgotes
that had been preincubated for l h
at 37C with 10 txM lactacystm
or clasto-lactacysnn. After 2 h i n-
cubation at 37C, the trypomas-
tlgotes were removed, and the
L6E9 cells were washed with
DMEM. One set of cells was
fixed with 4% paraformaldehyde
in PBS for 30 ram. The extracel-
lular trypomasUgotes were detected by lmmunofluorescence with a poly-
c]onal antibody to T cruzi, and the total number of parasites was deter-
maned by staining with Hoechst dye after permeabflization of the L6E9
cells with cold methanol for 10 man. The number of lntracellular parasites
was calculated by subtracung the extracellular from total number of para-
sites. The remoanmg infected cell cultures were remcubated at 37C. At
24, 48, and 72 h, mphcate wells were washed and stmned with May-Gmn-
wald-Glemsa. The slides were exarmned under light rmcroscopy and the
number of lntracellular amasugotes in 100 cells was counted. Results are
expressed as mean -+ SD.
par as i t es 2 h a f t e r i n f e c t i o n wa s n o t s i gni f i c a nt l y di f f e r e nt
f or t r y p o ma s t i g o t e s t r e a t e d wi t h l a c t a c ys t i n ( 41. 7 + 5. 4) o r
wi t h c l a s t o- l a c t a c ys t i n ( 41. 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 me a c t i vi t y was n o t r e q u i r e d f or cel l i n v a s i o n . Ne v e r t h e -
less, at 24, 48, a n d 72 h a f t e r i n f e c t i o n t h e n u mb e r o f i n t r a -
cel l ul ar a ma s t i g o t e s was mu c h l o we r i n cel l s i n f e c t e d wi 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 ma s t i g o t e s (Fi g. 4).
Ne x t , we s t u d i e d t h e ef f ect 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 ul a r t r a n s f o r ma t i o n o f t h e d i v i d i n g a ma s t i g o t e s i n t o t r y p o -
ma s t i got e s , a n e v e n t t h a t o c c u r s b e t we e n 40 a n d 48 h af t er
i n f e c t i o n . I n t h e f o l l o wi n g set o f e x p e r i me n t s , t h e my o -
bl ast s we r e t r e a t e d 48 h a f t e r i n f e c t i o n wi t h l a c t a c ys t i n o r
c l a s t o- l a c t a c ys t i n. Af t e r 2 h i n c u b a t i o n , t h e dr ugs we r e r e -
mo v e d , t h e cel l s we r e t h o r o u g h l y wa s h e d a n d r e i n c u b a t e d
at 37 C. At v a r i o u s t i me s t he r e a f t e r , t r y p o ma s t l g o t e s we r e
c o l l e c t e d i n t h e c u l t u r e s u p e ma 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 88 916
Concerdratlon of Lactacylltin (~tM) Time (h)
Fi gur e 5. Effect oflactacystm on amasugote/trypomasUgote mtracellu-
lar transforrnauon L6E9 irradiated myoblasts were infected with T. cruzt
trypomastlgotes At 48 h after infection, lactacystln or clasto-lactacystm
was added. After 2 h ofi ncubauon at 37C, the cultures were washed and
relncubated at 37C for various periods of time. The effect of the drugs
on parasite development was evaluated as follows. (A) By counting ,n a
Neubauer chamber the number oftrypomasugotes in the culture superna-
tants. This was measured 48 h after removal of the drugs. (B) By measur-
ing translahdase acuvlty in extracts of infected cells 72, 80, 88, and 96 h
after mfecuon, 1.e., 24, 32, 40, and 48 h after removal of the drugs. All
experiments were performed m tnphcate and values expressed as mean
+ SD
1912 Prot easome Cont r ol of Mor phol ogy of T. cruzi

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A
B
Fi gure 6. Morphology of T. cmzi infected cultures treated with lacta-
cystm L6E9-irra&ated myoblasts were infected with T. cruzi trypomas-
txgotes. At 48 h after infection, lactacysnn or clasto-lactacystin was added.
