A single-domain cyclophilin from Leishmania donovani reactivates soluble aggregates of adenosine kinase by isomerase-independent chaperone function

Disaggregation and reactivation of aggregated proteins by chaperones is well established. However, little is known regarding such kind of function of single-domain small cyclophilins (CyPs). Here we demonstrate that, with increasing concentrations, fully active adenosine kinase (AdK) of Leishmania donovani tends to form soluble aggregates, resulting in inactivation. Using this inactive enzyme as the substrate, it is shown that a CyP from L. donovani (LdCyP) alone can cause complete disaggregation, leading to reactivation of the enzyme. The reactivating ability of LdCyP remains unaffected even in the presence of cyclosporin A and macromolecular crowding agents. The reactivation occurs noncatalytically and is reversible. A truncated LdCyP, devoid of 88 amino acids from the N terminus, is found to be required in near stoichiometric proportion to reactivate AdK, suggesting essentiality of the C-terminal region. Gel filtration and light-scattering experiments together with protein cross-linking studies revealed that both full-length LdCyP and the truncated form directly interact with AdK and convert oligomeric forms of the enzyme to monomeric state. Homology modeling studies suggest that the exposed hydrophobic residues of LdCyP, by interacting with solvent-accessible hydrophobic surface of AdK, pull apart its aggregated inactive oligomers to functional monomers. Clearly, the results are consistent with the interpretation that the higher efficiency of the truncated LdCyP is most likely due to increased exposure of the hydrophobic residues on its surface. These observations, besides establishing L. donovani AdK as one of the model enzymes to study aggregation-disaggregation of proteins, raise the possibility that single-domain small CyPs, under physiological conditions, may regulate the activity of aggregation-prone proteins by ensuring their disaggregation.     

Amino acid residues of Leishmania donovani cyclophilin key to interaction with its adenosine kinase: biological implications

Cyclophilins (CyPs), by interacting with a variety of proteins, often modulate their biological activities and thus have been implicated in several cellular functions. However, mechanisms that determine such interactions are poorly understood. We earlier reported that an endoplasmic reticulum (ER)-located cyclophilin (LdCyP) from the purine auxotrophic parasitic protozoan Leishmania donovani reactivated its adenosine kinase (AdK). The AdK-reactivating property of LdCyP was however abolished at high ionic strength but not by nonionic detergents. Modeling of LdCyP, based on its crystal structure solved at 1.97 A resolution, revealed several solvent-exposed hydrophobic and charged residues. Mutagenesis of several of such solvent-exposed residues was performed and their corresponding activities with regard to their (i) AdK reactivation property, (ii) ability to form complex with the enzyme, (iii) capacity to induce red shift in the intrinsic tryptophan fluorescence maxima of AdK, and (iv) efficiency to withdraw the ADP inhibition from the AdK-mediated reaction were compared to the wild-type protein. Results indicated that while the replacement of R147 with either A or D severely impaired all of the above characteristics displayed by the wild-type LdCyP, the effect of mutating K114 and K153 was although relatively less but nevertheless noticeable. Alteration of other exposed hydrophobic and charged residues apparently did not have any discernible effect. Under the condition of cellular stress, the ER-located LdCyP is released into the cytoplasm with concomitant increase both in the specific activity of the cytosol-resident AdK and the uptake of radiolabeled Ado into the cells. These experiments, besides demonstrating the importance of the positive charge, identified R147 as the most crucial residue in the LdCyP-AdK interaction and provide evidence for the stress-induced retrograde translocation of LdCyP from the ER to the cytoplasm. A possible implication of this interaction in the life cycle of the parasite is proposed.     

Characterisation of a cyclophilin isoform in Trypanosoma cruzi

The immunosuppressive drug cyclosporin A (CsA) has shown antiparasitic activity against several protozoans and helminths, when complexed to proteins called cyclophilins (CyPs). In this paper, the molecular characterisation of one member of the CyP family in Trypanosoma cruzi is reported. TcCyP19 gene proved to be highly conserved compared to CyPs from other organisms and was highly homologous to a Trypanosoma brucei brucei CyPA. This gene was expressed in Escherichia coli and the purified recombinant protein exhibited a peptidyl prolyl cis-trans isomerase activity that was inhibited by CsA (IC(50) = 18.4 + /-0.8 nM). The TcCyP19 gene was located on two chromosomal bands in T. cruzi CL Brener clone.     

Cyclosporin A Treatment of Leishmania donovani Reveals Stage-Specific Functions of Cyclophilins in Parasite Proliferation and Viability

Background

Cyclosporin A (CsA) has important anti-microbial activity against parasites of the genus Leishmania, suggesting CsA-binding cyclophilins (CyPs) as potential drug targets. However, no information is available on the genetic diversity of this important protein family, and the mechanisms underlying the cytotoxic effects of CsA on intracellular amastigotes are only poorly understood. Here, we performed a first genome-wide analysis of Leishmania CyPs and investigated the effects of CsA on host-free L. donovani amastigotes in order to elucidate the relevance of these parasite proteins for drug development.

