From the Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India
Received for publication, November 5, 2002, and in revised form, January 23, 2003
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ABSTRACT |
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Hsp90 is important for normal growth and
development in eukaryotes. Together with Hsp70 and other accessory
proteins, Hsp90 not only helps newly synthesized proteins to fold but
also regulates activities of transcription factors and protein kinases.
Although the gene coding for heat shock protein 90 from
Plasmodium falciparum (PfHsp90) has been characterized
previously, there is very little known regarding its function in the
parasite. We have analyzed PfHsp90 complexes and addressed its role in
parasite life cycle using Geldanamycin (GA), a drug known to interfere
with Hsp90 function. Sedimentation analysis and size exclusion
chromatography showed PfHsp90 to be in 11 s20,w complexes of ~300 kDa in size.
Similar to the hetero-oligomeric complexes of Hsp90 in mammals, PfHsp70
was found to be present in PfHsp90 complexes. Homology modeling
revealed a putative GA-binding pocket at the amino terminus of
PfHsp90. The addition of GA inhibited parasite growth with
LD50 of 0.2 µM. GA inhibited parasite growth
by arresting transition from Ring to trophozoite. Transition from
trophozoite to schizonts and reinvasion of new erythrocytes were less
significantly affected. While inducing the synthesis of PfHsp70 and
PfHsp90, GA did not significantly alter the pattern of newly
synthesized proteins. Pre-exposure to heat shock attenuated GA-mediated
growth inhibition, suggesting the involvement of heat shock proteins. Specificity of GA action on PfHsp90 was evident from selective inhibition of PfHsp90 phosphorylation in GA-treated cultures. In
addition to suggesting an essential role for PfHsp90 during parasite
growth, our results highlight PfHsp90 as a potential drug target to
control malaria.
Plasmodium falciparum is responsible for the most
severe form of malaria in humans, causing approximately 2 million
deaths every year. During its asexual life cycle in human erythrocytes, the parasite progresses through three growth phases (1). The early form
following invasion called the Ring stage is the phase of establishment
in the erythrocyte. Trophozoite stage is the metabolically most active
biosynthetic phase, whereas the schizont stage represents the phase of
nuclear division before release of merozoites from the erythrocyte.
Heat shock proteins of the class Hsp60, Hsp70, and Hsp90 are known to
be expressed by the parasite during the intraerythrocytic stages in the
vertebrate host (2-4). Although these heat shock proteins share
significant homologies with their mammalian counterparts, there is very
limited information available regarding their functional roles in
parasite development. We have focused our study on the role of
parasite-heat shock protein 90 expressed during erythrocytic cycle in humans.
Previous reports have shown that the gene coding for Hsp90 from
P. falciparum is present on chromosome 7, has a single
intron of 800 bp, and encodes protein with 745 amino acids, giving a molecular mass of 86 kDa (5, 6). Sequence comparison shows 59%
identity and 69% similarity to human Hsp90. There is significant similarity at the NH2-terminal nucleotide-binding domain in
the central acidic hinge region as well as at the COOH-terminal
substrate-binding domain. The presence of EEVD motif at the COOH
terminus suggests cytosolic localization of heat shock protein 90 from
P. falciparum (PfHsp90)1 (7).
In higher eukaryotes, cytosolic Hsp90 is a highly abundant protein
organized in the form of a multi-chaperone complex. Two obvious roles
have been ascribed to Hsp90. 1) Together with Hsp70 and Hsp60,
Hsp90 helps newly synthesized proteins to fold and 2) it helps modulate
the activities of transcription factors (steroid hormone receptors and
nuclear receptors) and protein kinases (8-10). The latter activity of
Hsp90 puts it at the center stage of signal transduction events,
crucial for cell survival and growth. Indeed, experiments performed in
yeast, fruit fly, plant, and animal systems support the idea that in
addition to helping newly synthesized proteins to fold, Hsp90 also
regulates cell cycle and development (11-13).
In addition to the common approaches of gene disruption in yeast and
mutational analysis in Drosophila, a new approach of pharmacologically
interfering with Hsp90 function has been described in the literature
(14-16). The approach involves the use of a benzoquinone ansamycin
drug called Geldanamycin (GA), which specifically binds to Hsp90 and
interferes with its ATP-dependent chaperone function (14).
The specificity of GA interaction with Hsp90 has been described at the
molecular level through the crystal structure of human Hsp90 complexed
to GA (17). The use of GA in Drosophila and Arabidopsis has provided
important insights into the profound involvement of Hsp90 in various
cellular functions.
We have examined the complexes of PfHsp90 and analyzed its role in
parasite growth in human erythrocytes using GA. Using an antibody
specific to PfHsp90 and by employing sucrose gradient sedimentation as
well as gel filtration chromatography, we have characterized the
complexes of PfHsp90 in the parasite cytoplasm. In addition to
demonstrating its presence in a complex similar in size to mammalian
Hsp90, we also show PfHsp70 to be a part of the PfHsp90 multi-chaperone
complex. Most importantly, we show that GA inhibits parasite growth in
human erythrocytes. Modeling the NH2-terminal domain of
PfHsp90 based on its high degree of sequence identity to human Hsp90
uncovered a putative GA-binding pocket at its amino terminus. The
addition of GA to parasite cultures inhibited parasite growth with an
LD50 of 0.2 µM. The involvement of heat shock
proteins in GA-mediated growth inhibition was evident from the
observation that parasites pre-exposed to heat shock resisted growth
inhibitory effects of GA. The specificity of GA action on PfHsp90 was
evident from a selective inhibition of PfHsp90 phosphorylation in
GA-treated cultures without a significant change in overall protein
phosphorylation. Our results show that GA inhibits parasite growth
through its interaction with PfHsp90 and suggests an essential role for
PfHsp90 in parasite growth in human erythrocytes.
Reagents and Antibodies--
Antisera to PfHsp70 and PfHsp90
were generated against the recombinant COOH-terminal fragments of
proteins in rabbit (18). DSP was obtained from Pierce. GA, herbimycin A
(HA), and apyrase were purchased from Sigma.
