A Role for the Protease Falcipain 1 in Host Cell Invasion by the

Human Malaria Parasite

Doron C. Greenbaum et al.
Science 298, 2002 (2002);
DOI: 10.1126/science.1077426

This copy is for your personal, non-commercial use only.

If you wish to distribute this article to others, you can order high-quality copies for your
colleagues, clients, or customers by clicking here.

Downloaded from www.sciencemag.org on November 1, 2013

Permission to republish or repurpose articles or portions of articles can be obtained by
following the guidelines here.

The following resources related to this article are available online at

www.sciencemag.org (this information is current as of November 1, 2013 ):

Updated information and services, including high-resolution figures, can be found in the online
version of this article at:
http://www.sciencemag.org/content/298/5600/2002.full.html
Supporting Online Material can be found at:
http://www.sciencemag.org/content/suppl/2002/12/04/298.5600.2002.DC1.html
This article cites 23 articles, 9 of which can be accessed free:
http://www.sciencemag.org/content/298/5600/2002.full.html#ref-list-1
This article has been cited by 134 article(s) on the ISI Web of Science
This article has been cited by 22 articles hosted by HighWire Press; see:
http://www.sciencemag.org/content/298/5600/2002.full.html#related-urls
This article appears in the following subject collections:
Microbiology
http://www.sciencemag.org/cgi/collection/microbio

Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the
American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright
2002 by the American Association for the Advancement of Science; all rights reserved. The title Science is a
registered trademark of AAAS.
 REPORTS
are the target molecules that undergo the amount of TAF10, which probably forms the R. G. Roeder, Science 292, 1153 (2001); published
online 12 April 2001 (10.1126/science.1059188).
additional poly(A) extension by TPAP in functional TFIID complex, seems to be still
11. W. Deng, H. Lin, Dev. Cell 2, 819 (2002).
round spermatids. present in the nucleus of round spermatids in 12. K. C. Kleene, Development 106, 367 (1989).
It has been demonstrated that the deficiency Tpap–/– mice (Fig. 3, C and D). Even if this is 13. S. Y. Han et al., Biol. Reprod. 64, 507 (2001).
of cytoplasmic polyadenylation element–bind- so, round spermatids appear to be incapable of 14. L. Tora, Genes Dev. 16, 673 (2002).
15. S. Kashiwabara, Y. Arai, K. Kodaira, T. Baba, Biochem.
ing protein (CPEB) results in the arrests of producing enough mRNA to advance cell mor- Biophys. Res. Commun. 173, 240 (1990).
oogenesis and spermatogenesis at the pachytene phogenesis, because gene transcription (RNA 16. S. Kashiwabara et al., data not shown.
stages (17). Although expression of two cyto- synthesis) ceases around step-10 spermatids in 17. J. Tay, J. D. Richter, Dev. Cell 1, 201 (2001).
18. D. Metzger, E. Scheer, A. Soldatov, L. Tora, EMBO J.
plasmic polyadenylation element–containing the mouse (24). Our study shows a direct link 18, 4823 (1999).
mRNAs coding for the synaptonemal complex between the deficiency of a cytoplasmic 19. S. R. Albright, R. Tjian, Gene 242, 1 (2000).
proteins SCP1 and SCP3 are unaffected by the poly(A) polymerase, TPAP, and the arrest of 20. Z. Chen, J. L. Manley, Mol. Cell. Biol. 20, 5064 (2000).
21. R. N. Freiman et al., Science 293, 2084 (2001).
CPEB deficiency, the poly(A) tails are reduced mouse spermiogenesis, providing information 22. M. A. Hiller, T.-Y. Lin, C. Wood, M. T. Fuller, Genes
in length, and the protein products are missing in on the regulation of haploid-specific genes by Dev. 15, 1021 (2001).
CPEB-deficient mice. In this study, the expres- cytoplasmic polyadenylation in male germ 23. G. M. Fimia, A. Morlon, B. Macho, D. De Cesare, P.
sion levels and sizes of CPEB, SCP1, and SCP3 cells. Sassone-Corsi, Mol. Cell. Endocrinol. 179, 17 (2001).
24. A. L. Kierszenbaum, L. L. Tres, J. Cell Biol. 65, 258
mRNAs in Tpap–/– testes were similar to those (1975).
in Tpap⫹/⫹ and Tpap⫹/– testes (Fig. 3B). More- 25. We thank L. Tora, I. Davidson, and T. Tamura for
over, no significant difference of the poly(A) tail References and Notes providing antibodies. This study was partly supported
1. A. B. Sachs, P. Sarnow, M. W. Hentze, Cell 89, 831 by Grant-in-Aids for Scientific Research on Priority
size of SCP3 mRNA in pachytene spermato- (1997). Areas (A) and (B), Scientific Research (A), and Explor-
cytes was found between Tpap⫹/⫹ and Tpap–/– 2. M. Wickens, P. Anderson, R. J. Jackson, Curr. Opin. atory Research from the Japan Society for the Pro-
mice. These data suggest that CPEB is not in- Genet. Dev. 7, 220 (1997). motion of Science and the Ministry of Education,
3. N. B. Hecht, BioEssays 20, 555 (1998). Culture, Sports, Science and Technology in Japan.
volved in the TPAP-mediated cytoplasmic poly-
4. K. Steger, Anat. Embryol. 199, 471 (1999). Supporting Online Material
adenylation. The null mutation of the TPAP 5. S. Kashiwabara et al., Dev. Biol. 228, 106 (2000). www.sciencemag.org/cgi/content/full/298/5600/1999/
gene does not affect the size or expression level 6. Materials and methods are available as supporting DC1
of mRNAs encoding ovary-specific zona pellu- material on Science Online. Materials and Methods
7. F. Nantel et al., Nature 380, 159 (1996). Figs S1 to S4
cida 1 (ZP1) and ZP2 (6) (fig. S3), verifying the 8. J. A. Blendy et al., Nature 380, 162 (1996). References and Notes