Methodology/Principal Findings

Multiple sequence alignment and cluster analysis identified 17 Leishmania CyPs with significant sequence differences to human CyPs, but with highly conserved functional residues implicated in PPIase function and CsA binding. CsA treatment of promastigotes resulted in a dose-dependent inhibition of cell growth with an IC50 between 15 and 20 µM as demonstrated by proliferation assay and cell cycle analysis. Scanning electron microscopy revealed striking morphological changes in CsA treated promastigotes reminiscent to developing amastigotes, suggesting a role for parasite CyPs in Leishmania differentiation. In contrast to promastigotes, CsA was highly toxic to amastigotes with an IC50 between 5 and 10 µM, revealing for the first time a direct lethal effect of CsA on the pathogenic mammalian stage linked to parasite thermotolerance, independent from host CyPs. Structural modeling, enrichment of CsA-binding proteins from parasite extracts by FPLC, and PPIase activity assays revealed direct interaction of the inhibitor with LmaCyP40, a bifunctional cyclophilin with potential co-chaperone function.

Conclusions/Significance

The evolutionary expansion of the Leishmania CyP protein family and the toxicity of CsA on host-free amastigotes suggest important roles of PPIases in parasite biology and implicate Leishmania CyPs in key processes relevant for parasite proliferation and viability. The requirement of Leishmania CyP functions for intracellular parasite survival and their substantial divergence form host CyPs defines these proteins as prime drug targets.     

Crystal structure of the complex of brugia malayi cyclophilin and cyclosporin A

The resistance of the human parasite Brugia malayi to the antiparasitic activity of cyclosporin A (CsA) may arise from the presence of cyclophilins with relatively low affinity for the drug. The structure of the complex of B. malayi cyclophilin (BmCYP-1) and CsA, with eight independent copies in the asymmetric unit, has been determined at a resolution of 2.7 A. The low affinity of BmCYP-1 for CsA arises from incomplete preorganization of the binding site so that the formation of a hydrogen bond between His132 of BmCYP-1 and N-methylleucine 9 of CsA is associated with a shift in the backbone of approximately 1 A in this region.     

Delineation of the calcineurin-interacting region of cyclophilin B

The immunosuppressant drug cyclosporin A (CsA) inhibits T-cell function by blocking the phosphatase activity of calcineurin. This effect is mediated by formation of a complex between the drug and cyclophilin (CyP), which creates a composite surface able to make high-affinity contacts with calcineurin. In vitro, the CyPB/CsA complex is more effective in inhibiting calcineurin than the CyPA/CsA and CyPC/CsA complexes, pointing to fine structural differences in the calcineurin-binding region. To delineate the calcineurin-binding region of CyPB, we mutated several amino acids, located in two loops corresponding to CyPA regions known to be involved, as follows: R76A, G77H, D155R, and D158R. Compared to wild-type CyPB, the G77H, D155R, and D158R mutants had intact isomerase and CsA-binding activities, indicating that no major conformational changes had taken place. When complexed to CsA, they all displayed only reduced affinity for calcineurin and much decreased inhibition of calcineurin phosphatase activity. These results strongly suggest that the three amino acids G77, D155, and D158 are directly involved in the interaction of CyPB/CsA with calcineurin, in agreement with their exposed position. The G77, D155, and D158 residues are not maintained in CyPA and might therefore account for the higher affinity of the CyPB/CsA complex for calcineurin.     

A proposed molecular model for the interaction of calcineurin with the cyclosporin A-cyclophilin A complex.

Cyclosporin A (CsA) and FK506 are potent natural product immunosuppressants that induce their biological effects by forming an initial complex with cytosolic proteins termed immunophilins. These drug immunophilin complexes then bind to and inhibit the serine/threonine protein phosphatase calcineurin (CN). Two classes of immunophilin have been identified with cyclophilins (CyP's) being proteins specifically binding CsA and FKBPs specifically binding FK506. Solution and crystal structures of various CsA-CyP and FK506-FKBP complexes have been determined and show no apparent structural similarity between the two classes of drug protein complexes. These findings raise the question as to how, given their structural differences, these two complexes can both inhibit CN. While the crystal structure of the FK506-FKBP12-CN complex has been reported, no structure for a CsA-CyP CN complex has been determined. Here are reported studies that use various modelling strategies to construct a model for the interaction of the cyclosporin A- cyclophilin A complex with calcineurin. The first stage of constructing this model consisted of using conformational comparison of CsA and FK506, GRID and GROUP analysis and restrained molecular dynamics to dock CsA into the FK506 binding site of the FK506-FKBP12-CN structure. An initial model for the CsA-CyPA-CN complex was then constructed by superimposing the structure of the CsA-CyPA complex onto the docked CsA molecule. This model was then optimised with molecular dynamics simulations run on sterically clashing regions. The validity of the model for the CsA-CyPA-CN complex was then examined with respect to the effect of chemical modifications to CsA and amino acid substitutions within CyPA on the ability of the drug-immunophilin complex to inhibit calcineurin.     