After 2 h of incubation at 37C, the cultures were washed and remcu-
bated at 37C for another 48 h. The infected cultures were fixed and
stained with May-Grunwald-G]emsa and examined by light microscopy
(A) Myoblasts treated with lactacystin showing typical amasugotes. (B)
Myoblasts treated wath clasto-lactacystm showing trypomasngotes and in-
termediate forms.
cul t ur es t r e a t e d wi t h l act acys t i n at c o n c e n t r a t i o n s o f 3 a nd
1. 5 I zM, s i gni f i cant l y f e we r t r ypoma s t i got e s we r e r el eas ed
f r o m t he cel l s as c o mp a r e d wi t h c ont r ol s t r e a t e d wi t h
cl as t o- l act acys t i n o r me d i u m a l one (Fig. 5 A) . We also as-
sayed ext r act s o f i nf e c t e d cells f or t he pr e s e nc e o f t r ansi al i -
dase, an e n z y me expr es s ed onl y i n t r ypoma s t i got e s . I n c ul -
t ur es t r e a t e d wi t h cl as t o- l act acys t i n o r me d i u m al one, t he
1913 Gonzfilez et al.
A
B
15
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 s 0 0
lOOO ~
|
l i
0
4 5
lOO 1 o
S
a
o
010
0 05
0 O0
C o~o
o
o
=E 0 0 5
8
o 0 0 +
10 20 30
F r a c t i o n N u m b e r
=~ 0 8
o 6 ~
so ~ g
6
0 4 ~
.+.
0 O 0
40 50
10 20 30
F r l c U o n N u m b e r
120
|
8
J
i l - o
4 O
- 8 O 0 1 5
- 0 0 0
~o
- 4 0 0
- 2 0 0 ~
- 0 0 0
Figure 7. Purification and charactenzation of T. cmzi proteasomes. (A)
Gel filtrat]on on Superose 6. The chymotrypsm activity in fractions 17-24
was totally inhibited by lactacystm but unaffected by E-64. (B) Amon-
exchange chromatography of pooled fractions 17-24 on a Mono Q col-
umn. Bound proteins were eluted using a 0-1 mM KC1 hnear gra&ent.
Fractions that &splayed Ch-L actlv]ty that was mhlbltable by lactacystin,
but not by E-64, were eluted at approxamately 400-500 mM KC1. (C)
Gel Fdtration on Superose 6. Fracnons eluted from the Mono Q at 400-
500 mM KC1 were loaded onto Superose 6 16/ 30. Proteolytlc activities
under the major protein peak were measured wath the following fluoro-
gemc peptldes: Suc-Leu-Leu-Val -Tyr-MCA for Ch-L activity (Ch-L),
Boc-Leu-Arg-Arg-MCA for T-L actlvxty (T-L) and Z- Leu- Leu- Gl u-
[JNA for peptldylglutamyl peptide hydrolase (PGPH). All actlvmes were
strongly inhibited by lactacystm but not by E-64.
e xpr e s s i on of t r a ns i a l i da s e starts 80 h af t er i nf e c t i on, a nd i n -
creases unt i l t he e n d o f i nt r acel l ul ar par asi t e di f f er ent i at i on.
I n l a c t a c ys t i n- t r e a t e d cul t ur es , t he e xpr e s s i on o f t r ansi al i -
dase was i n h i b i t e d (Fig. 5 B). Fi nal l y, o n e set o f i nf e c t e d
cel l s was s t ai ned 90 h af t er i n f e c t i o n a nd e x a mi n e d b y l i ght
mi c r o s c o p y . Wh i l e 90% p e r c e n t o f cel l s t r e a t e d wi t h l act a-
cys t i n c o n t a i n e d t ypi cal amas t i got es , a b o u t 80% o f my o -
blasts t r e a t e d wi t h cl as t o- l act acys t i n c o n t a i n e d t r y p o ma s t i -
gor e - l i ke or i nt e r me di a t e fl agel l at e f or ms (Fig. 6). Ana l ogous

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A
B
A
1
8O
~ 8o
..~ 40
~ ~o
~ o
F i g u r e 9.
B
100
Control
C la s to q a c ~ llc y s tln
L a c t a c y s t t n
In wvo and m wtro mhibitaon of T. c m z i proteasomes by lac-
tacystm. (A). Trypomasugotes were incubated for 3 h in transformataon
medium containing 10 ~M lactacystln ( s o h d bars), or clasto-lactacystln
(smped bars) or with medium alone (open bars). Samples of parasites (3
107) were washed with PBS, resuspended in 200 pJ of 20 mM Trls, sonl-
cated, and centrifuged. Supernatants were lmmunopreclpltated wath 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 additional controls for the specificity of the lmmuno-
prec~patatlon reaction, untreated parasites were sonlcated, treated with
lactacysm ( s o h d bars), or c l a s t o - l a c t a c y s t m (striped bars), or medium (open
bars) and lrmnunoprecipltated as 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-dlrnenslonal gel analysis of T . cruzi 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) Electronnncroscopy of T. c m z i proteasomes. Bar,
100 nm
experiments were performed wi t h the cell-permeant cys-
teine proteinase inhibitors E-64d (31) and Cbz- Phe- Al a-
FMK at concentrations of 10 /~M. They had no effect on
the transformation of intracellular amastigotes i nt o trypo-
mastigotes, or on the expression of transialidase (data not
shown).