Sedimentation on Sucrose Gradient and Gel Filtration
Analysis--
[35S]Cys- and
[35S]Met-labeled trophozoites were cross-linked in
vivo using DSP (9). Cells were hypotonically lysed (18), and the
lysates were layered onto 5-25% continuous sucrose gradient. The
gradient was spun at 40,000 rpm for 15 h at 4 °C in SW41 rotor. 500-µl fractions were immunoprecipitated with PfHsp90 antiserum and
analyzed by SDS-PAGE and fluorography. For gel filtration analysis,
saponin-released parasites (18) were sonicated briefly in
phosphate-buffered saline containing protease inhibitors on ice. The
lysate was clarified by centrifugation at 20,000 × g for 20 min at 4 °C and passed through Superdex 200 column (Amersham Biosciences) at a flow rate of 0.5 ml/min. Manually collected fractions
(500 µl each) were trichloroacetic acid-precipitated and
immunoblotted for PfHsp90. Thyroglobulin (669 kDa), alcohol dehydrogenase (150 kDa), and bovine serum albumin (66 kDa) were used as
molecular size standards.
Metabolic Labeling, Radioiodination, and
Immunoprecipitation--
The peak fraction of PfHsp90 (corresponding
to elution volume 10.5 ml) was collected, and protein complexes were
cross-linked using DSP (see above). Iodination of cross-linked proteins
was carried out using [125I] by chloramine T method (19).
The iodinated fraction was precleared using protein A-agarose beads and
immunoprecipitated using anti-PfHsp90, anti-PfHsp70, or preimmune
serum. The immune pellets were solubilized in Laemmli buffer and
analyzed as above.
Parasites were metabolically labeled with [35S]Cys and
Met and lysed in 20 volumes of NETT buffer (150 mM sodium chloride, 1 mM EDTA, 10 mM Tris, and 1% Triton X-100) (18) containing 20 units/ml
apyrase, protease inhibitors, and phosphatase inhibitors. This lysate
was used for non-denaturing immunoprecipitation (IP). For denaturing
IP, cells were directly lysed in Laemmli buffer, heated at 95 °C for
5 min, and diluted to 0.1% SDS concentration with NETT buffer. PfHsp70
or PfHsp90 immunoprecipitated from these lysates were solubilized
in two-dimensional lysis buffer or Laemmli buffer for analysis. To
examine induction of heat shock proteins following GA treatment, cells
were treated with Me2SO or 10 µM GA
for 14h and metabolically labeled in the last 2 h. Cells were lysed in 2× Laemmli buffer, boiled at 95 °C for 5 min, and diluted with NETT buffer to 0.1% SDS concentration. Clarified lysate was used
for immunoprecipitation of PfHsp70 and PfHsp90.
Early Ring-infected erythrocytes at 2% hematocrit and with 1-2%
parasitemia were treated with Me2SO (as a control) or
various concentrations of GA (0.05, 0.5, 1, 2, and 5 µM)
for 24 h (21). At the end of 24 h,
[3H]hypoxanthine at 10 µCi/ml was added and incubated
for an additional 3 h. At the end of labeling, cells were lysed
and DNA was collected in glass fiber filters using a cell harvester and
the radioactivity incorporated was measured by liquid scintillation counting.
For phosphate labeling of proteins, parasites were cultured in
phosphate-free medium supplemented with 0.5% human serum,
[32P]- orthophosphoric acid, and 1 mCi/ml medium at
37 °C for 6 h in the absence or presence of GA. Cells were
lysed at the end of treatment in Laemmli buffer in presence of 2 mM NaF and 1 mM sodium orthovanadate and heated
at 95 °C for 5 min. The lysate was treated with DNase and RNase in
the presence of protease inhibitors. The lysate was diluted to 0.1%
SDS concentration with NETT buffer, which was used for IP or
measurement of cpm in a liquid scintillation counter.
Modeling the Amino Terminus of PfHsp90--
Amino-terminal
sequence of PfHsp90 (1-177 amino acids) was submitted to SWISS MODEL
program (www.expasy.ch) to obtain the three-dimensional structure using
the crystal structure of amino-terminal human Hsp90 (Protein Data Bank
code 1YET) as a template. This model was superimposed over the
structure of human Hsp90·GA complex using STAMP (Structure
Alignment of Multiple Protein) program (20).
Effect of GA on Parasite Growth--
Sorbitol-synchronized
Ring-infected cells were suspended in RPMI 1640 medium to 5%
hematocrit. 200-µl cell suspension was treated with Me2SO
or with GA in Me2SO at various concentrations for 24 h. Smears were taken at the end of treatment, Giemsa-stained, and
viewed under microscope. The same experiment was performed with early
trophozoites and also schizonts. To examine the effect of GA and HA on
parasitemia, Ring-infected erythrocytes were either treated with
Me2SO or with 5 µM drug for 48 h
(culture was replenished with medium and GA every 24 h) and
parasitemia was calculated. A change in percentage of parasitemia with
respect to control was plotted against the duration of drug treatment.
To analyze stage-specific expression of PfHsp90, synchronous cultures
of infected erythrocytes were harvested at 10 h (Ring), 20 h
(early trophozoite), 30 h (late trophozoite), and 40 h
(schizont) and lysed in 10 volumes of NETT buffer. The protein content
of the lysates was estimated by the Bradford method. Equal protein from the different stages was analyzed for the presence of PfHsp90 by
Western blotting.
Prior Heat Shock and GA Treatment--
An asynchronous culture
of infected erythrocytes at ~20% parasitemia was given heat shock at
41 °C for 1 h in complete RPMI 1640 medium and then recovered
at 37 °C for 2 h. As a control, an equal number of cells were
kept at 37 °C for 3 h. Parasitemia was calculated at the end of
recovery period in both control and heat-shocked cells following which
the cells were treated with either Me2SO alone or 1 µM GA for 24 h. Parasitemia was calculated again at
the end of treatment period.
In Vitro Kinase Assay--
Ring stage parasites were treated
with Me2SO or 0.5 µM GA for 6 h.
Parasites were released using 0.01% saponin in phosphate-buffered saline, and hypotonic lysates were prepared from these parasites. 6 µg of lysate from GA-treated or untreated parasites was used in a
kinase assay using 2 µCi of [ Two-dimensional Gel Electrophoresis--
Synchronous
Ring-infected erythrocytes were treated with 10 µM GA for
16 h at 37 °C followed by the addition of
[35S]Cys and Met at 100 µCi/ml for 3 h. As
a control, Ring stage parasites (normal Rings) were labeled with
[35S]Met at 100 µCi/ml for 3 h. The cells were
washed twice in phosphate-buffered saline, and parasites were released
by saponin lysis. Parasites were sonicated in 50 volumes of 20 mM Tris buffer containing 1% Triton X-100 supplemented
with protease inhibitors. Lysates containing equal cpm were
acetone-precipitated, solubilized in two-dimensional lysis buffer, and
analyzed on a pH 3.5-9.5 tube gel in the first dimension and 7.5%
SDS-PAGE in the second dimension followed by fluorography.
