KBP35 exhibited potent PPIase and protein folding activities against defined substrates in vitro, suggesting that it is a parasiticoth activities were inhibited by macrolide immunosuppressant drugs, ascomycin (a FK506 derivative) and rapamycin, but not by c, providing biochemical evidence of its inclusion in the FKBP family. Interestingly, FKBP35 inhibited purified plasmodial calc
protein phosphatase 2B) in the absence of any drug. In the parasite’s cell, FKBP35 exhibited a stage-specific nucleocytoplasmic sid not co-localize with calcineurin. FKBP35 associated with plasmodial heat shock protein 90 (Hsp90), another member of theuperfamily, via the TPR domain. Geldanamycin, a Hsp90 inhibitor, and ascomycin inhibitedP. falciparumgrowth in a synergistic fashioxtensive search of theP. falciparumgenome revealed no other FKBP sequence, implicating PfFKBP35 as a highly significant antimrug target. Thus, the single FKBP ofPlasmodiumis an essential parasitic chaperone with a novel drug-independent calcineurin-inhctivity.2005 Elsevier B.V. All rights reserved.
The immunophilin superfamily consists of highly con-erved, ubiquitously expressed proteins that possess rotamaser peptidyl prolylcis–trans isomerase (PPIase) activity andre considered to play an essential role in the folding of clientroteins by accelerating the isomerization of X-Pro peptideonds, a rate-limiting step in protein folding pathway[1–3].
� While our paper was in the final stages of acceptance, it came tour attention that Monaghan and Bell have recently published their re-ults on PfFKBP35 that are very similar to ours: Monaghan P, Bell A. Alasmodium falciparum FK506-binding protein (FKBP) with peptidyl pro-
The majority of immunophilins can be classified into tlarge families, viz. cyclophilins (CyPs) and FK506-bindproteins (FKBPs), and each family binds to specific immusuppressant molecules of fungal origin[4]. The CyPs bincyclosporin A (CsA), a cyclic undecapeptide[5,6]; in con-trast, the FKBPs specifically bind macrolides such as FK(tarcolimus) and rapamycin (sirolimus) that are structurunrelated to CsA[7]. In both families, the drugs bind to tPPIase domain that is roughly 100 amino acids long, leato inhibition of the PPIase activity. Thus, the immunophPPIase domain is synonymous with drug-binding domthe FKBP PPIase domain, for example, is also referredFK506-binding domain or FKBD (Figs. 1 and 2).
The immunosuppression activity of the drugs, howeinvolves a mechanism different from PPIase inhibitin which the cytosolic CsA–CyP or FK506–FKBP co
164 R. Kumar et al. / Molecular & Biochemical Parasitology 141 (2005) 163–173
Fig. 1. The PPIase catalytic domain (PPIase/FKBD), the three TPR motifs (-1, -2, -3), and prospective CaM-binding domain (CaM-BD) of relevant FKBPsare shown. The extra C-terminal sequence in human FKBP38 (accession number: AAO39020) contain the underlined hydrophobic mitochondrial targetingsequence[54], absent in the others. Within the PPIase domain, the asterisked residues are conserved in all four FKBPs; the shaded ones are important forFK506-binding and thus, for inhibition of PPIase activity and calcineurin (see text for details). Within the TPR domain, residues important for Hsp90-bindingare shaded; those that are absolutely essential for binding are additionally marked by overhead dots[50]. For CyP40, only the TPR domain is shown, because itsPPIase domain has no homology with the FKBP PPIase domains. Due to the degeneracy of TPR motifs, their amino acid identities are poor, and are not marked.The plasmodial sequences are described in detail under Section3; accession numbers of FKBP 12 and CyP40 are NP463460 and Q08752, respectively. Thedeletions of PfFKBP35 are demarcated with triangles (also seeFig. 2). All recombinants, including the deletions contained N-terminal His-tag for purification.
Fig. 2. Domain arrangement in FKBPs and the PfFKBP35 deletion mutants. The modular PPIase/FK506-binding domain, TPRs (box) and CaM-BD areindicated, respectively, by white, speckled and striped boxes. The approximate lengths and domain content of a few representative FKBPs are indicatedgraphically; overhead numbers are average lengths of the domains in number of amino acids. FKBPs may contain additional domains that are not shown.Recombinant mutants of PfFKBP35 were constructed in which the deletions extended from the C-terminus and included the third TPR (�TPR3) or all threeTPRs (�TPR123). The N-terminal deletion (�FKBD) removed the PPIase/FKBD domain only, and therefore, contained the rest of the protein including theTPRs and CaM-BD. The exact deletion points are shown inFig. 1.
R. Kumar et al. / Molecular & Biochemical Parasitology 141 (2005) 163–173 165
plex actively binds to and inhibits calcineurin (CN), aCa2+-calmodulin (CaM)-dependent protein phosphatase,also known as protein phosphatase 2B (PP2B) or proteinphosphatase 3[4,8,9]. This leads to an increase in net phos-phorylation of the transcription factor (NF-AT) which thenfails to translocate from the cytoplasm to the nucleus. Thus,NF-AT-dependent early lymphokines are not expressed,resulting in the suppression of T-cell and B-cell function.Rapamycin, in contrast, functions through a different mech-anism in which the Rpm–FKBP complex does not inhibitcalcineurin but instead inhibits a immunologically criticalSer/Thr protein kinase, hence named “target of rapamycin”(TOR) [4,10,11]. Crystal structures have been solved for anumber of drug–immunophilin complexes as well as theirco-complexes with calcineurin, leading to identificationof conserved amino acid residues of immunophilins andcalcineurin important for such interactions[5,6,7,12]. Dueto their immunosuppressive action, these drugs and a newgeneration of their synthetic analogs are routinely prescribedto transplant recipients to prevent tissue rejection. Someof the other clinical effects of these drugs, however, mayinvolve novel mechanism(s); for example, the neuropro-tective effect of FK506 is also manifested by syntheticnon-immunosuppressant (non-calcineurin-binding) FK506derivatives (V-10, 367 and GPI-1046)[13] as well as inFKBP-knockout mice[14]. It is evident that these drugs havep uesa eled.
alls terizeb 5F s theg ee com-p in them t se-q cifics s arev ya ith ap anda hus,t P12o 12,c s arekT t arep re-c ns.S rs,l sig-n fi a pairo nc s),s there
are three TPR motifs, such as in HsFKBP38 and HsFKBP51,TPRl and TPR2 are separated by 15–16 amino acid residues,while TPR2 and TPR3 are either connected or separated byone residue only (Fig. 1B). As in the PPIase sequence, theTPR motifs of FKBP resemble each other more than thosein CyPs [1,16]. The heat shock protein 90 (Hsp90) is anabundant molecular chaperone that plays an essential rolein protein chaperoning and often uses the immunophilins asinteracting partners[16]. In fact, specific residues in the TPRdomain of FKBP have been shown to interact with the con-served MEEVD sequence at the C-terminus of Hsp90, under-scoring the role of the FKBP TPRs in forming the chaperonecomplex[16–18]. In general, chaperones and co-chaperonesrarely act alone and often form multipartite complexes thatare recruited for folding, maturation and regulation of spe-cific cellular proteins[19,20]. In the most extensively studiedexample, the mammalian steroid hormone receptor is regu-lated by its reversible association with Hsp90, FKBP, CyP40,p23 and protein phosphatase 5 (PP5), a unique Ser/Thrphosphatase with a N-terminal TPR domain[2,21–24].