testis-specific function of TPAP. 9. I. Martianov et al., Mol. Cell 7, 509 (2001).
Westen blot analysis of cytoplasmic and 10. D. Zhang, T.-L. Penttila, P. L. Morris, M. Teichmann, 3 June 2002; accepted 16 October 2002
nuclear protein extracts from testicular tis-
sues revealed that the level of TAF10 was
reduced only in the nuclear fraction of
Tpap–/– testes, whereas TBP, TAF12,
A Role for the Protease
TAF13, and TRF2 (16) were equally present
in the cytoplasmic or nuclear fractions of
Falcipain 1 in Host Cell Invasion
Tpap⫹/⫹ and Tpap–/– testes (Fig. 3C). The
specific reduction of TAF10 was verified by by the Human Malaria Parasite
immunoprecipitation analysis of the nuclear
extracts with antibody to TBP (Fig. 3D). Doron C. Greenbaum,1* Amos Baruch,2 Munira Grainger,4
These data demonstrate that TPAP affects the Zbynek Bozdech,2 Katlin F. Medzihradszky,1 Juan Engel,3
transport of at least TAF10 into the nucleus, Joseph DeRisi,2 Anthony A. Holder,4 Matthew Bogyo2*
possibly by additional polyadenylation-de-
pendent translational activation of dormant Cysteine proteases of Plasmodium falciparum are required for survival of the
mRNA encoding a transporter protein or pos- malaria parasite, yet their specific cellular functions remain unclear. We used a
sibly by TPAP itself. Moreover, the poly(A) chemical proteomic screen with a small-molecule probe to characterize the pre-
tails of TAF10, TAF12, and TAF13 mRNAs dominant cysteine proteases throughout the parasite life cycle. Only one protease,
are unlikely to contribute to stability and falcipain 1, was active during the invasive merozoite stage. Falcipain 1–specific
translational control, because the levels of inhibitors, identified by screening of chemical libraries, blocked parasite invasion of
these three mRNAs and cytoplasmic proteins host erythrocytes, yet had no effect on normal parasite processes such as hemo-
in Tpap–/– testes are comparable to those in globin degradation. These results demonstrate a specific role for falcipain 1 in host
Tpap⫹/⫹ testes, despite the incomplete elon- cell invasion and establish a potential new target for antimalarial therapeutics.
gation of poly(A) tails (Fig. 3, A and C).
Several TAFs, including TAF10, as a com- Malaria is a devastating disease that affects lines and antifolates are the most common
ponent of the TFIID complex have been dem- 300 to 500 million people and kills about 2 drugs for disease prevention and cure.
onstrated to be dispensable for general RNA million people per year. Currently, quino- However, multidrug resistance is a major
polymerase II–mediated transcription and to be issue, highlighting the need for new anti-
essential for selective expression of specific malarial drugs to combat this parasite. Pro-
1
Department of Pharmaceutical Chemistry, 2Depart-
genes (14, 18 –22). Because the TFIID complex ment of Biochemistry and Biophysics, 3Department of teases represent one of the largest families
containing TAF10 is severely impaired in Pathology, Veterans Affairs Medical Center, Universi- of potential therapeutic targets, and cys-
Tpap–/– testes by insufficient transport of ty of California, San Francisco, CA 94143, USA. 4Di- teine proteases have been shown to be es-
TAF10 into the nucleus (Fig. 3, C and D), it is vision of Parasitology, National Institute for Medical sential for the survival of several human
Research, Mill Hill, London NW7 1AA, UK.
conceivable that TAF10 may play an important parasites (1–3). Cysteine proteases have
role in the expression of a subset of haploid- *To whom correspondence should be addressed at M. been specifically implicated in several cel-
Bogyo, University of California, San Francisco, 513
specific genes, possibly as a CREM coactivator Parnassus Avenue, San Francisco, CA 94043, USA.
lular functions during the P. falciparum life
(23), required for the morphogenetic program E-mail: [email protected] (D.C.G.) and mbogyo@ cycle, including hemoglobin degradation
during spermatogenesis. However, a small biochem.ucsf.edu (M.B.) (4–5), cleavage of red blood cell ankyrin to