Complexes of PfHsp90--
Mammalian Hsp90 is known to be organized
in a 11 s20,w hetero-oligomeric complex
consisting of Hsp70 and other accessory co-chaperones. To examine
whether PfHsp90 is organized in a complex similar to that of mammalian
Hsp90, we employed techniques of sucrose density gradient
ultracentrifugation and size exclusion chromatography. Synchronous
cultures of P. falciparum were metabolically labeled with
radiolabeled amino acids for 24 h, and the lysates were subjected
to centrifugation on sucrose gradients as described under "Materials
and Methods." Fractions collected after run were immunoprecipitated
with antibodies to PfHsp90 and analyzed by SDS-PAGE and fluorography.
As shown in Fig. 1A (top
panel), PfHsp90 was found in complexes ranging in size from 4.5 to
9 s20,w, indicative of heterogeneous
populations ranging from monomeric forms to complexes as high as 150 kDa in size. To examine whether our lysis protocol was disrupting
complexes of PfHsp90 in the cell, we used an approach of in
vivo cross-linking to freeze complexes of PfHsp90 before preparing
parasite lysates. We used DSP, which is a membrane-permeable,
reversible cross-linker to cross-link PfHsp90 complexes. As seen in
Fig. 1A, upon cross-linking (+DSP), PfHsp90 was found in
complexes ranging from 5 s20,w to as
high as 11 s20,w, corresponding to
approximately 300 kDa in size. Fig. 1A, bottom
panel, shows quantitation of these results.
We also employed size exclusion chromatography using HPLC to analyze
PfHsp90 complexes. Lysates were prepared from synchronous cultures of
the parasite as described under "Materials and Methods" and
analyzed on a gel filtration column Superdex 200 interfaced to HPLC.
Fractions were collected up to a one-bed volume of the column and
analyzed for the presence of PfHsp90 and PfHsp70 by trichloroacetic
acid precipitation of fractions and Western blotting using specific
antibodies. As shown in Fig. 1B (top panel),
PfHsp90 eluted as a single peak at a volume of 10-11 ml. A comparison with the elution volumes of standards indicated that the complex corresponded to ~300 kDa in size. This was in agreement with the 11 s20,w complexes observed on sucrose
gradient analysis of cross-linked samples. On the other hand, PfHsp70
was found to elute at a volume of 10.5-13.5 ml, ranging in size from
monomeric forms to complexes as high as 300 kDa (bottom
panel). Quantitation of this profile is presented in the
bottom panel of Fig. 1B.
To analyze the composition of the 300-kDa complex seen on HPLC, we
iodinated the peak fractions corresponding to elution volumes 10-10.5
ml after cross-linking and immunoprecipitated the labeled complex using
antibodies to PfHsp90 and PfHsp70. An equal volume of the labeled
fraction was also incubated with protein A beads and pre-immune serum
to ascertain the specificity of immunoprecipitation. Fig. 1C
shows an analysis of the immunoprecipitate by SDS-PAGE and
fluorography. As expected, a clear band corresponding to PfHsp90 (lane 2) was visible on SDS-PAGE, which was absent in the
control (lane 1). In addition to PfHsp90, three other bands
corresponding in size to ~75-, 60-, and 50-kDa proteins were also
seen in PfHsp90 immunoprecipitate. To examine whether the 75-kDa
protein was PfHsp70, an equal volume of iodinated fraction was
immunoprecipitated using antibodies to PfHsp70. As shown in Fig.
1C (lane 3) in addition to a signal corresponding
to PfHsp70, we also found a signal for PfHsp90. The result indicated
that PfHsp70 was included in the 300-kDa complex of PfHsp90 in the
parasite cytoplasm.
Interaction of PfHsp90 and PfHsp70--
To further examine
possible interactions between PfHsp70 and PfHsp90, we used a more
direct approach of co-immunoprecipitation from labeled parasite
lysates. Synchronous cultures of P. falciparum were
metabolically labeled for 2 h with radiolabeled amino acids and
lysed with detergent containing buffer in the presence of apyrase as
described under "Materials and Methods." Apyrase was included in
the lysis buffer to stabilize possible association of PfHsp70 and
PfHsp90 (9). The lysates were divided into three equal aliquots. One
aliquot was immunoprecipitated with antibodies to PfHsp90. The second
aliquot was immunoprecipitated with antibodies to PfHsp70; whereas the
third aliquot was incubated with preimmune serum alone to serve as a
control. The immunoprecipitates were analyzed by SDS-PAGE and
fluorography as described under "Materials and Methods." As seen in
Fig. 2A, in samples
immunoprecipitated with antibodies to PfHsp70 (lane 2), we
could detect a band corresponding to itself but, in addition, a band
corresponding in size to PfHsp90 was also evident. Similarly, in sample
immunoprecipitated with antibodies to PfHsp90, we could find a band
corresponding to PfHsp70 in addition to itself (lane 3).
Preimmune serum control (lanes 1 and 4) did not
show any specific signal. Immunoprecipitation done under denaturing
conditions for Pfhsp70 and PfHsp90 pulled down only the respective
proteins (lanes 5 and 6).
To ascertain that the co-precipitating bands indeed correspond to
PfHsp70 and PfHsp90, we also analyzed the immunoprecipitates by
two-dimensional gel electrophoresis. We have previously defined the
positions of PfHsp70 and PfHsp90 spots on two-dimensional gels (18). As
shown in Fig. 2B, in PfHsp70 immunoprecipitates, we found a
spot of PfHsp70 (arrowhead). In PfHsp90 immunoprecipitate (Fig. 2C) in addition to a spot corresponding to PfHsp90
(arrow) at a position expected from its size (90 kDa) and pI
(4.8), we also found a distinct 75-kDa spot (arrowhead)
corresponding in size (74.3 kDa) and pI (5.4) to PfHsp70. The preimmune
serum control on the other hand did not show the presence of either of
these spots on two-dimensional gels (data not shown). When a small
aliquot of PfHsp90 IP (apyrase) was mixed with denaturing PfHsp70 IP
(which pulls down only PfHsp70 as shown in Fig. 2A,
lane 5), there was an increase in the intensity of the
75-kDa spot (compare PfHsp90:PfHsp70 ratios between panels C
and D) showing that PfHsp70 is co-precipitated with PfHsp90
under non-denaturing conditions. The results confirmed that PfHsp90
and PfHsp70 were present in a common complex.