Plasmodium falciparum, the agent of severe cerebralmalaria, is a lower eukaryotic, protozoan parasite that claims1–2 million human lives annually[25]. The rapid develop-ment of parasitic resistance to drugs that were once effectivehas led to a global demand for new antimalarial compoundstargeting potentially novel plasmodial gene products. At leastt andr rizedi sed activ-i izedt isi e ofC /orP anti-m , nos plas-m firste romt tima-i n-ac antf activ-i basiso in int ethert ours s alsoe sd ant,b oth-e olog[ ncea PRso he
otentially multiple targets and pathways in various tissnd organisms, the identities of which need to be unrav
Multiple immunophilin sequences have been found inpecies examined to date, and many have been characiochemically [1]. Homo sapiens, for example, have 1KBPs and at least 16 CyP-like sequences, whereaenome of the lower eukaryote,Saccharomyces cerevisia,ncodes 8 CyPs and 4 FKBPs. Multiple sequencearisons have revealed that the PPIase domains withembers of each family (FKBP or CyP) share significanuence homology and contain recognizable family-speignature motifs; however, the PPIase domains of FKBPery dissimilar from those of the CyPs[1]. In the generallccepted nomenclature, the immunophilins are named wrefix of one or two letters indicating the species of originsuffix to reflect the calculated molecular mass in kDa. T
he archetypal FKBP is the 12 kDa human enzyme, hFKBr HsFKBP12. The small immunophilins, such as FKBPontain a single PPIase domain while the larger onenown to contain up to four such domains in tandem (Fig. 1).he large ones may additionally contain domains tharimarily important for protein–protein interaction and/orruitment of the immunophilin to specific cellular locatiouch domains include WW, WD40, modified RING finge
eucine zippers, CaM-binding domain, ER localizationals and tetratricopeptide repeat (TPR)[1]. The TPR moti
s a degenerate 34 amino acid sequence that folds intof antiparallel�-helices[15], and large immunphilins ofteontain multiple TPR motifs C-terminal to the FKBD(eparated by a characteristic length of peptide. When
d
hree small CyPs, highly related in primary structureanging in size from 19 to 24 kDa, have been characten P. falciparum[26–28]. All three had a conserved PPIaomain, and the recombinant proteins exhibited PPIase
ties that were inhibited by CsA. We have also characterhe P. falciparumcalcineurin (PfCN) mid shown that itnhibited by two of the plasmodial CyPs in the presencsA[29–31]. Thus, inhibition of the PPIase activities andfCN offers potential mechanism(s) of the observedaiarial activity of the various cyclosporins. In contrast
ystematic study has been undertaken to characterizeodial FKBP(s) and their biochemical properties. The
vidence for a plasmodial FK506-binding protein came fhe observation that FK506 and rapamycin possess anarial activity [32]. In a pioneering preliminary report, Moghan and Bell[33] described the firstP. falciparumFKBPDNA and showed that the partially purified recombinorm of the protein possessed FK506-sensititve PPIasety. These authors named the protein FKBP35 on thef its size and also recognized a C-terminal TPR doma
he predicted sequence. It was, however, not known whhe putative FKBP was expressed in the parasite. Whiletudies were in progress, apparently the same protein waxpressed recombinantly by Braun et al.[34]; these authorid not investigate the PPIase activity of the recombinut detected the protein in the parasite. Finally, we andrs have recently characterized a plasmodial Hsp90 orth
35–37]and shown that it has the invariant MEEVD sequet the C-terminus that can potentially interact with the Tf FKBP [17,18]. In this report, we confirm and extend t
166 R. Kumar et al. / Molecular & Biochemical Parasitology 141 (2005) 163–173
previous findings by providing a detailed characterization ofthe FKBP35 ofP. falciparum, and show that the TPR-domainof PfFKBP35 indeed associates with PfHsp90. We show thathighly purified recombinant PfFKBP35 not only has a PPI-ase activity that is sensitive to both FK506 and rapamycin butalso refolds denatured proteins. Interestingly, it significantlyinhibits PfCN in vitro in the absence of any drug; however, invivo the protein exhibits a stage-specific nucleocytoplasmictranslocation that largely segregates it from PfCN. Together,these results establish PfFKBP35 as a true co-chaperone. Se-quence homology search as well as FK506-affinity bindingindicated that PfFKBP35 is the only FKBP-like protein inthePlasmodiumgenome, making it an attractive target forfunctional analysis and antimalarial drug discovery.
2. Materials and methods
2.1. Materials
Ascomycin (ethyl-FK506) and rapamycin were purchasedfrom Calbiochem (San Diego, CA) and cyclosporin A, bovinecyclophilin and RNase T1 (fromAspergillus oryzae) werefrom Sigma (St. Louis, MO). Geldanamycin was a kindgift from NCI, NIH [35]. The peptideN-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide (abbreviated as ALPF.pNA) was pur-c use)a nA-2 -b romD ti-b nti-b r-r di N-t ithp yc ionm rimerm -r
2
ry-t -n ra-tI wasc eicaT ruc-t
(r gGi d
agarose (Sigma) at 10◦C for 1 h. The immune complexeswere collected by centrifugation, washed, heated in SDSsample buffer, centrifuged at 10,000×g for 5 min, andthe supernatant analyzed by SDS-PAGE, followed by im-munoblot using appropriate primary antibody and HRP-coupled secondary antibody (Sigma). Immunoblot was de-veloped with Super Signal Ultra chemiluminescence (Pierce,Rockford, IL).
2.3. PPIase and protein folding assays
PPlase assay was carried out according to Nair et al.[42]. Stock solutions (0.5 mM) of all inhibitors were madein DMSO, and further diluted in 35 mM HEPES (pH 7.9) asneeded. Briefly, 1.5 ml reactions contained purified wild typeand mutant His-tagged PfFKBP35 proteins (at a final con-centration of 130 nM), 250�g TLCK-treated chymotrypsin(Sigma), 35 mM HEPES (pH 7.9), 0.014% Triton X-100,20�M substrate peptide ALPF.pNA. When used, the in-hibitor was preincubated with FKBP (60 min). The assaywas started by the simultaneous addition of chymotrypsinand substrate peptide. Inhibitor-free control reactions con-tained equivalent amount of DMSO only. Reactions wereperformed at 10◦C and the rise in A400 was recorded ev-ery 15 s for 4 min. The catalytic efficiency (kcat/Km) was cal-culated from the relationshipK = (k /K )[FKBP] +K ,wn KBP,r err e dif-f Thek t of[
wAg ing5 r-im 0 nMw ho-t
2
ndt ex-t(w pre-e ar-a atedw for1 e col-u hedw re
hased from Bachem (Torrance, CA). Monoclonal (montibody, reactive to PfHsp90 and peptide antibody (C) against PfCN have been described[30,35]. Rabbit antiody against recombinant PfFKBP35 was a kind gift fr. Thomas Wandless[34], and monoclonal His-tag anody was purchased from Novagen (La Jolla, CA). Aody against the peptide QKQYDEKKKPLFEKRDEII, coesponding to residues 55–73 of PfARP[38], was raisen mice. Recombinant FKBP35 was expressed with aerminal His-tag from pET-15b clone in BL21(DE3) wMICO plasmid[39] and purified by Ni2+-agarose affinithromatography using the kit from Novagen. All deletutants were constructed by the PCR-based Megapethod[40] and also purified by Ni2+-affinity chromatog
aphy.