I d e n t i f i c a t i o n o f t h e L a c t a c y s t i n T a r g e t i n T . c r u z i . We used
two approaches to identify the target oflactacystin in T . c m z i .
First, we isolated the lactacystin-inhibitable chymotrypsin
activity from crude extracts of parasite. As shown in Fig. 7 A,
a broad peak of chymotrypsin activity was detected follow-
i ng filtration of the extracts i n a Superose 6 col umn. How-
ever, only the activity in the shoulder peak (fractions 17-24),
cont ai ni ng proteins of higher molecular mass, was i nhi b-
itable by lactacystin, but not by E-64. In later fractions the
chymotryptic activity was i nhi bi t ed by E-64 but not by lac-
tacystin. The lactacystin-inhibitable fractions were t hen
subjected to ani on-exchange chromatography i n a Mono
Q col umn. A peak of chymotrypsin activity that was i nhi b-
ited by lactacystin eluted at 400-450 mM of KC1 (Fig. 7 B).
Pool ed fractions from this peak were t hen filtered t hrough
another Superose 6 column. A major symmetrical OD peak
of 670 kD was eluted from the col umn. It cont ai ned the
three characteristic peptidase activities of eukaryotic pro-
teasomes, T-L, Ch-L, and PGPH (Fig. 7 C). All activities
were inhibitable by lactacystin. Usi ng Suc- Leu- Leu- Val -
Tyr - AMC as a substrate, the specific activity of the Ch-L
activity was 1.5 ~M/ mg/ h. At concentrations up to 50
p~M, the cruzain inhibitors Cbz- Phe- Al a- FMK and Cbz-
( S- Bz) Cys- Phe- CHN 2 did not affect the Ch-L activity of
the purified proteasomes.
Using SDS-PAGE under denaturing conditions the 670 kD
molecules were resolved into subunits with molecular masses
bet ween 25-35 kD. By isoelectrofocusing, their isoelectric
points varied bet ween 4.5 and 8.5 (Fig. 8 A). The NHz-t er-
minal prot ei n sequence of the prot ei n from one band (TSI-
MAVTFKD) is identical to that of the [3-subunit of PRE3,
a PGPH activity from yeast proteasomes (32). Electron mi -
croscopy of negatively stained preparations revealed charac-
teristic images ofproteasomes, i.e., hol l ow cylinders 18 nm
i n length and 12-15 nm i n diameter (Fig. 8 B).
To identify the target of lactacystin in vivo, we i ncu-
bated samples of trypomastigotes for 2 h i n transformation
medi um i n the presence oflactacystin, clasto-lactacystin, or
medi um alone. The parasites were washed, and sonicated
1914 Proteasome Control of Morphology of T . c r u z i

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Published November 1, 1996
extracts were i mmunopreci pi t at ed wi t h a rabbit antiserum
to purified T. cruzi proteasomes, or wi t h normal rabbit se-
rum. Immunoprecipitates were t hen assayed for chymo-
trypsin activity. As shown in Fig. 9 A, the i mmunopreci pi -
tared proteasomes from parasites that had been incubated
with lactacystin were inactive. The control i rnmunopreci p-
itates from parasites treated with medi um alone or clasto-
lactacystin had Ch- L activity that was inhibited by lactacys-
tin, but not by E-64. No enzymatic activity was detected in
samples immunoprecipitated with normal rabbit serum. As
additional controls of the specificity of the i mmunopreci pi -
tation, trypomastigote extracts were treated with lactacystin
or clasto-lactacystin and then immunoprecipitated as de-
scribed above. The immunoprecipitates originating from
extracts treated wi t h lactacystin were inactive (Fig. 9 B)
D i s c u s s i o n
We show here that the proteasome inhabitors MG132
and lactacystin prevent ed the transformation of trypomas-
tigotes into amastigotes an axenic medi um. MG132, a pep-
tide aldehyde, also potently inhibits cysteine proteases, but
lactacystin selectively inhibits the peptidase activity of pro-
teasomes. The transient intermediate of lactacystin, clasto-
lactacystin 13 lactone, binds tightly to threonines in the active
site of the [3 subunits ofprot easomes (24, 33). Clasto-lacta-
cystin di hydroxy acid (Fig. 2 B), the product of hydrolysis
of the active 13 lactone, had no activity in parasite transfor-
mation. Lactacystin does not inhibit serine or cysteine pro-
teases of mammalian cells (24), and did not affect the activ-
aty of cruzain, the major T. cruzi lysosomal enzyme. We
further ascertained that proteasomes are the targets of lacta-
cystin in trypomastigotes by t wo i ndependent criteria. First,
proteasomes were isolated to apparent homogenei t y from
crude extracts of parasites using a lactacystin-based assay to
follow purification. Second, while immunoprecipitates of
proteasomes present in extracts of clasto-lactacystin-treated
parasites had Ch- L activity, the immunoprecipitates from
lactacystin-treated parasites were inactive.