A Putative GA-binding Domain at the Amino Terminus of
PfHsp90--
GA, a benzoquinone ansamycin drug with antitumor
properties, is known to specifically bind the amino-terminal domain of
human Hsp90. The structure of the complex of human Hsp90 with GA has shown that the drug interacts with the amino-terminal ATP-binding domain of Hsp90 (17). To examine the presence of putative GA binding
site in PfHsp90, we aligned sequences corresponding to the
amino-terminal GA-binding domain of human Hsp90 with PfHsp90 (see Fig.
3A). The amino-terminal domain
of PfHsp90 shows 69% identity with human Hsp90 and contains a
GXXGXG motif essential for ATP binding.
The contact-making residues, critical in the binding to GA, were
conserved in PfHsp90 (indicated by asterisk). Based on the
high degree of sequence similarity between human Hsp90 and PfHsp90 in
this region, we modeled the structure of the PfHsp90. We used the
structure of human Hsp90 in complex with GA as a template for this
purpose (Protein Data Bank code 1YET). Modeling the amino-terminal
domain of PfHsp90 (residues 3-177) indicated that the overall fold in
this region was highly similar to that in human Hsp90 (Fig.
3B). The contact-making residues, namely Lys-58, Asp-93,
Gly-97, Lys-112, Phe-138, and Gly-183, were similarly positioned in
PfHsp90 (Fig. 3C). The structures could be superimposed with
a root mean square deviation of 0.07. The analysis indicated that GA is
likely to interact with PfHsp90 in a manner similar to its association
with human Hsp90.
GA Inhibits Parasite Growth--
To examine the effect of GA on
parasitemia, synchronous Ring stage parasites were treated with 5 µM GA as described under "Materials and Methods" and
percentage parasitemia was determined every 12 h until 48 h.
We also examined the effect of a closely related benzoquinone
ansamycin, HA, on parasite growth. The results were plotted as
percentage parasitemia with respect to control versus hours
of drug treatment. As shown in Fig.
4A, there was a progressive
decline in the percentage parasitemia in cultures exposed to 5 µM GA. Although 5 µM HA treatment also
resulted in reduction in parasitemia, the effect was less drastic.
[3H]Hypoxanthine incorporation in GA-treated cultures
(Fig. 4B) confirmed growth inhibitory effects of GA, and the
LD50 value was determined to be 0.2 µM.
GA Blocks Ring to Trophozoite Stage Progression--
To examine
the effect of GA on growth of the parasite, we used highly synchronous
cultures of P. falciparum in Ring stage and treated them
with different concentrations of GA. At the end of 24 h of GA
treatment, smears were taken to determine the stage of the parasite and
percent parasitemia. The results were compared, in parallel, with
mock-treated Ring stage culture. As expected, in the absence of GA
treatment, Ring stage cultures progressed to the trophozoite stage (see
Fig. 5A, left
panel, arrow). Fig. 5A, middle
panel, also shows a smear from culture treated with 10 µM GA. Most of the parasites persisted in the Ring stage
(see arrow) even at the end of 24 h in the presence of
the drug. In cultures treated with GA at a concentration of 0.5, 1, 5, or 10 µM for 24 h, we found a progressive decline in
the number of parasites progressing to the trophozoite stage.
Right panel shows the extent of progression from Ring to
trophozoite stage at increasing concentrations of GA. A similar
experiment was carried out with synchronous cultures in the trophozoite
stage progressing to schizont stage in the presence of different
concentrations of GA. As shown in Fig. 5B, in the absence of
GA treatment (left panel), trophozoite stage culture
effectively progressed to schizonts (arrow), but in the presence of GA (middle panel), there was persistence of
trophozoites (arrow) even after 24 h. The degree of
inhibition in stage progression increased with an increase in GA
concentration. Fig. 5B, right panel, shows
quantitation of the inhibitory effect of GA on progression from
trophozoite to schizont stages. The inhibitory effect of GA was more
pronounced for progression from Ring to trophozoite stage than from
trophozoite to schizont stage. Importantly, GA did not significantly
affect the release of merozoites from schizonts, and their reinvasion
to gave rise to new Rings (5C). To examine the levels of PfHsp90
present in different stages of parasite growth, we prepared lysates
from equal number of Rings, early trophozoites, late trophozoites, and
schizonts and examined equal amounts of protein from different stages
by SDS-PAGE and Western blotting. Fig. 5D, top
panel, shows Western blot of PfHsp90 in the Ring (lane
1), early trophozoite (lane 2), late trophozoite (lane 3), and schizont (lane 4) stages, and the
quantitation is presented in the bottom panel. The result
indicated that PfHsp90 was present in maximal amounts during
trophozoite stage.
GA Induces Heat Shock Protein Synthesis--
To examine the effect
of GA treatment on overall protein synthesis and heat shock protein
induction, we used two aliquots of synchronous Ring stage culture. One
was cultured for 14 h in the presence of GA and labeled for 2 h (see "Materials and Methods"). Giemsa staining of smears from the
cultures confirmed that GA-treated aliquot was blocked in the Ring
stage (as also described above). The second Ring stage culture was
labeled for 2 h without any treatment and lysed as described under
"Materials and Methods." Aliquots of lysates from both the samples
were precipitated with acetone, and radioactivity incorporated was
counted. As shown in Fig. 6A,
the amount of radioactivity incorporated in GA-treated culture was not
drastically different from that seen in untreated Ring stage culture
(normal Rings). When lysates of normal Ring and GA-blocked Ring
containing equal cpm were analyzed by two-dimensional gel
electrophoresis, overall protein profile looked similar in both (Fig.
6B). The result suggested that the overall profile of
proteins made in the presence of GA was qualitatively and
quantitatively similar to that in controls.
To examine the effect of GA on the levels of PfHsp70 and PfHsp90, we
immunoprecipitated these proteins from lysates containing equal cpm
from GA-treated and untreated cultures using specific antibodies. As
shown in Fig. 6C, top panel, higher amounts of PfHsp70 and PfHsp90 were seen in GA-treated parasites (lanes
2 and 4) compared with untreated cultures (lanes
1 and 3). Bottom panel shows quantitation of
these data, indicating ~3-fold induction of PfHsp70 and 5-fold
induction of PfHsp90 upon GA treatment.