.2. Parasite growth, cell lysis and immunodetection
P. falciparum3D7 was grown on A-positive human ehrocytes; drug treatments, measurement of IC50, synchroization with sorbitol, purification of parasite and prepa
ion of cell-free lysate were all carried out as described[35].mmunofluorescence staining of parasite-infected RBCarried out and the cells were viewed in a three-laser LCS SP2 confocal microscope, followed by 3D reconst
ion as described[41].For each immunoprecipitation (IP) experiment, 500�g
protein) parasitic lysate was incubated with 3�g of theabbit anti-PfFKBP35 antibody (or non-immune rabbit In “control”) and 20�l suspension of protein A-couple
obs cat m uhereKobs andKu are the first-order rate constants ofp-itroanilide release in the presence and absence of the Fespectively, and (kcat/Km)[FKBP] is the pseudo-first-ordate constant for PPIase-catalyzed isomerization. Threerent concentrations of the FKBPs were employed.cat/Km value was calculated from the slope of the ploFKBP] versusKobs−Ku.
For the RNase T1 refolding assay[43], RNase T1 (50�M)as first unfolded by incubating at 25◦C for 3 h in buffer(50 mM Tris–Cl (pH 8.0), 1 mM EDTA) containing 6 M
uanidine hydrochloride. Refolding was initiated by dilut0�l of this solution with 2.2 ml of buffer A containing va
ous amounts of FKBP. Refolding was followed at 10◦C byeasuring the increase in tryptophan fluorescence at 32ith excitation at 268 nm in a Hitachi UV–vis spectrop
ometer[43].
.4. Affinity selection of PfFKBP35
Ethyl-FK506 (ascomycin) was coupled to Affi-gel ahe matrix used for affinity chromatography of parasiteract following published procedures[44]. Briefly, 500�gprotein) ofP. falciparumextract (described in Section2.2)as passed through 0.2 ml of ascomycin affinity columnquilibrated with buffer A containing 0.15 M NaCl. In a pllel experiment, the plasmodial extract was preincubith 1 mM ascomycin or 1 mM CsA at room temperature0 min and the treated extract then passed through thmn in an identical manner. All columns were then wasith 2 ml of buffer A plus 0.4 M NaCl. Bound proteins we
R. Kumar et al. / Molecular & Biochemical Parasitology 141 (2005) 163–173 167
eluted with 1 mM ascomycin in buffer A and analyzed bySDS-PAGE[29]. The gel was silver-stained to detect the pro-teins. The corresponding band in a parallel gel was transferredto lmmobilonPSQ membrane and subjected to standard mi-crosequencing[29].
2.5. Calcineurin phosphatase assay and inhibition byFKBP-FK506
Recombinant plasmodial calcineurin (PfPP2B or PfCN)was purified [30] and assayed as described[45] withsome modifications as follows. Reactions (50�l) contained50 mM Tris–Cl (pH 7.5), 100 mM NaCl, 6 mM Mg-acetate,0.5 mM CaCl2, 0.1 mg/ml BSA, 0.5 mM DTT, 0.025% NP-40, 0.2 M bovine brain CaM (Sigma), 10 nM recombinantPfCN (holoenzyme), 150 nM FKBP, 2�M drug (ascomycinor rapamycin), 0.5�M RII phosphopeptide (a specific CNsubstrate). Reaction mixtures were incubated for 10 minat 30◦C prior to the addition of the substrate. Reactionswere then initiated by the addition of the substrate pep-tide, and dephosphorylation was allowed to proceed at30◦C. The liberated inorganic phosphate was measuredby a Malachite Green color assay using the Calbiochemkit (San Diego, CA). All reactions were followed withtime to ensure linearity, and results were corrected by sub-traction of the corresponding values from a no-enzymer
little more than the FKBD/PPIase domain. The HsFKBP38and thePlasmodiumproteins are similar in size and con-tain three TPRs with significant similarity. Furthermore, thespacing between the three TPR motifs of PfFKBP35 was alsocharacteristic of the FKBP family (Figs. 1 and 2). As men-tioned before, members of the FKBP family are distinguishedby the conserved sequence features of their PPIase domainand this is also true of PfFKBP35 (Fig. 1). Because the PPI-ase domains of the CyPs are very different in sequence, theywere not included inFig. 1.
Upon closer examination of the PfFKBP35 sequence,functionally important amino acid residues were also foundto be conserved. The asterisked residues within the PPIasedomain of PfFKBP35 are in fact the most conserved residuesin all FKBPs [1,46]. Of particular mention are the shadedresidues (Fig. 1), shown to be important for PPIase activityand FK506-binding by mutational analysis as well as crys-tal structures[5,46]. Thus, it seems that PfFKBP35 containsall the important FK506-interacting residues in its putativePPIase domain.
The various TPR motifs found in proteins have poorlyconserved primary structure but all contain appropriatelypositioned amino acids of conserved physical propertiessuch that each motif forms antiparallel helices[15,47].This is illustrated by the sequence comparison inFig. 1,whereby the three TPRs of the FKBP are included alongw cy-c n ofC rlier,C r ins
3r
Pf-F ncT e ac-t -l ovedft ence(t ltedi PI-a s 1-1 oftc -t , butl ll( thePt ivityi
eaction.
. Results
.1. Identification and characterization of PfFKBP35equence
To discover ifP. falciparumhas a FKBP homolog, wearched the predicted proteins of the parasite in theoDB repository (www.plasmoDB.org) by BLAST homol-gy using the HsFKBP12 amino acid sequence as qhis led to an intronless gene on Chromosome 12 (nucle955110-1956024) with a predicated protein of 304 amcids (chr12.phat472). The cDNA sequence was furtherertained by reverse transcription and PCR using sperimers, which confirmed the preliminary report of this p
ein mentioned previously[33]. The predicted primary struure of this prospective FKBP and that of a similarlyained sequence (GenBank PY02360) fromP. yoelii yoeliiPyFKBP35) are shown inFig. 1. The PyFKBP35 gene wlso intronless. The calculated MW of theP. falciparumandP.oelii yoelii proteins were 34,826 and 34,983, respectivpon rounding up of the numbers to the nearest thou35 k), the proteins were named PfFKBP35 and PyFKBhe two proteins were 81% identical in primary struct
ending credence to the authenticity of both sequenceig. 1, we compare the two parasitic sequences withepresentative human homologs: FKBP12 and FKBP38KBP12 is one of the smallest FKBPs known and con
ith the similarly spaced TPRs of a representativelophilin, namely human CyP40. The PPIase domaiyP40 was not included because, as mentioned eayP and FKBP PPIase domains are highly dissimilaequence.