2002 6 DECEMBER 2002 VOL 298 SCIENCE www.sciencemag.org

 REPORTS
facilitate host cell rupture (6 ), and the con- protease was identified as falcipain 1. Al- soluble and -insoluble lysates from each
comitant release of parasites from the para- though falcipain 1 was the first cysteine stage with 125I-DCG-04 (Fig. 1C). The ac-
sitophorous vacuole (7 ). Furthermore, cys- protease gene cloned from the P. falcipa- tivity profiles of falcipains 1, 2, and 3
teine proteases are important for host cell rum genome (18), difficulties in recombi- indicated that regulation of these enzymes
invasion by P. falciparum and other related nant expression (19) have prevented its de- is highly divergent (Fig. 1D). Falcipain 2
parasites (8, 9). However, the lack of a tailed biochemical and functional charac- and 3 activities peaked at the trophozoite
technically facile method to genetically terization. Thus, falcipain 1 is an ideal stage, which is consistent with previously
manipulate the parasite has made function- target for in situ studies with pharmacolog- reported protein levels and with a role for
al analysis of key proteases difficult. ical tools to perturb its function. these enzymes in hemoglobin degradation
P. falciparum has a complex life cycle To gain insight into the functional roles (4, 5). In contrast, falcipain 1 activity
involving two distinct sexual and asexual of specific cysteine proteases, we used peaked during the merozoite stage and was
stages of growth. The human asexual eryth- highly synchronized parasite populations to the predominant protease activity in both
rocytic phase (blood stage) is the cause of profile protease activities throughout the the merozoite and the ring stages (Fig. 1D).
most malaria-associated pathology and multiple developmental stages of the para- This activity profile differs from the report-
therefore is the focus of this study. The site. For each stage, extracts were generat- ed expression of falcipain 1 mRNA only in
blood stage begins when merozoites (ini- ed at pH 5.5, although analysis of falcipain ring-stage parasites. This difference can be
tially released from the liver) invade red 1, 2, and 3 activity indicated a broad pH explained by both translational and post-
blood cells. Over the next 48 hours, inter- activity profile. Cysteine protease activity translational mechanisms that control the
nalized parasites differentiate (ring stage), was determined by labeling both detergent- maturation of falcipain 1 throughout the
metabolize hemoglobin (trophozoite stage),
and replicate (schizont stage) to produce
expanded populations of invasive merozo-
ites that are released upon rupture of the
host cell (10). Merozoites have a limited
life-span outside the host cell and must
immediately find new cells to invade and
start the cycle anew.
Initially, we used a functional proteomic
method to identify and biochemically ana-
lyze all cysteine protease activity in ex-
tracts of P. falciparum (11–13). Four pro-
tease activities were detected in whole-cell
lysates from mixed blood stages of P. fal-
ciparum parasites with the radiolabeled
cysteine protease probe 125I-DCG-04 (Fig.
1A). One protease activity was enriched in
an NP-40 –insoluble pellet fraction, where-
as the remaining protease activities re-
mained soluble, suggesting distinct local-
ization and/or biochemical properties of
these enzymes.
The biotin affinity tag of DCG-04 af-
forded a single-step purification of all la-
beled proteins and their subsequent identi-
fication by mass spectrometry– based se-
quencing (Fig. 1B). Each of the proteases
identified was a member of the papain fam-
ily of cysteine proteases. Human calpain 1
was isolated from the NP-40 –insoluble
Fig. 1. Identification and biochemical characterization of active cysteine proteases within the
fraction but is not thought to play a func- erythrocytic cycle of P. falciparum. (A) Asynchronous cultures of P. falciparum parasites were
tional role in P. falciparum (14 ). It is un- isolated after sorbitol lysis of host red blood cells. Parasite pellets were lysed by addition of
likely that this protease was purified as a 0.2% NP-40, and total cellular extracts were labeled with 125I-DCG-04. Homogenates were
result of contamination by red blood cells, separated on a 15% polyacrylamide gel, and labeled polypeptides were visualized with a
because red blood cells were lysed before PhosphorImager (total lysate lane). DCG-04 –labeled parasite extracts were separated into
isolation of proteases with the probe. The 0.2% NP-40 soluble and insoluble fractions. Fractions were separated on 15% polyacrylamide
gel and visualized by phosphoimaging. (B) Identification of affinity-labeled proteases. Parasite
purified cathepsin C–like protease matched culture (1 liter) was harvested, lysed, and labeled with nonradioactive DCG-04. Probe-labeled
a sequence found in the P. falciparum ge- proteases were affinity-purified by a single-step affinity purification protocol (9). Isolated
nome database (locus IDMal12P1.457) proteases were excised by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and identified
(15–17 ); however, no biological function by in-gel trypsin digestion followed by mass spectrometry–based sequencing. Targets were
for this enzyme has been reported. Falci- identified by database (PlasmoDB) matching of peptide sequences. Cat C, cathepsin C. (C)
pains 2 and 3 were also isolated from the Profiling protease activity throughout the erythrocytic life cycle. Synchronous parasites were
harvested at the ring, trophozoite (troph), schizont (schiz), and merozoite (mer) stages. Lysates
NP-40 –soluble extracts. These proteases were separated into 0.2% NP-40 soluble and insoluble fractions and labeled with 125I-DCG-04.
reside in the food vacuole, where they play (D) Falcipain 1 and 2/3 protease bands from (C) were quantified with the ImageQuant software
a critical role in hemoglobin degradation (Molecular Dynamics) and plotted for each of the major blood stages. Data for falcipain 1
(4, 5). The remaining, detergent-insoluble represent a composite of insoluble and soluble activities.

www.sciencemag.org SCIENCE VOL 298 6 DECEMBER 2002 2003

 REPORTS
life cycle of the parasite. This activity pro-
file suggests a primary function for falci-
pain 1 either in red blood cell rupture or
during reinfection of new host red blood
cells.
In addition to having a distinct activity
profile, falcipain 1 also changed its subcel-
lular location during the late schizont and
merozoite stages, as indicated by the ap-
pearance of an NP-40 –soluble active form
(Fig. 1C, far right lane). This soluble activ-
ity was most abundant in the merozoite
stage and absent at the ring stage. During
merozoite development, several new sub-
cellular compartments are created that com-
pose the apical organelles. These organelles
participate in the process of host cell inva-
sion through a controlled secretion of their
contents (20). Localization of falcipain 1 to
Fig. 2. Localization of falcipain 1 in merozoites by immunofluorescence imaging. Samples of
merozoites were smeared and fixed in 1% paraformaldehyde. The slides were incubated with specialized compartments such as these
an antibody to falcipain 1 or with antibodies specific to the rhoptry (RopH2) and microneme may explain its change in detergent solu-
(EBA-175) proteins. Nuclei were stained with 4⬘,6-diamidino-2-phenylindole (DAPI). The lack bility. Furthermore, the short-lived, soluble
of colocalization indicates that falcipain 1 is found in compartments at the apical end of the falcipain 1 activity in merozoites could be
merozoite that are distinct from the rhoptries and micronemes. explained by a mechanism in which active
falcipain 1 is secreted during invasion.
To better understand the location and