Prior Heat Shock Counteracts GA-mediated Growth Inhibition--
To
analyze the involvement of heat shock proteins in GA-mediated parasite
growth inhibition, we examined whether prior heat shock is able to
attenuate GA-mediated growth arrest. An asynchronous culture of
P. falciparum was divided into two aliquots. One aliquot was
exposed to heat shock for 1 h at 41 °C, whereas the other aliquot was used as a control at 37 °C. The culture was allowed to
recover for 2 h following heat shock, and percent parasitemia was
determined in both of the aliquots. Following recovery, both of the
aliquots were incubated with 1 µM GA or Me2SO
for 24 h. At the end of 24 h of GA treatment, parasitemia was
again determined in both the cultures. Fig.
7 shows quantitation of the drop in parasitemia upon GA treatment in control culture and culture exposed to
prior heat shock. Although there was >50% drop in parasitemia upon GA
treatment in control cells, the decrease was significantly attenuated
in culture pre-exposed to heat shock. The result suggests that
pre-induction of heat shock proteins as a result of prior heat shock
may confer protection against GA-mediated growth inhibition.
GA Blocks PfHsp90 in a Dephosphorylated State--
Chaperoning
function of mammalian Hsp90 has been linked to its phosphorylation
state in the cell, and GA has been shown to abrogate Hsp90
phosphorylation in mammalian cells (22-24). To examine whether
phosphorylation state of PfHsp90 was affected in GA-treated parasites,
we labeled Ring stage culture with [32P]phosphoric acid
with or without GA treatment (0.5 µM) as described under
"Materials and Methods" and measured total cpm incorporated in the
cell lysates. Lysates corresponding to equal cpm were
immunoprecipitated with antibodies to PfHsp90 or PfHsp70, and the
immunoprecipitates were analyzed by SDS-PAGE and fluorography. As shown
in Fig. 8A (top
panel), both PfHsp70 (lane 3) and PfHsp90 (lane
5) immunoprecipitates showed labeled bands in GA-untreated
cultures, confirming phosphorylation of these proteins in the parasite.
In parasite cultures labeled in the presence of 0.5 µM GA, while the signal corresponding to PfHsp70
(lane 4) was similar to control that for PfHsp90 were significantly reduced (lane 6). Quantitation of bands
indicated a 60% drop in signal for phosphorylated PfHsp90 in
GA-treated cultures (Fig. 8A, bottom panel).
To rule out the possibility that GA was inhibiting overall kinase
activity in the parasite, we also examined the ability of lysates from
GA-treated parasites to phosphorylate exogenously added bovine milk
casein. Ring stage parasites were treated with 0.5 µM GA
or Me2SO for 6 h. Hypotonic lysates prepared from
saponin-released parasites were incubated with casein in the presence
of [ Among the different heat shock proteins described in
biological systems, heat shock protein 90 plays a particularly
important role in cell growth and development. In addition to
participating in the folding and assembly events of newly synthesized
proteins, Hsp90 also regulates signal transduction events by
interacting with transcription factors and protein kinases. In
mammalian cells, Hsp90 is one of the most abundant proteins in the
cytoplasm, existing as a multi-chaperone complex associated with Hsp70,
Hsp60, p23, and cyclophilin (25). The lethal phenotype of Hsp90
disruption in yeast underlines its pivotal role in cell function (26). Mutations in Hsp90 from Drosophila melanogaster are known to
result in a variety of developmental abnormalities (12). Similarly, defects in Hsp90 function in Arabidopsis thaliana result in
phenotypic changes (13). The results have been interpreted to suggest
that normal functioning of Hsp90 is essential to buffer phenotypic variations.
Although the gene coding for Hsp90 in P. falciparum has been
cloned (27), there is very limited information available regarding its
function in the parasite. At the level of primary structure (as
predicted from the nucleotide sequence), Hsp90 from P. falciparum is highly homologous to Hsp90 in mammalian cells.
Previous studies indicate that PfHsp90 is predominantly cytoplasmic and
is expressed in all three intraerythrocytic stages of human host (4).
To begin to understand the biochemical role of PfHsp90, we have
initiated studies on its size and composition of its complexes in the
parasite cytoplasm. Furthermore, by using GA, a drug known to inhibit
Hsp90 function (14, 15), we show that PfHsp90 is essential for parasite stage progression during intraerythrocytic growth.
Sedimentation analysis on sucrose gradients from in vivo
cross-linked samples indicated that PfHsp90 was present in complexes ranging in size from 5 to 11 s20,w in
the cell. The sedimentation coefficient of 5 s20,w corresponded to monomeric 90-kDa form of PfHsp90, whereas 11 s20,w
corresponded to complexes of size 300 kDa. This was in agreement with
the size of multi-chaperone complex reported for mammalian Hsp90 (28).
Gel filtration analysis confirmed a size range up to ~300 kDa for
PfHsp90. Similar to the Hsp90 complex reported in mammalian cells, we
found PfHsp70 to be a part of the PfHsp90 complex. In addition, at
least two other proteins of size 60 and 50 kDa could be seen in the
PfHsp90 complexes. The identities of these proteins remain to be
established. It is probable that these are parasite counterparts of
Hop60 and cyclophilin known to be present in the mammalian Hsp90
multi-chaperone complex. Recently published genome sequences of the
parasite indeed show the presence of Hop60 and cyclophilin homologs
(29).
GA has been reported to interact with the nucleotide-binding domain of
human Hsp90 (30). The crystal structure of the complex between
amino-terminal domain of Hsp90 and GA has been reported previously
(17). The high degree of sequence similarity between human Hsp90 and
PfHsp90 allowed us to model the amino-terminal domain of PfHsp90 and
compare its structure with that of human Hsp90. Superimposition of
PfHsp90 amino-terminal domain with Hsp90 in complex with GA using
STAMP program revealed that all of the points of contact could be
juxtaposed. The model of PfHsp90 suggested that it possessed a putative
GA-binding domain at the amino terminus.
The addition of GA to P. falciparum culture resulted in
inhibition of its intraerythrocytic stage progression. Inhibition of
parasite growth occurred in a narrow time window, affecting progression
from Ring to trophozoite stage maximally. Progression from trophozoites
to schizont stage release of merozoites from schizonts and reinvasion
by newly released merozoites were affected less drastically by GA.