.2. PPIase and protein chaperone activities ofecombinant PfFKBP35
As predicted by the conserved PPIase domain inKBP35 (Fig. 1), the recombinant enzyme, expressed iE.oli was indeed found to possess PPIase activity (Fig. 3A).ested against the synthetic peptide as substrate, thivity exhibited akcat/Km of 21 mM−1 s−1. To further deineate the PPIase domain, various portions were remrom the C-terminal end of the protein (Fig. 2). Deletionhat removed TPR3 along with all the downstream sequ�TPR3) had no detectable effect on activity (Fig. 3A). Fur-her deletion removing the two remaining TPRs resun a protein (�TPR123) that essentially contained the Pse domain only, corresponding to N-terminal residue41 (Figs. 1 and 2); this fragment retained nearly 90%
he PPIase activity of the full-length enzyme (Fig. 3A). Inontrast, the C-terminal 163 residues (�FKBP) that conained all three TPRs and the rest of the C-terminusost the PPIase domain (Fig. 2), showed no activity at aFig. 3A). These results provide enzymatic evidence forPIase domain of PfFKBP35 as delineated inFig. 1, and show
hat the TPR domains play no direct role in PPIase actn vitro.
168 R. Kumar et al. / Molecular & Biochemical Parasitology 141 (2005) 163–173
Fig. 3. PPIase (A) and chaperone (B) activities of PfFKBP35. The assayswere performed as described in Section2 using recombinant full-length Pf-FKBP35 and its various deletion mutants as follows: (seeFig. 2); full-lengthPfFKBP35 (white circle);�TPR1 (white triangle);�TPR123 (rectangle);�FKBD (black triangle); None (black circle).
A clear relationship between PPIase and protein foldingactivity of the immunophilins is yet to be established. BothCyP40 and FKBP52 have been shown to display chaper-one function that is independent of PPIase activity[48,49].Thus, to directly test if recombinant PfFKBP35 has pro-tein folding activity, its ability to refold a model substrate(viz. RNase T1) was examined, and significant refoldingactivity was indeed detected (Fig. 3B). As with PPIase,deletion of all three TPRs did not significantly destroy thefolding activity, whereas deletion of the PPIase domaindid. Thus, it appears that the PPIase and folding activi-ties of PfFKBP35 are related and encoded by the samedomain.
The drug sensitivity of PfFKBP35 was examined next.The PPIase activity of the recombinant enzyme was sen-sitive to rapamycin and the FK506 derivative, ascomycin.The IC50 of the two drugs were about 0.4 and 0.8�M, re-spectively (data not shown). Characteristic to the FKBP fam-ily, PfFKBP35 was resistant to CsA. It was also resistant toGA, an ansamycin antibiotic that binds to the N-terminaldomain of the Hsp90 chaperone and inhibits PfHsp90 aswell as parasite growth[35,36]. Thus, PfFKBP35 has the
enzymatic and sequence hallmarks of a TPR-containingFKBP.
3.3. PfFKBP35 associates with PfHsp90 via TPR
As mentioned in the Section1, mammalian FKBP part-ners with Hsp90. This results in the regulation of steroidreceptors and likely a battery of other important cellu-lar proteins. Biochemical studies and mutational analysisof recombinant CyP40 have identified specific amino acidresidues in the TPR domain that are important for Hsp90association[50]. Residues essential for Hsp90-binding andthose that increase the efficiency of binding are indicatedin Fig. 1. It is seen that all these residues are either identi-cal or conservatively replaced in PfFKBP35. The C-terminalMEEVD motif that interacts with FKBP[17,18] is also con-served in the plasmodial Hsp90. Thus, we anticipated thatPfFKBP35 may also complex with Hsp90, and proceededto test this. Indeed, FKBP–Hsp90 association in the par-asite was revealed in co-immunoprecipitation experiments(Fig. 4A). Specificity of the association was ascertainedby controls such as non-immune sera and the lack of as-sociation of PfARP, a non-relevant protein, previously de-scribed as a regulator of plasmodial protein phosphatase2A [38].
We then tested if the PfFKBP35–PfHsp90 association canb dP dt oundpw etionm notb rolei
3s
p yticc ,FN eeni thatd lgi-c . Asa as-cc as-s nd0 as-c on-c plot( in-d druge pose
e reproduced in vitro. Recombinant full-length His-taggefFKBP35 was immobilized on a Ni2+-agarose column an
otal parasite extract was passed through. When the broteins were analyzed by immunoblot (Fig. 4B), PfHsp90as indeed detected. In contrast, when the TPR-delutant of HFKBP35 was immobilized, PfHsp90 didind. Thus, the TPR domain must play an important
n PfFKBP35–PfHsp90 interaction.
.4. Inhibition of PfHsp90 and PfFKBP35 hasynergistic antimalarial effect
We had shown earlier that GA, an inhibitor ofP. falci-arumHsp90, has potent antimalarial effect in erythroculture with an IC50 of about 20 nM[35]. As mentionedK506 and rapamycin also inhibited parasite growth[32].ow that the parasitic targets of GA and FK506 have b
dentified as separate molecular entities, we reasonedrugs of the two families may improve the pharmacoal prospect of each other when used in combinationfirst step, we determined the antimalarial activity of
omycin and rapamycin againstP. falciparumin our cultureonditions. Using standard hypoxanthine incorporationay, the IC50 of the two drugs were found to be 0.15 a.2�M, respectively (data not shown). We then testedomycin and GA together in a range of combinatorial centrations and presented the results in an isobologramFig. 5A). The concave nature of the isobologram clearlyicates a synergistic relationship, which means that eachnhanced the antiparasitic activity of the other. We pro
R. Kumar et al. / Molecular & Biochemical Parasitology 141 (2005) 163–173 169
Fig. 4. Interaction between PfFKBP35 and PfHsp90. Immunodetection pro-cedures and the antibodies are described in Section2.2. The antibodies wereagainst the following: H = Hsp90; F = PfFKBP35; A = PfARP (A) associa-tion in vivo. In the first three lanes immunoprecipitation (IP), followed byimmunoblot (IB) analysis of the pellet, was done with the indicated an-tibodies. The first lane is control IP using non-immune rabbit IgG. Theblot was developed with HRP-conjugated rabbit antibody against mouseIgG. The “IB only” lanes are straightforward immunoblots (no IP) of 50�gplasmodial extract, using the indicated antibodies, followed by appropriateHRP-conjugated secondary antibody to demonstrate antibody specificity.(B) Association in vitro. His-tagged recombinant full-length PfFKBP35 oronly its FKBD segment (�TPR123) was first immobilized on Ni2+-column,and then parasite extract was passed through the column. After washing toremove unbound material, the matrix was divided into two equal parts andthe bound proteins subjected to immunoblot with the indicated antibodies(F or H). The size markers (in kDa) are indicated on the left and the detectedproteins on the right.
that combination therapy targeting the two major chaperonesof Plasmodium, namely Hsp90 and FKBP35, is a promisingantimalarial regimen worth further exploration.