We also studied the effect oflactacystin on the infectivity
of T. cmzi trypomastigotes to myoblasts. In these experi-
ments, we tried to minimize or exclude possible effects of
the drug on the target cells. For example, when studying
the attachment and penetration phases of infection, drug-
treated parasites were washed before incubation with the
myoblasts. We found that lactacystin had no effect on inva-
sion, an active process that requires parasite energy (34),
and is associated wi t h calcium fluxes in the parasite (35).
However , the intracellular devel opment of the lactacystin-
treated parasites was arrested. It cannot be deduced from
these results whet her lactacystin inhibited only the t rypo-
mastigote/amastigote transformation. Ther e is a distinct
possibility that lactacystin inhibited amastigote proliferation
as well, since the eukaryotic cell cycle is regulated by pr o-
teasomes. In any case, these experiments also show that the
effects oflactacystin persisted during the intracellular devel-
opment of the parasite. Lactacystin is an irreversible inhibi-
t or ofprot easomes, and the half-life of T. cruzi proteasomes
may be long. Alternatively, drug treatment may have irre-
versibly affected a prot easome-dependent essential parasite
function.
Lactacystin also prevent ed the transformation of amasti-
gotes into trypomastigotes that occurs at the end of the in-
tracellular phase. In these experiments, myoblasts infected
48 h previously with trypomastigotes were exposed for 2 h
to 1-3 p~M of lactacystin. The effect was striking: as com-
pared with clasto-lactacystin-treated cells, the lactacystin-
treated cells released fewer trypomastigotes into the culture
medium, contained more amastigotes in their cytoplasm,
and displayed much less transialidase activity. In contrast,
higher concentrations of cell-permeant inhibitors of crnzi-
pain had no effect on the amastigote/trypomastigote trans-
formation. The small concentrations oflactacystin used, the
short durataon of drug treatment, the specificity of the ob-
served effects, and the lack of effect of cysteine protease in-
hibitors argue strongly that the prime targets of lactacystin
are the transforming parasites rather than the myoblasts.
These results show that proteasome activity is necessary
for remodeling, but the substrates that are degraded have
not been identified. They probably include proteins that
maintain the old shape, most likely cytoskeletal elements,
a set of proteins and enzymes i nvol ved in the old met a-
bolic pathways, and stage-specific surface proteins. In addi-
tion to these housekeeping functions, the cleavage of key
regulatory proteins by proteasomes may provi de the cen-
tral switching mechanism that initiates the stage-specific
changes (36).
In eukaryotic cells, the substrates destined for degrada-
tion are recognized by specific E2- E3 Ub- pr ot ei n ligases
(37). However , very little is known about the Ub- pr ot ea-
some system in prot ozoan parasites. Southern and Nor t h-
ern blots of DNA and RNA from various strains of T. cruzi
revealed large variations in the number of Ub genes (38).
Its genome may contain more than 100 Ub coding se-
quences, a number much larger than in ot her organisms.