Trophozoite being biosynthetically most active phase, the progression
from Ring to trophozoite, may depend heavily on PfHsp90 function,
thereby showing an obvious sensitivity to GA. Indeed, Hsp90 synthesis
was found to be maximal during Ring to trophozoite transition.
Examination of cultures beyond one generation (>48h) revealed a
significant decrease in the number of parasites in GA-treated cultures.
This was probably a result of lysis of Ring stage-arrested parasites
upon GA treatment.
Attenuation of GA-mediated growth arrest in parasites pre-exposed to
heat shock provided direct evidence for the involvement of heat shock
proteins in GA-mediated growth arrest. Such "cross-tolerance" conferred by pre-exposure to heat stress has been well documented in
the literature (31). That GA-mediated arrest in parasite growth was a
result of its effect on PfHsp90 function was evident from a specific
drop in the levels of phosphorylated PfHsp90 without any decrease in
overall protein phosphorylation in GA-treated parasites. A similar
GA-mediated drop in Hsp90 phosphorylation has been reported in
mammalian cells. Such a decrease in mammalian Hsp90 phosphorylation has
been shown to be linked to inhibition of its chaperoning function
(22).
GA has been shown to affect the growth of another protozoan parasite
belonging to the genus Leishmania (32). GA arrests the
growth of the promastigote stage (insect stage) of Leishmania donovani in culture. GA is also thought to mimick heat stress experienced by the parasite during transmission from the insect to the
vertebrate host and induce its progression to amastigote stage, which
is normally found only in the vertebrate host. Overexpression of
cytosolic Hsp90 has been shown to overcome such growth inhibitory effect of GA in Leishmania. GA has also been reported to
inhibit tumor growth in animal cells by interfering with tumor-inducing factors like mutated p53, Raf kinase src kinase, and steroid
receptors, which are known substrates of cytosolic Hsp90 (14, 33, 34). GA is thought to bring about tumor inhibitory effects by abrogating interactions of the above tumor-promoting factors with Hsp90. The
anti-tumor potential of GA is currently being evaluated under clinical
trials in humans (33).
In all, our study shows PfHsp90 to be present in hetero-oligomeric
complexes essential for parasite growth. Specific mechanisms of
parasite growth inhibition by GA need to be addressed in future. Identification of PfHsp90 client proteins, i.e. nuclear
receptors and protein kinase orthologs in the parasite, becomes an
important priority for malaria biologists. The study of GA effects on
gene expression profiles during stage progression will help uncover detailed mechanisms of parasite stage progression and may lead to
identification of novel drug targets against malaria.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]ATP with or
without 5 µg of dephosphorylated bovine milk casein as substrate in
1× kinase buffer (10 mM Tris, pH 7.5, 10 mM
NaCl, 5 mM MgCl2, 1 mM
phenylmethylsulfonyl fluoride, and 5 µM
ZnSO4) in the presence of 1 mM sodium
orthovanadate and 1 mM NaF. The kinase assay was performed
at 37 °C for 30 min and terminated by the addition of Laemmli buffer
and boiling. Phosphorylation of casein was analyzed by 10% SDS-PAGE
and fluorography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Analysis of PfHsp90 complexes.
Panel A, sedimentation analysis. Two aliquots of P. falciparum infected erythrocytes were labeled with
[35S]Cys and [35S]Met. One aliquot was
lysed as such, and the other aliquot was lysed after cross-linking.
Lysates were layered on sucrose gradients and centrifuged as described
under "Materials and Methods." 500-µl fractions were collected
from the top and immunoprecipitated using anti-PfHsp90 antibody. The
immunoprecipitates were analyzed by SDS-PAGE and fluorography. Bovine
serum albumin (4.5 s20,w), IgG (7.1 s20,w), and catalase (11 s20,w) were used as sedimentation
standards. The bottom panel shows quantitation of these
results. Panel B, gel filtration analysis. Lysates from
saponin pellets were clarified and analyzed using Superdex 200 gel
filtration column. 500-µl fractions were collected and immunoblotted
for PfHsp90 and PfHsp70. Thyroglobulin (669 kDa), alcohol dehydrogenase
(150 kDa), and bovine serum albumin (BSA) (66 kDa) were used
as molecular size standards. Bottom panel shows quantitation
of the profile. Panel C, radioiodination and
immunoprecipitation analysis. HPLC fractions corresponding to
10-10.5-ml elution volume were pooled, cross-linked, and iodinated.
Iodinated fractions were immunoprecipitated using preimmune serum
(lane 1), anti-PfHsp90 (lane 2), or anti-PfHsp70
antisera (lane 3). Immunoprecipitates were analyzed by
SDS-PAGE and fluorography.
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Fig. 2.
Co-immunoprecipitation of PfHsp70 with
PfHsp90. Panel A, P. falciparum infected
erythrocytes in early trophozoite stage were metabolically labeled with
[35S]Cys and [35S]Met for 2 h. At the
end of labeling period, cells were lysed in 20 volumes of NETT buffer
in the presence of apyrase (apy) or under denaturing
condition using SDS (den.). Clarified lysates were
immunoprecipitated using antibodies to PfHsp70 (lanes 2 and
5) or PfHsp90 (lanes 3 and 6) and
analyzed by SDS-PAGE and fluorography. Lanes 1 and
4, protein A controls. Panels B-D, analysis of
the immunoprecipitates by two-dimensional gel electrophoresis (2D
GE). Panel B, PfHsp70 IP. Panel C,
PfHsp90 IP. Panel C, PfHsp90 IP mixed with denaturing
PfHsp70 IP. Arrow, PfHsp90 spot; arrowhead,
PfHsp70 spot.
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Fig. 3.
A putative GA-binding domain in
PfHsp90. Panel A, amino terminus of human Hsp90 and PfHsp90
are aligned. Identical residues are indicated in black
color, whereas blue-colored residues correspond to
similar amino acids and dissimilar residues are denoted by red
color. Residues marked with an asterisk are involved in
hydrogen bonding with GA. GXXGXG motif is
indicated in boldface. Panel B, amino terminus
domain structure of PfHsp90 (modeled using SWISS MODEL) is superimposed
on that of human Hsp90 with GA (Protein Data Bank code 1YET) using
STAMP program. Blue line corresponds to PfHsp90, and
yellow line corresponds to human Hsp90. GA is shown in the
center in yellow color. Panel C, a magnified view
of the superposed model proximal to GA is shown. Thick and
thin lines correspond to human Hsp90 and PfHsp90,
respectively. Amino acid residues crucial for hydrogen bonding with GA
are indicated.