To determine whether there are other plasmodial proteinsthat may bind FK506 (ascomycin), we performed an affin-ity chromatography in which the parasitic extract was passedthrough an ascomycin-column and the bound proteins specif-ically eluted with ascomycin (as described in Section2.4).The results are presented inFig. 5B. PfFKBP35 was in factthe only protein that specifically eluted from the column (lane1). The specificity of ascomycin-PfFKBP35 binding was fur-ther underscored by the fact that preincubation of the extractwith ascomycin (lane 2) but not with CsA (lane 3) stronglyreduced the binding of PfFKBP35 to the column. These re-
Fig. 5. (A) Antiplasmodial activity of a combination of ascomycin (ethyl-FK506) and geldanamycin. Note that the experimental line is concave, in-dicating synergism, as compared to the hypothetical straight line that wouldhave been obtained if there were no interaction between the drugs. The num-bers on both axes are drug concentrations expressed as fractions of their indi-vidual IC50 values. (B) FK506-affinity selection of PfFKBP35. Plasmodialcell-free extract was pre-incubated with no drug (lane 1) or with 1 mMascomycin (lane 2) or 1 mM CsA (lane 3) and affinity-chromatographedthrough ascomycin column (as described in Section2.4). The bound pro-teins were eluted with 1 mM ascomycin and analyzed by SDS-PAGE andsilver-staining.
sults suggest that PfFKBP35 is the major – if not the only –target of FK506 inP. falciparum.
3.5. PfFKBP35 inhibits parasitic calcineurin in theabsence of drug
In light of our previous finding thatP. falciparumcalcineurin could be inhibited by the CsA–CyP complex, itwas only logical to test if such inhibition is effected by theFK506–PfFKBP35 complex as well. Indeed, the phosphataseactivity of purified PfCN was inhibited by about 70% inthe presence of ascomycin (FK506 derivative) and purifiedPfFKBP35 (Fig. 6). To our surprise, when control reactionswere done in the absence of ascomycin, PfCN was stillinhibited to essentially the same extent. Clearly, PfFKBP35possesses an inherent calcineurin-inhibitory activity that isdrug-independent. Essentially similar inhibition was alsoobserved when commercial bovine CN was used (data notshown), suggesting that this unique property is characteristicof PfFKBP35 and not specific for a particular CN.
170 R. Kumar et al. / Molecular & Biochemical Parasitology 141 (2005) 163–173
Fig. 6. Inhibition of PfCN phosphatase by PfFKBP35. Phosphatase activ-ity was assayed in the presence or absence of drugs (FK = Et-FKBP, i.e.,ascomycin; Rm = rapamycin) and the following recombinant FKBPs: Pf-FKBP = full-length;�TPR =�TPR123;�FKBD = only the FKBD deleted;FKBP12 = recombinantBrugia malayiFKBP13 (New England Biolabs,Beverly, MA), very similar to human FKBP12 (Figs. 1 and 2). The deletionsare described in detail inFig. 2. All phosphatase activities are expressedas percentage of the drug-free, FKBP-free activity and are average of threeexperiments with the error bars as shown.
3.6. PfFKBP35 exhibits nucleocytoplasmic shuttlingand does not co-localize with PfCN
To investigate the possible physiological consequence ofPfFKBP35’s ability to inhibit PfCN, we tested their rela-tive amount and distribution in the parasite cell through theintraerythrocytic development cycle of the parasite. First,immunoblot analysis (Fig. 7A) showed that PfFKBP35is constitutively present at all times. In contrast, PfCNis barely detected in the ring stage, but becomes abun-dant in trophozoites and schizonts, coinciding with themetabolic and synthetic activity of the parasite. We notethat the protein results matched the mRNA levels detectedby microarray analysis[51], suggesting that expression ofboth proteins is primarily regulated at the transcriptionallevel. Next, indirect immunofluorescence staining (Fig. 7B)revealed that PfFKBP35 is predominantly cytoplasmic inthe ring stage. In sharp contrast, a significant amount ofPfFKBP35 translocated to the nucleus as the rings dif-ferentiated into trophozoites and schizonts. PfCN, on theother hand, remained essentially in the cytoplasm when ex-pressed. We draw two important conclusions from these re-sults: (i) PfFKBP35 plays a role in the parasitic nucleusas well as in the cytoplasm, depending on the life stageof the parasite; and (ii) when calcineurin appears in theparasitic cytoplasm, much of the FKBP moves from cy-t lk oft of-f byP
Fig. 7. Relative quantity and location of PfFKBP35 and PfCnA (cal-cineurin). The antibodies have been described in Section2. (A) Immunoblotof 40�g totalP. falciparumprotein in each lane: R = Ring; T = trophozoite;S = schizont; U = a similarly isolated fraction from uninfected RBC. (B)Stage-specific subcellular localization of PfFKBP35 and PfCnA proteins.Stages of parasite growth are indicated on the right. Both proteins (green)were probed with their primary antibody followed by FITC-conjugated sec-ondary antibody. Nuclei (red) were stained with TO-PRO-3 iodide. All pan-els are super-impositions of the two stains, showing intracellular locationof the proteins relative to the nucleus. Note that yellow or orange indicatesco-localization of green and red, signifying nuclear location of the protein,whereas green indicates cytoplasmic location. “Control” pre-immune sera ofthe corresponding rabbits did not react with any protein, as expected; henceno green color.
4. Discussion
The most important findings of this paper can be sum-marized as: (a) PfFKBP35 is the sole FKBP ortholog inP.falciparum, as judged by genome-wide homology search andby in vitro FK506 affinity-binding studies (Figs. 1 and 5B);(b) It is the only TPR-domain protein authenticated in thisparasite since the discovery of the TPR-domain phosphatase,PfPP5; (c) It is a true chaperone with rotamase and proteinfolding activities (Fig. 3); (d) It is a novel FKBP with aninherent calcineurin-inhibitory activity that does not requireFK506 (Fig. 6); (e) It associates with PfHsp90, a major cel-lular chaperone, and its TPR domain is essential for this as-sociation (Fig. 4); (f) The inhibitors of these two proteins aresynergistically antimalarial (Fig. 5A). In what follows, wewill discuss the implications of these findings.
oplasm to the nucleus. It thus appears that the buhe two proteins rarely see each other, which mayer a mechanism by which PfCN evades inhibitionfFKBP35.