These are encoded in five pol yUb genes and five Ub fusion
genes, whose transcription is altered under stress condi -
taons. Ther e is a significant increase in steady-state levels of
Ub mRNA bet ween the midlog phase cultures of noni n-
fective epimastigotes of T. cruzi, and the stationary phase
cultures that contain the morphologically distinct, infective
metacyclics (39). It is not ewor t hy that heat -shock elements
are present in the intergenic regions preceding the pol yUb
genes. Perhaps the expression of the Ub genes in T. cruzi is
regulated by the shifts in environmental pH and tempera-
ture, and by ot her stress conditions that lead to stage-spe-
cific remodeling. In yeasts that bear mutations in protea-
somes, sensitwity to stress is increased, and under stress
conditions the mutants accumulate ubiqmtinated proteins.
Ot her proteases have been identified in T. cruzi (40-42).
One of them, cruzain, a lysosomal cathepsin L-like cysteine
protease, also plays a role in growt h and differentiation of
the parasate (28-30). Studies in different laboratories have
shown that synthetic inhibitors of cruzain, including Cbz -
1915 Gonzfilez et al.

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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 nhi bi t
T. cruzi i nf ect i vi t y. H o w e v e r , di f f er ent f r o m l act acyst i n, t he
c ys t e i ne pr ot e a s e i nhi bi t or s p r e v e n t par as i t e p e n e t r a t i o n
i nt o t he h e a r t mu s c l e cells (28). As s h o wn he r e , r el at i vel y
h i g h c o n c e n t r a t i o n s o f C b z - P h e - A l a - F N I K a nd C b z - ( S -
B z ) - C y s - P h e - C H N 2 di d n o t affect t he r e mo d e l i n g o f T.
cruzi i n a xe ni c m e d i u m o r i nsi de cells. Al t h o u g h o u r f i nd-
i ngs do n o t e x c l u d e a r ol e f or c r uz a i n a nd o t h e r l ys os os mal
e n z y me s i n t he e xt e ns i ve pr ot e ol ys i s t ha t mu s t a c c o mp a n y
r e mo d e l i n g , t h e y a r gue t ha t t he r ol e o f c r uz a i n is n o t p i v -
ot al d u r i n g t hes e phas es o f par as i t e d e v e l o p me n t .
S o me publ i c a t i ons r e p o r t t he p r e s e n c e o f p r o t e a s o me s i n
Trypanosoma (43, 44) a nd Entamoeba (45), b u t t h e i r f u n c t i o n
has n o t b e e n st udi ed. We f o u n d t ha t t he s t r uct ur al f eat ur es
a n d a r c h i t e c t u r e o f t he T. cruzi p r o t e a s o me s we r e s i mi l a r
t o t hos e o f o t h e r speci es. By S D S - P A G E t he cyl i ndr i cal
20S s t r uc t ur e wa s r e s ol ve d i n t o t he t ypi cal 6 - 8 ba nds o f
2 5 - 3 5 k D. H o w e v e r , mo r e t h a n 20 pr ot e i ns , wi t h wi d e l y
di ve r s e pl s , we r e s e e n i n T. cruzi pr ot e a s or ne s a na l yz e d b y
t wo - d i me n s i o n a l PAGE. I t as gener al l y accept ed t hat t he 20S
p r o t e a s o r n e is a d i me r o f 14 s ubuni t s a r r a nge d otT[37137oL 7. I n
t he ye a s t Saccharomyces cerevisiae t h e r e ar e f o u r t e e n ge ne s e n -
c o d i n g 7 ot a nd 7 [3 s ubuni t s , a nd t he d e n d r o g r a m r e p r e -
s e nt i ng t he a l i g n me n t s o f all e u k a r y o t i c p r o t e a s o me s e -
q u e n c e s yi el ds o n l y 14 s u b g r o u p s c o n t a i n i n g a si ngl e yeas t
me mb e r . T h e e x p l a n a t i o n f o r t he l ar ge n u m b e r o f T. cruzi
p r o t e a s o me - a s s o c i a t e d pr ot e i ns ma y b e trivial: s o me e xt r a
spot s c o u l d r e p r e s e n t pos t t r a ns l a t i ona l mo 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 mpl y c o n t a mi n a n t s . Al t e r na t i ve l y, an u n -
usual f e a t ur e o f T. cruzi is t ha t its pr ot e i ns ar e f r e q u e n t l y
e n c o d e d b y sever al t a n d e ml y a r r a nge d ge ne s t ha t ar e p o l y -
cl s t r oni cal l y t r a ns c r i be d f r o m a si ngl e p r o mo t e r a nd ar e
c o n c u r r e n t l y expr es s ed. Se q u e n c e va r i a t i on o f ge ne s f o u n d
i n o n e s uch t r a ns c r i pt i on uni t c o u l d add t o t he 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 . Fu r t h e r st udi es ar e neces s ar y t o cl ar -
i f y t hi s issue.