Fig. 3. Identification of
falcipain 1–specific in-
hibitors by screening
peptide epoxide posi-
tional scanning libraries
(PSLs) in crude cell ex-
tracts. (A) Structures of
the general inhibitor
scaffolds used for
screening. A single ami-
no acid position (P2)
was held constant
whereas the remaining
positions contained an
isokinetic mixture of natural amino ac-
ids (Mix positions). A set of 19 natural
and 41 nonnatural amino acids were
used at the P2 position to generate the
libraries. A second set of P2 diverse
sublibraries were generated with the
enantiomeric form (2R, 3R in place of
2S, 3S) of the epoxide building block.
(B) Colorimetric cluster display of inhi-
bition data. PSLs were screened against
P. falciparum lysates by pretreatment
of samples with individual constant P2
libraries followed by labeling with 125I-
DCG-04. Labeling intensity of each tar-
get relative to the control untreated
sample was used to generate percent
competition values. The resulting data
were clustered and visualized with pro-
grams designed for analysis of microar-
ray data. Proteases are arrayed along
the y axis and inhibitors are arrayed
along the x axis. Natural amino acids
are indicated by standard one-letter
codes and nonnatural amino acids are
labeled with the NN prefix [for struc-
tures and corresponding number as-
signment see (12, 13)]. Libraries generated with natural amino acids in the P2 and YAH-Eps(S,S)] identified from library screening. Compounds were added to
position using the (R, R) scaffold are indicated by the single-letter code followed total parasite extract for 30 min followed by labeling with 125I-DCG-04 for 1
by (R, R). (C) Competition analysis of a negative control compound [YAG- hour. Samples were separated on a 15% SDS-PAGE gel. Comp, compound; Conc,
Eps(S,S)] and three falcipain 1–specific compounds [YA2-Eps(S,S), YA29-Eps(S,S), concentration; Falc, falcipain; hCalp, human calpain.