R. Kumar et al. / Molecular & Biochemical Parasitology 141 (2005) 163–173 171
FKBP12, the best characterized member of the FKBP fam-ily of immunophilins (Fig. 1), inhibits calcineurin only inthe presence of FK506. This is also true for FKBP51[52]which, like PfFKBP35, contains a TPR domain (Fig. 2).The mechanism of how drug-immunophilin complexes in-hibit CN remains poorly understood. Nonetheless, structuraland mutational analyses have identified at least 25 aminoacid residues that span the composite surface of CN holoen-zyme formed by the CNA and CNB subunits and inter-act with the FKBP12–FK506 complex[7]. Essentially, allthe FKBP residues known to be important for the inhibi-tion of CN by FKBP–FK506 are conserved in PfFKBP35(Fig. 1). It is relevant to add here that both FKBP12 andFKBP51 can bind CN in the absence of drug[52,53], sug-gesting that the FKBPs in general may have an inherentbinding affinity for CN. However, as mentioned above, bothof them must complex with the drug to inhibit the phos-phatase activity CN. Thus, it is unclear why PfFKBP35 doesnot require any drug to inhibit CN. Determination of struc-ture of PfFKBP35–CN co-crystals may shed light on thisissue.
To date, the only FKBP that has been reported to pos-sess a drug-independent CN-inhibitory activity is the humanFKBP38[54], which is included inFig. 1 partly because ofthis commonality and partly because of comparable size anddomain arrangement. However, there are important differ-e acidi inF nga , buta manF ers):Y 38d dingd maya 8a ableo les aF ith-o P38s n thata os ringt -d TMd notf ha bed inP asa
tino-m hus,t al-t f-F N,w asite.
A logical query is how PfCN can ever function as a phos-phatase when there is an apparently abundant pool of intra-cellular FKBP present at all times. Natural cellular inhibitors,in fact, exist for many enzymes including phosphatases; inP. falciparum, we have shown that an aspartate-rich protein,PfARP, is a potent inhibitor for protein phosphatase 2A[38].In general, the interaction between an inhibitor and its tar-get enzyme can be regulated by a variety of mechanismsthat are in turn regulated by physiological signals. As forPfFKBP35, the finding that it moves to the nucleus whenPfCN appears in the cytoplasm suggests that cellular com-partmentalization may serve as the predominant mechanismto segregate the two. Any remaining cytoplasmic pool of Pf-FKBP35 in the trophozoite and schizont stages may continueto keep the activity of PfCN in check, perhaps because ex-cessive dephosphorylation of PfCN substrates is detrimentalto cell cycle progression. The sequences of some medium-size FKBPs contain apparent nuclear localization signals(NLS) or domains that may interact with nuclear proteinsor DNA, although their exact function remains unknown[1].Mammalian FKBP25 was localized in the nucleus but a por-tion of it was also found in the cytoplasm[56]. Moreover,the sequence of mammalian FKBP25 also contains mul-tiple potential phosphorylation sites that may regulate itsfunction or localization[1]. PfFKBP35 sequence does notcontain an easily discernible NLS, and its phosphorylations yto-p es-t c cellc
aceo rme-a -d ilinsi t-r plas-mb ll oft im-m g-i f all1 antg nti-m hers tiono ants ycinw iono
duald r-i -u ationa d ra-p inh sive
nces between FKBP38 and PfFKBP35. First, the aminodentity between the two is only 26% overall. As shownig. 1, all the residues critically important for FK506-bindind PPIase activity are present in the plasmodial proteinnumber of them are non-conservatively replaced in huKBP38. These substitutions are (in PfFKBP35 numb44L, F54Q and F118L. It is thus quite likely that FKBPoes not bind FK506. Moreover, because the drug-binomain coincides with the PPIase domain, FKBP38lso lack PPIase activity[54]. We speculate that FKBP3nd FKBP35 are members of a novel class of FKBPs capf attaining a three-dimensional structure that resembK506-complexed FKBP, and thus inhibit calcineurin wut the need to bind the drug. Second, the human FKBequence has a C-terminal transmembrane (TM) domaillows it to locate to the mitochondria[54]. These studies alsuggested that FKBP38 may inhibit apoptosis by anchohe anti-apoptotic proteins, Bcl-2 and Bcl-xL, to mitochonrial membranes. In contrast, PfFKBP35 is devoid of theomain (Fig. 1), and homologs of Bcl-family members are
ound in the genome of anyPlasmodiumsp. Thus, althougn apoptosis-like phenomenon has been recently descrilasmodium[55], we consider it unlikely that FKBP35 hdirect role in this process.The immunosuppressant FK506 is produced by ac
ycetes and is not naturally present in mammals. The FK506-dependent CN-inhibitory activity of FKBPs,hough clinically important, is physiologically irrelevant. PKBP35, in contrast, is a physiological inhibitor of Chich may have important consequences for the par
tatus remains unknown. Clearly, the mechanism of clasmic to nuclear shuttling of PfFKBP35 needs to be
ablished, but it seems to be connected to the parasitiycle.
A variety of plasmodial proteins traffic to the outer surff the infected erythrocytes and determine nutrient pebility, antigenicity and adhesiveness[57,58]. Recent evience from mammalian cells suggests that immunoph
nteract with cytoplasmic dynein[2], a motor protein for reograde transport to the nucleus and a key player inodial merozoite release and invasion[59]. It remains toe seen whether PfFKBP35 participate in some or a
hese pathways. It is to be noted that although theunophilins display chaperone activity in vitro their biolo
cal importance remains unresolved. In yeast, deletion o2 immunophilins singly or together produced no significrowth defect[60]. Thus, the exact mechanism of the aalarial activity of FK506 and rapamycin must await furt
tudies although PfFKBP35 is a likely target. Identificaf the client proteins of PfFKBP35 is clearly an importtep in this endeavor. In either case, FK506 and rapamill be highly useful and specific tools for further dissectf the cellular role of PfFKBP35.
Regardless of its exact mechanism, the success ofrug therapy in malaria[61] and the synergistic antimala
al activity of FK506 and GA (Fig. 5A) suggest that moleclar chaperones may serve as new targets for combinntimalarials, Chaperone inhibitors such as GA, CsA anamycin are already approved by FDA for clinical useumans for their anti-proliferative and immunosuppres
172 R. Kumar et al. / Molecular & Biochemical Parasitology 141 (2005) 163–173
activities and low toxicity. Within the past few years, an im-pressive number of parasitic chaperones and co-chaperoneshave been either characterized or conjectured by sequence ho-mology, confirming the assumption that these parasites pos-sess thriving protein chaperone machinery. InP. falciparum,well-documented components of the machinery include:three CyPs, one Hsp90, a p23 ortholog, and a Hip ortholog[35,62]. Homology search of theP. falciparumgenome re-veals potentially additional chaperone-family members suchas a second Hsp90[35], an Hsp70 (chr7000093.phat2 ofPlasmoDB) and a fourth CyP of 26 kDa (chrl2.gen518).We note that the C-terminus of the predicted PfHsp70 se-quence has the conserved motif GPTVEEVN which, like theC-terminal MEEVD motif of Hsp90, is known to interactwith TPR [17,18]. Thus, PfFKBP35 may also interact withPfHsp70, further suggestive of its involvement in the chap-erone complex. We suggest that any of these chaperones orco-chaperones can be antimalarial targets.