T h e p r e s e n t p a p e r d e mo n s t r a t e s t ha t p r o t e a s o me act i vi t y
is essent i al f o r T. cruzi r e mo d e l i n g . Ve r y si mi l ar resul t s
we r e r e c e n t l y o b t a i n e d wi t h o t h e r p r o t o z o a n parasi t es. I n a
r o d e n t mal ar i a mo d e l Si nni s, P. , B. Gu t i e r r e z , M. Br i ones,
and V. Nu s s e n z we r g ( manuscr i pt i n pr epar at i on) s h o we d t ha t
l act acys t i n di d n o t p r e v e n t t he p e n e t r a t i o n o f t he 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 ongl y i n h i b i t e d t h e i r t r a n s f o r ma t i o n i nt o t he r o u n d
h e p a t o c y t e st ages a nd s u b s e q u e n t d e v e l o p me n t . Ei c hi nge r ,
D. , V. Nu s s e n z we i g , a nd J. Go n z a l e z ( ma n u s c r i p t i n p r e p a -
r at i on) d e mo n s t r a t e d t ha t l act acys t i n p r e v e n t e d t he e nc ys t a -
t i o n o f Entamoeba invadens. Trypanosoma, Entamoeba, a nd
Plasmodium b e l o n g t o p h y l a wi d e l y s e pa r a t e d i n e v o l u t i o n .
Th e r e f o r e , i t is l i kel y t ha t t he me c h a n i s ms g o v e r n i n g st age-
speci fi c mo r p h o l o g i c a l changes i n p r o t o z o a ar e c o n s e r v e d ,
a nd p r o t e a s o me - d e p e n d e n t , a nd t hat p r o t e a s o me i nhi bl t or s
ma y h a v e a b r o a d r a n g e o f t ar get s. En c o u r a g i n g f eat ur es f o r
a t t e mp t i n g t o d e v e l o p t hi s class o f c h e mo t h e r a p i c agent s
ar e t ha t s o me parasi t es, s uc h as Plasmodium, u n d e r g o c o n -
st ant a nd r a pi d r e mo d e l i n g i n t he ma mma l i a n host . Th u s ,
ef f ect i ve dr ugs n e e d n o t b e a d mi n i s t e r e d f o r p r o l o n g e d p e -
r i ods o f t i me t o ar r est par asi t e d e v e l o p me n t . F u r t h e r mo r e ,
t he a c c ur a t e di s c r i mi na t i on b e t we e n t he ol d a nd n e w p r o -
t ei ns t ha t c oe xi s t wi t h i n t he s a me cel l d u r i n g r e mo d e l i n g o f
p r o t o z o a ma y r e q u i r e s peci al i zed f eat ur es o f t he p r o t e a -
s o m e / U b s ys t em. P r o t e a s o me s f r o m i nt r acel l ul ar p r o t o z o a n
par asi t es ma y al so di f f er s i gni f i cant l y i n s t r uc t ur e f r o m t hos e
o f t he hos t cell, r e n d e r i n g t he i nf e c t e d cells s us cept i bl e t o
d e s t r u c t i o n b y cells o f t he i m m u n e s ys t em. Ho p e f u l l y ,
s o me o f t hes e a p p r o a c h e s t o t h e r a p y wi l l yi e l d t o e x p e r i -
me n t a l at t ack.
We are grateful to Drs. Christopher Cardozo and Martin Rechst ei ner for helpful discussions and providing
reagents. We thank Dr. Daniel E~chmger for helping wi t h the transialidase assays and for revtewing the
manuscript. We thank Mrs. Bessy Gutierrez for the preparation o f figures.
This wor k was supported by grants from the National Institutes of Health to V. Nussenzweig, and E.J. Co-
rey. J. Gonzfilez has been supported by a postdoctoral fellowship from the Pew Latin American Fellows Pr o-
gram. F.J. Ramal ho- Pi nt o was supported by CNPq (Brazil).
Address correspondence to Dr. Jorge Gonzfilez, Depart ment of Pathology, Michael Helldelberger Division
of Immunol ogy, 550 First Ave., Ne w York, NY 10016.
Received for publication 19 August 1996 and in revised form 5 September 1996.
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1918 Proteasome Control of Morphology of T. cruzi

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