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Fig. 4.
Effect of GA on parasite growth.
Panel A, synchronous culture in Ring stage was treated with
5 µM GA or 5 µM HA or Me2SO (as
a control) for 48 h, and percent parasitemia was calculated every
12 h. Solid circles represent percent parasitemia in
HA-treated culture, whereas open circles represent that in
the GA-treated culture. Panel B, P. falciparum
Ring-infected erythrocytes were treated with various concentrations of
GA (0.05, 0.5, 1, 2, and 5 µM) for 24 h and
incubated with [3H]hypoxanthine for 3 h. Samples
were prepared as described under "Materials and Methods," and cpm
incorporation was estimated. The experiment was done in triplicates,
and the mean values of cpm incorporation against concentrations of GA
were plotted to calculate the LD50.
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Fig. 5.
GA blocks ring to trophozoite
transition. P. falciparum cultures were treated with
various concentrations of GA (0.5, 1, 5, and 10 µM) or
with Me2SO (DMSO) as control for 24 h at
early Ring stage (A, left panel), at early
trophozoite stage (B, left panel), or schizont
stage (C, left panel). Smears were taken at the
end of 24 h and stained using Giemsa stain. Right
panels of A-C show the quantitation of progression
from Ring to trophozoite (A, right panel),
trophozoite to schizont (B, right panel), or from
schizont to Ring stage (C, right panel) in the
presence of GA. D, synchronous culture of infected
erythrocytes were harvested at 10 h (Ring), 20 h (early
trophozoite), 30 h (late trophozoite), and 40 h (schizont)
and lysed as described under "Materials and Methods." Equal protein
from the different stages was analyzed for the presence of PfHsp90 by
Western blotting. In the top panel, lane 1, Ring;
lane 2, early trophozoite; lane 3, late
trophozoite; and lane 4, schizont. The bottom
panel shows quantitation of the levels of PfHsp90 in all of the
four stages plotted as percentage of total.
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Fig. 6.
PfHsp70 and PfHsp90 are induced by GA
treatment. Ring stage parasites were labeled metabolically
(Normal rings) or treated with 10 µM GA and
labeled as described under "Materials and Methods." Normal and
GA-treated parasites were lysed, and an aliquot of each was used to
measure cpm incorporation. A, total cpm incorporated in
normal Rings and GA-treated parasites. B, two-dimensional
profiles of 35S-labeled normal Rings and GA-treated
parasites. IEF, isoelectric focusing. C, equal
cpm from GA-treated (lanes 2 and 4) and untreated
(lanes 1 and 3) parasite lysates were
immunoprecipitated with antibodies to PfHsp70 and PfHsp90 and analyzed
by SDS-PAGE and fluorography. In top panel, lanes
1 and 2, PfHsp70 immunoprecipitates; lanes 3 and 4, PfHsp90 immunoprecipitates. Quantitation of PfHsp70
and PfHsp90 signals is shown in bottom panel.
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Fig. 7.
Prior heat shock counteracts GA-mediated
growth arrest. An asynchronous culture of infected erythrocytes
was given heat shock at 41 °C for 1 h and recovered for 2 h. Control or heat shocked cells (prior HS) were treated
with Me2SO (DMSO) or 1 µM GA for
24 h. Parasitemia was calculated in both sets before and after GA
treatment. Effect of GA on parasitemia is shown in the form of a
bar diagram.
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Fig. 8.
GA blocks PfHsp90 in a dephosphorylated
state. Trophozoites were labeled with
[32P]phosphoric acid in the presence of 0.5 µM GA or Me2SO for 6 h. Cells were lysed
at the end of treatment, and the lysates were used for
immunoprecipitation. Panel A, immunoprecipitates of PfHsp70
and PfHsp90 from GA-treated and untreated lysate were analyzed by 7.5%
SDS-PAGE and autofluorography. Top panel, lanes 1 and 2, immunoprecipitates using preimmune serum; lanes
3 and 4, PfHsp70 immunoprecipitates; and lanes
5 and 6, PfHsp90 immunoprecipitates. Lanes
1, 3, and 5, untreated lysates;
lanes 2, 4, and 6, GA-treated lysates.
Quantitation for PfHsp90 phosphorylation levels is shown in
bottom panel. Panel B, lysates from GA-treated or
untreated parasites were incubated with [ -32P]ATP and
milk casein as described under "Materials and Methods." The lysates
were analyzed by SDS-PAGE and fluorography to visualize the band
corresponding to labeled casein.
-32P]ATP for 30 min (see "Materials and
Methods"). Casein in the reaction mixture was then analyzed by
SDS-PAGE and fluorography. As shown in Fig. 8B, casein was
phosphorylated to a similar extent by lysates from both GA-treated and
untreated parasites. These results indicated that GA did not affect
total kinase activity in the infected erythrocytes and that decrease in
phosphorylation of PfHsp90 seen in in vivo
phosphate-labeling experiment was a result of a specific effect of GA
on PfHsp90.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We acknowledge Dr. Sekar and P. Ananthalakshmi (Interactive Graphics Centre, Department of Physics, Indian Institute of Science, Bangalore, India) for their help in superposition of PfHsp90 on host-Hsp90.
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FOOTNOTES |
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* This work was supported by a grant from Department of Biotechnology, New Delhi, India and Indo-French Centre for Promotion of Advanced Research (IFCPAR), New Delhi, India (to U. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of Junior Research Fellowship from the Council of
Scientific and Industrial Research, New Delhi, India.
§ Both authors contributed equally to this work.
¶ To whom correspondence should be addressed. Tel.: 91-080-3942823; Fax: 91-080-3600814; E-mail: tatu@biochem.iisc.ernet.in.
Published, JBC Papers in Press, February 12, 2003, DOI 10.1074/jbc.M211309200
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ABBREVIATIONS |
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The abbreviations used are: PfHsp90, heat shock protein 90 from P. falciparum; GA, geldanamycin; HA, herbimycin A; DSP dithiobissuccinimidyl propionate, IP, immunoprecipitation; STAMP, Structure Alignment of Multiple Protein; HPLC, high pressure liquid chromatography.