Acknowledgements
This research was supported in part by NIH grantAI045803 from the National Institute of Allergy and In-fectious Diseases (to S.B.). We are deeply indebted to Dr.Thomas J. Wandless (Stanford University, CA) for the gen-e
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[11] Harding MW. Immunophilins, mTOR, and pharmacodynamic strate-gies for a targeted cancer therapy. Clin Cancer Res 2003;9:2882–6.
[12] Van Duyne GD, Standaert RF, Karplus PA, Schreiber SL, Clardy J.Atomic structures of the human immunophilin FKBP-12 complexeswith FK506 and rapamycin. J Mol Biol 1993;229:105–24.
[13] Gold BG, Villafranca JE. Neuroimmunophilin ligands: the develop-ment of novel neuroregenerative/neuroprotective compounds. CurrTop Med Chem 2003;3:1368–75.
[14] Guo X, Dawson VL, Dawson TM. Neuroimmunophilin ligands ex-ert neuroregeneration and neuroprotection in midbrain dopaminergicneurons. Eur J Neurosci 2001;13:1683–93.
[15] D’Andrea LD, Regan L. TPR proteins: the versatile helix. TrendsBiochem Sci 2003;28:655–62.
[16] Ratajczak T, Ward BK, Minchin RF. Immunophilin chaperones insteroid receptor signalling. Curr Top Med Chem 2003;3:1348–57.
[17] Brinker A, Scheufler C, Von Der Mulbe F, et al. Ligand dis-crimination by TPR domains. Relevance and selectivity of EEVD-recognition in Hsp70× Hop× Hsp90 complexes. J Biol Chem2002;277:19265–75.
[18] Chen S, Sullivan WP, Toft DO, Smith DF. Differential inter-actions of p23 and the TPR-containing proteins Hop, Cyp40,FKBP52 and FKBP51 with Hsp90 mutants. Cell Stress Chaperones1998;3:118–29.
[19] Duina AA, Chang HC, Marsh JA, Lindquist S, Gaber RF. A cy-clophilin function in Hsp90-dependent signal transduction. Science1996;274:1713–5.
[20] Tesic M, Marsh JA, Cullinan SB, Gaber RF. Functional interactionsbetween Hsp90 and the co-chaperones Cns1 and Cpr7 inSaccha-romyces cerevisiae. J Biol Chem 2003;278:32692–701.
[21] Pratt WB, Toft DO. Steroid receptor interactions with heat shockprotein and immunophilin chaperones. Endocr Rev 1997;18:306–60.
[ ep-ling
[ K,mpo-s of30.
[ ro-cep-SA
[ ncet
[ d
hem
[ -Mol
[ ilin
[ ro--–
[ st-rger
[ech-yn-ess,
rous gift of the PfFKBP35 antibody.
eferences
[1] Galat A. Peptidylprolylcis/trans isomerases (immunophilins): bilogical diversity—targets—functions. Curr Top Med Chem 2001315–47.
[2] Pratt WB, Galigniana MD, Harrell JM, DeFranco DB. Role of hsand the hsp90-binding immunophilins in signalling protein moment. Cell Signal 2004;16:857–72.
[3] Houry WA. Chaperone-assisted protein folding in the cell cytoplaCurr Protein Pept Sci 2001;2:227–44.
[4] Blankenship JR, Steinbach WJ, Perfect JR, Heitman J. Teachindrugs new tricks: reincarnating immunosuppressants as antifdrugs. Curr Opin Investig Drugs 2003;4:192–9.
[5] Huai Q, Kim HY, Liu Y, et al. Crystal structure of calcineuricyclophilin-cyclosporin shows common but distinct recogniof immunophilin-drug complexes. Proc Natl Acad Sci U2002;99:12037–42.
[6] Jin L, Harrison SC. Crystal structure of human calcineurin cplexed with cyclosporin A and human cyclophilin. Proc Natl ASci USA 2002;99:13522–6.
[7] Kissinger CR, Parge HE, Knighton DR, et al. Crystal structof human calcineurin and the human FKBP12-FK506-calcinecomplex. Nature 1995;378:641–4.
[8] Liu J, Farmer Jr JD, Lane WS, Friedman J, Weissman I, SchrSL. Calcineurin is a common target of cyclophilin-cyclosporinand FKBP-FK506 complexes. Cell 1991;66:807–15.
[9] Feske S, Okamura H, Hogan PG, Rao A. Ca2+/calcineurin signallingin cells of the immune system. Biochem Biophys Res Com2003;311:1117–32.
10] Weisman R. The fission yeast TOR proteins and the rapamresponse: an unexpected tale. Curr Top Microbiol Imm2004;279:85–95.
22] Riggs DL, Roberts PJ, Chirillo SC, et al. The Hsp90-binding ptidylprolyl isomerase FKBP52 potentiates glucocorticoid signain vivo. EMBO J 2003;22:1158–67.
23] Silverstein AM, Galigniana MD, Chen MS, Owens-Grillo JChinkers M, Pratt WB. Protein phosphatase 5 is a major conent of glucocorticoid receptor.hsp90 complexes with propertiean FK506-binding immunophilin. J Biol Chem 1997;272:16224–
24] Wu B, Li P, Liu Y, et al. 3D structure of human FK506-binding ptein 52: implications for the assembly of the glucocorticoid retor/Hsp90/immunophilin heterocomplex. Proc Natl Acad Sci U2004;101:8348–53.
25] Kremsner PG, Krishna S. Antimalarial combinations. La2004;364:285–94.
26] Hirtzlin J, Farber PM, Franklin RM, Bell A. Molecular anbiochemical characterization of aPlasmodium falciparumcy-clophilin containing a cleavable signal sequence. Eur J Bioc1995;232:765–72.
27] Reddy GR. Cloning and characterization of aPlasmodium falciparum cyclophilin gene that is stage-specifically expressed.Biochem Parasitol 1995;73:111–21.
28] Berriman M, Fairlamb AH. Detailed characterization of a cyclophfrom the human malaria parasitePlasmodium falciparum. BiochemJ 1998;334:437–45.
29] Dobson S, May T, Berriman M, et al. Characterization of ptein Ser/Thr phosphatases of the malaria parasite.Plasmodium falciparum: inhibition of the parasitic calcineurin by cyclophilincyclosporin complex. Mol Biochem Parasitol 1999;99:167–81.
30] Kumar R, Musiyenko A, Oldenburg A, Adams B, Barik S. Potranslational generation of constitutively active cores from laphosphatases in the malaria parasite.Plasmodium falciparum: im-plications for proteomics. BMC Mol Biol 2004;5:6.
31] Kumar R, Musiyenko A, Barik S.Plasmodium falciparumcal-cineurin and its association with heat shock protein 90: manisms for the antimalarial activity of cyclosporin A and sergism with geldanamycin. Mol Biochem Parasitol, in prdoi:10.1016/j.molbiopara.2005.01.012.