2004 6 DECEMBER 2002 VOL 298 SCIENCE www.sciencemag.org

 REPORTS
cellular function of falcipain 1, we used 1 are distinct from the rhoptries and mi- typic evaluation at specific stages within
immunofluorescence-based localization cronemes, yet localized to the apical end of the life cycle of the parasite.
studies with an antibody generated with a the merozoite (Fig. 2, right panels). This To identify falcipain 1–specific inhibi-
unique, COOH-terminal peptide sequence unique staining of falcipain 1 implies local- tors, we screened a positional scanning li-
found in the mature enzyme. Immunoblots ization to the third set of apical organelles, brary of peptidyl epoxides (12, 13) in
confirmed that these antibodies specifically the dense granules, or to a subset of these whole parasite lysates. We generated librar-
recognized both the mature 26-kD enzyme organelles. The complement of subcellular ies by fixing a single amino acid residue on
and the larger proenzyme (21). The anti- compartments that compose the apical or- a tripeptide inhibitor scaffold while varying
bodies were then used in immunofluores- ganelles may be more diverse than previ- the remaining two positions (Fig. 3A). This
cence assays of synchronized cultures to ously thought, and falcipain 1 might be method produces a series of sublibraries
visualize the location of falcipain 1. Mero- located in a previously unidentified com- made up of several hundred compounds,
zoites stained with an antibody to falcipain partment. Ultimately, falcipain 1 localiza- each having a single different fixed amino
1 revealed punctate staining in distinct tion to the apical end of the merozoite acid residue (Fig. 3A). Inhibitor potency
vesicular structures at the apical end of the suggests a potential function in the invasion and selectivity were assessed by incubation
parasite, opposite the nucleus (Fig. 2, left of red blood cells. of lysates with each inhibitor sublibrary,
panels). This punctate falcipain 1 staining A more definitive analysis of falcipain 1 followed by reaction with the general cys-
pattern was also observed at the schizont function requires application of methods teine protease activity– based probe 125I-
stage, although it was excluded from the that allow inhibition or disruption of its DCG-04. The potency of specific inhibitor
area of the food vacuole, further supporting enzymatic activity in live parasite cultures. scaffolds was measured as a ratio of the
a functional divergence from its close rel- Gene ablation or knockout techniques have percent residual labeled proteases after in-
atives falcipains 2 and 3 (22). In addition, proven difficult in P. falciparum, and dele- hibitor treatment relative to an untreated
co-staining was performed with antibodies tion of many genes from the haploid ge- control. For analysis, the inhibition data
to the rhoptry protein RopH2 and to the nome results in a lethal phenotype. An were displayed in a colorimetric format and
micronemal protein EBA-175, two of three alternative chemical approach with a selec- clustered on the basis of similarities in
invasion-specific organelles present only in tive inhibitor that renders a specific target inhibitor profiles using software for mi-
merozoites (Fig. 2, middle panels). Merg- protease inactive allows dissection of the croarray analysis (23) (Fig. 3B). Several
ing of the two staining patterns indicated protease’s biochemical function. Such a falcipain 1–specific P2 amino acid residues
that the compartments containing falcipain “chemical genetic” approach allows pheno- were identified (Fig. 3B, black rectangles)
and were used to design a series of specific
inhibitors. These optimized inhibitors and a
control, inactive P2 glycine-containing in-
hibitor (Fig. 3B, yellow rectangle), were
assayed in parasite extracts over a range of
concentrations (Fig. 3C). The most selec-
tive of the resulting inhibitors, YA29-
Eps(S,S), showed greater than 25-fold se-
lectivity for falcipain 1 over all other cys-
teine proteases. Furthermore, at optimal
concentrations of 5 to 10 ␮M, we observed
selective inhibition of falcipain 1. The
compound YAG-Eps(S,S) showed no activ-
ity toward any of the protease targets and
therefore served as a control for in situ
inhibitor studies.
Effects of cysteine protease inhibition in
live cultures of P. falciparum were deter-
mined by treating parasites with the general
papain family protease inhibitor E-64d,
with the falcipain 1–specific inhibitor
YA29-Eps(S,S), or with the control inhib-
itor YAG-Eps(S,S) at 40 hours after inva-
sion. After rupture (10 to 12 hours later),
the percentage of surviving cells was cal-
culated by counting parasites. As predicted,
the control inhibitor YAG-Eps(S,S) had no
Fig. 4. Functional evaluation of falcipain 1–specific compounds in vivo. (A) Synchronous
parasites (5% parasitemia) were treated with E-64d (maroon bars), with the falcipain 1–
effect on parasite growth, as measured by a
specific inhibitor YA29-Eps(S,S) (blue bars), or with the control inhibitor YAG-Eps(S,S) (gray constant number of schizonts and levels of
bars) at the indicated concentrations 40 hours after invasion (late schizont stage). Smears were ring-stage parasites comparable to those of
prepared at 58 hours after invasion. Ring- and schizont-infected red blood cells were counted dimethyl sulfoxide–treated cultures (Fig.
as a percentage of total red blood cells. (B) Representative images of the parasites treated 4A, gray bars, and 4B, left panel). Inhibi-
at the highest concentration (10 ␮M) of inhibitors from (A). The control compound YAG- tion of all papain family proteases with
Eps(S,S) had no effect on the parasites, as indicated by the presence of rings. The enlarged
food vacuole of the schizonts in the E-64d–treated parasites (black arrowheads) is apparent.
E-64d resulted in a marked decrease in the
Falcipain 1–specific inhibitors such as YA29-Eps(S,S) blocked invasion but not host cell number of new ring-stage parasites (Fig.
rupture, as seen by the lack of ring-stage parasites with normal release of merozoites (open 4A, maroon bars). This decrease was dose
arrowheads). dependent and resulted from a block of