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---|
1. | Bannister, L. H., Hopkins, J. M., Fowler, R. E., Krishna, S., and Mitchell, G. H. (2000) Parasitol. Today 6, 427-433[CrossRef] |
2. | Das, A., Syin, C., Fujioka, H., Zheng, H., Goldman, N., Aikawa, M., and Kumar, N. (1997) Mol. Biochem. Parasitol. 88, 95-104[CrossRef][Medline] [Order article via Infotrieve] |
3. | Kumar, N., Koski, G., Harada, M., Aikawa, M., and Zheng, H. (1991) Mol. Biochem. Parasitol. 48, 47-58[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Jendoubi, M.,
Dubois, P.,
and Silva, L. P.
(1985)
J. Immunol.
134,
1941-1945 |
5. | Su, Q., and Wellems, T. E. (1994) Gene (Amst.) 151, 225-230[CrossRef][Medline] [Order article via Infotrieve] |
6. | Bonnefoy, S., Attal, G., Langsley, G., Tekaia, F., and Puijalon, O. M. (1994) Mol. Biochem. Parasitol. 67, 157-170[CrossRef][Medline] [Order article via Infotrieve] |
7. | Buchberger, A., and Bakau, B. (1997) in Guidebook to Molecular Chaperones and Protein-folding Catalysts (Gething, M. J., ed) , p. 147, Oxford University Press, Oxford, United Kingdom |
8. | Neckers, L., Mimnaugh, E., and Schulte, T. W. (1998) in Stress Proteins: Handbook to Experimental Pharmacology (Latchman, D. S., ed), Vol. 136 , pp. 9-32, Springer-Verlag, Heidelberg, Germany |
9. | Eggers, D. K., Welch, W. J., and Hansen, W. J. (1997) Mol. Biol. Cell 8, 15559-15573 |
10. | Csermely, P., Schnaider, T., Soti, C., Prohaszka, Z., and Nardai, G. (1998) Pharmacol. Ther. 79, 129-168[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Nathan, D. F.,
Vos, M. H.,
and Lindquist, S.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
12949-12956 |
12. | Rutherford, S. L., and Lindquist, S. (1998) Nature 396, 336-342[CrossRef][Medline] [Order article via Infotrieve] |
13. | Queitsch, C., Sangster, T. A., and Lindquist, S. (2002) Nature 417, 618-624[CrossRef][Medline] [Order article via Infotrieve] |
14. | Whitesell, L., Mimnaugh, E. G., Costa, B., Myers, C. E., and Neckers, L. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8324-8328[Abstract] |
15. |
Schneider, C.,
Lorenzino, L.,
Nimmesgern, E.,
Ouerfelli, O.,
Danishefsky, S.,
Rosen, N.,
and Hartl, U. F.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
14536-14541 |
16. | Sollars, V., Lu, X., Wang, X., Garfinkel, M. D., and Ruden, D. M. (2003) Nature Genet. 33, 70-74[CrossRef][Medline] [Order article via Infotrieve] |
17. | Stebbins, C. E., Russo, A. A., Schneider, C., Rosen, N., Hartl, F. U., and Pavletich, N. P. (1997) Cell 89, 239-250[Medline] [Order article via Infotrieve] |
18. |
Banumathy, G.,
Singh, V.,
and Tatu, U.
(2002)
J. Biol. Chem.
277,
3902-3912 |
19. | Hermanson, G. T. (ed) (1996) Bioconjugate Techniques , pp. 400-405, Academic Press, San Diego, CA |
20. | Russell, R. B., and Barton, G. J. (1992) Proteins Struct. Funct. Genet. 14, 309-323[Medline] [Order article via Infotrieve] |
21. | Schlichtherle, M., Wahlgren, M., Perlmann, H., and Scherf, A. (2000) in Methods in Malaria Research (Schlichtherle, M. , Wahlgren, M. , Perlmann, H. , and Scherf, A., eds), 3rd ed. , p. 30, Malaria Research and Reference Reagent Resource Center, Manassas, VA |
22. | Szyszka, R., Kramer, G., and Hardesty, B. (1989) Biochemistry 28, 1435-1438[Medline] [Order article via Infotrieve] |
23. |
Zhao, Y. G.,
Gilmore, R.,
Leone, G.,
Coffey, M. C.,
Weber, B.,
and Lee, P. W. K.
(2001)
J. Biol. Chem.
276,
32822-32827 |
24. |
Mimnaugh, E. G.,
Worland, P. J.,
Whitesell, L.,
and Neckers, L. M.
(1995)
J. Biol. Chem.
270,
28654-28659 |
25. | Buchner, J. (1999) Trends Biochem. Sci. 24, 136-141[CrossRef][Medline] [Order article via Infotrieve] |
26. | Borkovich, K. A., Farrelly, F. W., Finkelstein, D. B., Taulien, J., and Lindquist, S. (1989) Mol. Cell. Biol. 9, 3919-3930[Medline] [Order article via Infotrieve] |
27. | Jendoubi, M., and Bonnefoy, S. (1988) Nucleic Acids Res. 16, 10928[Medline] [Order article via Infotrieve] |
28. |
Murphy, P. J. M.,
Kanelakis, K. C.,
Galigniana, M. D.,
Morishima, Y.,
and Pratt, W. B.
(2001)
J. Biol. Chem.
276,
30092-30098 |
29. | Gardner, M. J., Shallom, S. J., Carlton, J. M., et al.. (2002) Nature 419, 531-534[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Grenert, J. P.,
Sullivan, W. P.,
Fadden, P.,
Haystead, T. A. J.,
Clark, J.,
Mimnaugh, E.,
Krutzsch, H.,
Ochel, H.-J.,
Schulte, T. W.,
Sausville, E.,
Neckers, L. M.,
and Toft, D. O.
(1997)
J. Biol. Chem.
272,
23843-23850 |
31. | Mizzen, L. A., and Welch, W. J. (1988) J. Cell Biol. 106, 1105-1116[Abstract] |
32. |
Wiesgigl, M.,
and Clos, J.
(2001)
Mol. Biol. Cell
12,
3307-3316 |
33. | Blagosklonny, M. V. (2002) Leukemia 16, 455-462[CrossRef][Medline] [Order article via Infotrieve] |
34. |
Bagatell, R.,
Khan, O.,
Paine-Murrieta, G.,
Taylor, C. W.,
Akinaga, S.,
and Whitesell, L.
(2001)
Clin. Cancer Res.
7,
2076-2084 |