R. Kumar et al. / Molecular & Biochemical Parasitology 141 (2005) 163–173 173
[32] Bell A, Wernli B, Franklin RM. Roles of peptidy 1-prolylcis–transisomcrase and calcineurin in the mechanisms of antimalarial ac-tion of cyclosporin A, FK506, and rapamycin. Biochem Pharmacol1994;48:495–503.
[33] Monaghan P, Bell A. Molecular Parasitology Meeting XIII (Abstract#281B). Woods Hole, MA: Marine Biological Laboratory; 2002.
[34] Braun PD, Barglow KT, Lin YM, et al. A bifunctional moleculethat displays context-dependent cellular activity. J Am Chem Soc2003;125:7575–80.
[35] Kumar R, Musiyenko A, Bank S. The heat shock protein 90 ofPlasmodium falciparumand antimalarial activity of its inhibitor, gel-danamycin. Malar J 2003;2:30.
[36] Banumathy G, Singh V, Pavithra SR, Tatu U. Heat shock protein 90function is essential forPlasmodium falciparumgrowth in humanerythrocytes. J Biol Chem 2003;278:18336–45.
[37] Bonnefoy S, Attal G, Langsley G, Tekaia F, Mercereau-PuijalonO. Molecular characterization of the heat shock protein 90 gene ofthe human malaria parasitePlasmodium falciparum. Mol BiochemParasitol 1994;67:157–70.
[38] Dobson S, Kumar R, Bracchi-Ricard V, et al. Characterization ofa unique aspartate-rich protein of the SET/TAF-family in the hu-man malaria parasite,Plasmodium falciparum, which inhibits proteinphosphatase 2A. Mol Biochem Parasitol 2003;126:239–50.
[39] Cinquin O, Christopherson RI, Menz RI. A hybrid plasmid for ex-pression of toxic malarial proteins inEscherichia coli. Mol BiochemParasitol 2001;117:245–7.
[40] Burke E, Barik S. Megaprimer PCR: application in mutagenesis andgene fusion. Methods Mol Biol 2003;226:525–32.
[41] Kumar R, Musiyenko A, Cioffi E, et al. A zinC-binding dual-specificity YVH1 phosphatase in the malaria parasite,Plasmodiumfalciparum, and its interaction with the nuclear protein, pescadillo.
[ hu-with94–
[ si-sy-J
[ cificSA
[ a2.
[ pro-m
[47] Das AK, Cohen PW, Barford D. The structure of the tetratricopep-tide repeats of protein phosphatase 5: implications for TPR-mediatedprotein–protein interactions. EMBO J 1998;17:1192–9.
[48] Bose S, Weikl T, Bugl H, Buchner J. Chaperone function of Hsp90-associated proteins. Science 1996;274:1715–7.
[49] Freeman BC, Toft DO, Morimoto RI. Molecular chaperone ma-chines: chaperone activities of the cyclophilin Cyp-40 and the steroidaporeceptor-associated protein p23. Science 1996;274:1718–20.
[50] Ward BK, Allan RK, Mok D, et al. A structure-based mutationalanalysis of cyclophilin 40 identifies key residues in the core tetra-tricopeptide repeat domain that mediate binding to Hsp90. J BiolChem 2002;277:40799–809.
[51] Bozdech Z, Llinas M, Pulliam BL, Wong ED, Zhu J, DeRisi JL.The transcriptome of the intraerythrocytic developmental cycle ofPlasmodium falciparum. PLoS Biol 2003;1:5.
[52] Li TK, Baksh S, Cristillo AD, Bierer BE. Calcium- and FK506-independent interaction between the immunophilin FKBP51 and cal-cineurin. J Cell Biochem 2002;84:460–71.
[53] Cardenas ME, Hemenway C, Muir RS, Ye R, Fiorentino D, Heit-man J. Immunophilins interact with calcineurin in the absence ofexogenous immunosuppressive ligands. EMBO J 1994;13:5944–57.
[54] Shirane M, Nakayama KI. Inherent calcineurin inhibitor FKBP38targets Bcl-2 to mitochondria and inhibits apoptosis. Nat Cell Biol2003;5:28–37.
[55] Al-Olayan EM, Williams GT, Hurd H. Apoptosis in the malaria pro-tozoan,Plasmodium berghei:a possible mechanism for limiting in-tensity of infection in the mosquito. Int J Parasitol 2002;32:1133–43(Erratum in: Int J Parasitol 2003;33:105).
[56] Riviere S, Menez A, Galat A. On the localization of FKBP25 inT-lymphocytes. FEBS Lett 1993;315:247–51.
[57] Haldar K, Mohandas N, Samuel BU, et al. Protein and lipid traf-Cell
[ lood–86.
[ H,
00.[ nd
ns-i
[ an-ents
[ itslaria
Mol Biochem Parasitol 2004;133:297–310.42] Nair SC, Rimerman RA, Toran EJ, et al. Molecular cloning of
man FKBP51 and comparisons of immunophilin interactionsHsp90 and progesterone receptor. Mol Cell Biol 1997;17:5603.
43] Suzuki Y, Haruki M, Takano K, Morikawa M, Kanaya S. Posble involvement of an FKBP family member protein from a pchrotrophic bacteriumShewanellasp. SIB1 in cold-adaptation. EurBiochem 2004;271:1372–81.
44] Luan S, Albers MW, Schreiber SL. Light-regulated, tissue-speimmunophilins in a higher plant. Proc Natl Acad Sci U1994;91:984–8.
45] Sewell TJ, Lam E, Martin MM, et al. Inhibition of calcineurin bynovel FK-506-binding protein. J Biol Chem 1994;269:21094–10
46] Kay JE. Structure-function relationships in the FK506-bindingtein (FKBP) family of peptidylprolylcis–trans isomerases. BiocheJ 1996;314:361–85.
ficking induced in erythrocytes infected by malaria parasites.Microbiol 2002;4:383–95.
58] Cooke BM, Mohandas N, Coppel RL. The malaria-infected red bcell: structural and functional changes. Adv Parasitol 2001;50:1
59] Fowler RE, Smith AM, Whitehorn J, Williams IT, Bannister LMitchell GH. Microtubule associated motor proteins ofPlasmodiumfalciparummerozoites. Mol Biochem Parasitol 2001;117:187–2
60] Dolinski K, Muir S, Cardenas M, Heitman J. All cyclophilins aFK506 binding proteins are, individually and collectively, dispeable for viability in Saccharomyces cerevisim. Proc Natl Acad ScUSA 1997;94:13093–8.
61] Srivastava IK, Vaidya AB. A mechanism for the synergistictimalarial action of atovaquone and proguanil. Antimicrob AgChemother 1999;43:1334–9.
62] Wiser MF. A Plasmodiumhomologue of co-chaperone p23 anddifferential expression during the replicative cycle of the maparasite. Parasitol Res 2003;90:166–70.