www.sciencemag.org SCIENCE VOL 298 6 DECEMBER 2002 2005

 REPORTS
parasite development at the late schizont ing the process of host cell invasion during the 12. D. Greenbaum et al., Mol. Cell Proteomics 1, 60 (2002).
stage, as measured by an increase in schi- erythrocytic cycle of P. falciparum. The prima- 13. D. Greenbaum et al., Chem. Biol., 9, 1085 (2002).
14. M. Hanspal, V. K. Goel, S. S. Oh, A. H. Chishti, Mol.
zonts. High concentrations of E-64d also ry sequence of falcipain 1 is well conserved Biochem. Parasitol. 122, 227 (2002).
induced a massive enlargement of the food across the Plasmodium genus (26), making it a 15. M. J. Gardner et al., Science 282, 1126 (1998).
vacuole within parasites, which is consis- potentially useful new target for design of ther- 16. S. Bowman et al., Nature 400, 532 (1999).
tent with previously reported effects of apeutic drugs in all four plasmodial species that 17. A. Bahl et al., Nucleic Acids Res. 30, 87 (2002).
18. P. J. Rosenthal, R. G. Nelson, Mol. Biochem. Parasitol.
general cysteine protease inhibitors (24 ) cause malaria in humans. Therapeutic agents 51, 143 (1992).
(Fig. 4B, middle panel). In contrast, falci- that are able to specifically prevent or slow the 19. F. Salas, J. Fichmann, G. K. Lee, M. D. Scott, P. J.
pain 1–specific inhibitors caused a similar process by which merozoites infect new cells Rosenthal, Infect. Immun. 63, 2120 (1995).
dose-dependent decrease in the percentage are likely to disrupt the development cycle and 20. C. E. Chitnis, M. J. Blackman, Parasitol. Today 16, 411
(2000).
of new ring-stage parasites (Fig. 4A, blue allow time for the host immune response to 21. D. C. Greenbaum, M. Bogyo, unpublished data.
bars), but did not block schizont develop- destroy the extracellular parasite. 22. D. C. Greenbaum, J. Engel, A. A. Holder, M. Bogyo,
ment and subsequent rupture as indicated unpublished data.
23. Cluster, version 2.11; TreeView, version 1.50, M. B.
by the appearance of merozoites (Fig. 4B, References and Notes Eisen, P. T. Spellman, P. O. Brown, D. Botstein, Proc.
right panel). Furthermore, schizont rupture 1. P. J. Rosenthal, G. K. Lee, R. E. Smith, J. Clin. Invest. Natl. Acad. Sci. USA. 95, 14863 (1998) (available at
was not affected, as assessed by the con- 91, 1052 (1993). http://rana.lbl.gov).
2. J. H. McKerrow, J. C. Engel, C. R. Caffrey, Bioorg. Med. 24. P. J. Rosenthal, J. H. McKerrow, M. Aikawa, H. Naga-
stant number of schizonts at all concentra- Chem. 7, 639 (1999). sawa, J. H. Leech, J. Clin. Invest. 82, 1560 (1988).
tions of falcipain 1–specific inhibitor (Fig. 3. J. C. Engel, P. S. Doyle, I. Hsieh, J. H. McKerrow, J. Exp. 25. D. Greenbaum, A. Holder, M. Bogyo, unpublished data.
4A) and by measurement of the release of Med. 188, 725 (1998). 26. P. J. Rosenthal, P. S. Sijwali, A. Singh, B. R. Shenai,
the parasitophorous vacuolar protein SERA 4. B. R. Shenai, P. S. Sijwali, A. Singh, P. J. Rosenthal, Curr. Pharm. Des. 8, 1659 (2002).
J. Biol. Chem. 275, 29000 (2000). 27. We thank J. McKerrow, P. Rosenthal, and C. C. Wang
(serine repeat antigen) (25). These results 5. P. S. Sijwali, B. R. Shenai, J. Gut, A. Singh, P. J. (University of California, San Francisco) for critical
suggest that falcipain 1 is not involved in Rosenthal, Biochem. J. 360, 481 (2001). evaluation of the manuscript. Supported by funding
hemoglobin degradation or red blood cell 6. P. Raphael et al., Mol. Biochem. Parasitol. 110, 259 (2000). from the Sandler Program in Basic Sciences (M.B.).
7. B. L. Salmon, A. Oksman, D. E. Goldberg, Proc. Natl.
rupture at the end of schizogony, but rather Acad. Sci. U.S.A. 98, 271 (2001). Supporting Online Material
has a specific role in the invasion of red 8. R. Mayer et al., J. Med. Chem. 34, 3029 (1991). www.sciencemag.org/cgi/content/full/298/5600/2002/
blood cells by extracellular merozoites. 9. X. Que et al., J. Biol. Chem. 277, 25791 (2002). DC1
10. L. H. Bannister, G. H. Mitchell, G. A. Butcher, E. D. Materials and Methods
We have shown, using a functional pro- References and Notes
Dennis, S. Cohen, Cell Tissue Res. 245, 281 (1986).
teomics screen combined with a chemical 11. D. Greenbaum, K. F. Medzihradszky, A. Burlingame, M.
genetic approach, that falcipain 1 functions dur- Bogyo, Chem. Biol. 7, 569 (2000). 16 August 2002; accepted 15 October 2002

Signaling of Rat Frizzled-2 Through corresponding regions of the hamster ␤2-

adrenergic receptor (␤2AR) (19). The Rfz2

Phosphodiesterase and Cyclic GMP chimeric receptor, whose cytoplasmic do-

mains display no similarity to that of ␤2AR
(fig. S1A) (20), is functional insofar as it
Adriana Ahumada,1 Diane C. Slusarski,2 Xunxian Liu,1 couples to calcium mobilization (19) and to
Randall T. Moon,3 Craig C. Malbon,1 Hsien-yu Wang4* rapid activation of calcium-calmodulin– de-
pendent kinase II (16), as does the wild-type
The Frizzled-2 receptor (Rfz2) from rat binds Wnt proteins and can signal Rfz2.
by activating calcium release from intracellular stores. We show that wild- For functional analysis of Rfz2 signal-
type Rfz2 and a chimeric receptor consisting of the extracellular and trans- ing, we expressed the Rfz2 chimera in
membrane portions of the ␤2-adrenergic receptor with cytoplasmic domains mouse totipotent F9 teratocarcinoma cells
of Rfz2 also signaled through modulation of cyclic guanosine 3⬘,5⬘-mono- and in Chinese hamster ovary (CHO) cells
phosphate (cGMP). Activation of either receptor led to a decline in the that lack endogenous ␤2AR. We identified
intracellular concentration of cGMP, a process that was inhibited in cells stable transfectants expressing the Rfz2
treated with pertussis toxin, reduced by suppression of the expression of the chimera in CHO clones by reverse tran-
heterotrimeric GTP– binding protein (G protein) transducin, and suppressed scription (RT) and polymerase chain reac-
through inhibition of cGMP-specific phosphodiesterase (PDE) activity. More- tion (PCR) amplification (fig. S1B) (19,
over, PDE inhibitors blocked Rfz2-induced calcium transients in zebrafish 20), immunoblotting with antibodies to
embryos. Thus, Frizzled-2 appears to couple to PDEs and calcium transients ␤2AR (fig. S1C) (19, 20), and specific
through G proteins. binding of the ␤2AR antagonist [125I]iodo-

The Wnt proteins are secreted signaling pro- tion (10 –14). The Rfz2 receptor, by itself, 1
Department of Molecular Pharmacology, Diabetes
teins that play diverse roles in cell polarity, transduces binding of Wnt-5A to increases in and Metabolic Diseases Research Center, University
cell proliferation, and specification of cell intracellular calcium release (15) and activa- Medical Center, SUNY–Stony Brook, Stony Brook, NY
11794 – 8651, USA. 2Department of Biological Scienc-
fate (1–3). Wnt proteins signal through friz- tion of calcium-calmodulin– dependent pro- es, University of Iowa, Iowa City, IA 52242, USA.
zled (Fz) gene products (4, 5), members of tein kinase II (16, 17) and protein kinase C 3
Howard Hughes Medical Institute, Department of
the superfamily of G-protein–coupled recep- (18) without appreciably activating the ca- Pharmacology and Center for Developmental Biology,
tors (GPCRs) (6 – 8). Wnt-Fz family mem- nonical Wnt–␤-catenin pathway. Because pu- University of Washington School of Medicine, Seattle,
WA 98195, USA. 4Department of Physiology and
bers can be grouped into functionally distinct rified, active Wnt proteins are not available Biophysics, Diabetes and Metabolic Diseases Research
classes. Activation of the Wnt–␤-catenin for analysis of Rfz2 receptor function, we Center, University Medical Center, SUNY–Stony
pathway increases nuclear accumulation of engineered a chimeric receptor to substitute Brook, Stony Brook, NY 11794 – 8661, USA.
the Lef-Tcf transcriptional coactivator the three cytoplasmic loops and the COOH- *To whom correspondence should be addressed. E-
␤-catenin (1, 2, 9), thus activating transcrip- terminal tail of the Rfz2 receptor for the mail: [email protected]

2006 6 DECEMBER 2002 VOL 298 SCIENCE www.sciencemag.org