Reversal of ADP-mediated aggregation of adenosine kinase by cyclophilin leads to its reactivation

Cyclophilins have been implicated in several important cellular functions. Our earlier results showed that reactivation of adenosine kinase (AdK) by CyP (LdCyP) from the parasitic protozoa Leishmania donovani is accompanied with disaggregation of the enzyme [Chakraborty, A., et al. (2002) J. Biol. Chem. 277, 47451-47460; Chakraborty, A., et al. (2004) Biochemistry 43, 11862-11872]. However, it remained to be known why the enzyme displayed progressive inhibition during the time-dependent reaction and what LdCyP does to prevent and/or reverse the inhibition. Herein, we demonstrate that one of its reaction products, ADP but not AMP, facilitates the formation of AdK aggregates, leading to its inactivation. Further studies revealed that LdCyP reactivates the enzyme by withdrawing the ADP inhibition. To investigate the molecular mechanism, the intrinsic tryptophan fluorescence and polarization of AdK were monitored in the presence of either LdCyP or ADP and in combination thereof. Whereas in the presence of LdCyP the tryptophan fluorescence emission maxima of AdK exhibited a red shift, ADP had a quenching effect. However, both the red shift and quenching became less noticeable when one (W234) of the two tryptophan residues of AdK was altered, indicating W234 fluorescence is relatively more sensitive to both LdCyP and ADP binding. Kinetic measurements indicated that LdCyP-facilitated reactivation of AdK is accompanied with a concomitant increase in the KD of ADP but not of AMP. Interestingly, addition of myokinase (MK) and pyruvate kinase (PK) along with phosphoenolpyruvate, either singly or in conjunction, to the AdK reaction mixture led to its reactivation. The effect of PK but not of MK could be substituted by CyP and vice versa. Taken together, the results suggest that LdCyP-induced reactivation occurs due to conformational reorientation of AdK in a manner that decreases the affinity of the enzyme for ADP with consequent relief from the ADP-mediated aggregation.     

Active-site residues of cyclophilin A are crucial for its incorporation into human immunodeficiency virus type 1 virions.

Human immunodeficiency virus type 1 (HIV-1) incorporates the cellular peptidyl-prolyl cis-trans isomerase cyclophilin A (CyPA), the cytosolic receptor for the immunosuppressant cyclosporin A (CsA). CsA inhibits the incorporation of CyPA and reduces HIV-1 virion infectivity but is inactive against closely related primate lentiviruses that do not interact with CyPA. The incorporation of CyPA into HIV-1 virions is mediated by a specific interaction with a proline-containing, solvent-exposed loop in the capsid (CA) domain of the Gag polyprotein. CsA, which disrupts the interaction with CA, binds at the active site of CyPA. To test whether active-site residues are also involved in the interaction with HIV-1 CA, we used a panel of previously characterized active-site mutants of human CyPA. Expression vectors for epitope-tagged wild-type and mutant CyPA were transfected into COS-gamma cells along with HIV-1 proviral DNA, and the virions produced were analyzed for the presence of tagged proteins. Cotransfection of the wild-type expression vector led to the incorporation of readily detectable amounts of epitope-tagged CyPA into HIV-1 virions. One CyPA mutant with a substantially decreased sensitivity to CsA was incorporated with wild-type efficiency, demonstrating that the requirements for binding to CsA and to HIV-1 CA are not identical. The remaining six CyPA mutants were incorporated with markedly reduced efficiency, providing in vivo evidence that HIV-1 CA interacts with the active site of CyPA.     

NMR studies of [U-C]cyclosporin A bound to human cyclophilin B

NMR data (¹H and ¹³C chemical shifts, NOEs) on [[U-¹³C]cyclosporin A bound to cyclophilin B were compared to previously published data on the [U-¹³C]CsA/CyPA complex [Fesik et al., (1991) Biochemistry 30, 6574–6583]. Despite only 64% sequence identity between CyPA and CyPB, the conformation and active site environment of CsA when bound to CyPA and CyPB are nearly identical as judged by the similarity of the NMR data.     

环孢霉素A通过ROS-Cyclophilin A-ERK1/2信号途径抑制人脐静脉内皮细胞与中性粒细胞粘附

为研究环孢霉素A(cyclosporin A,CsA)对缺氧／复氧诱导人脐静脉内皮细胞(ECV-304)与中性粒细胞粘附的影响,本工作以缺氧／复氧诱导粘附为模型,采用D-N-乙酰氨基己糖苷酶比色法检测粘附率,流式细胞术检测ECV-304细胞表面粘附分子E-选择素(E-selectin)、细胞间粘附分子-1(ICAM-1)的表达,Fenton反应测定活性氧(reactive oxygen species,ROS)的含量,Westera-blot法检测ECV-304细胞亲环素A(cyclophilin A,CyPA)、磷酸化及总细胞外信号调节激酶(ERK1／2)蛋白的表达。结果发现,ECV-304细胞经缺氧／复氧处理后,ROS释放增多,E-selectin、ICAM-1的表达上调,其表面中性粒细胞的粘附增加,CsA能显著抑制缺氧／复氧的上述作用。缺氧／复氧后,CyPA蛋白表达明显上调,ERK1/2显著活化,细胞总ERK1/2蛋白表达无明显改变。CyPA抑制剂CsA以及CyPA反义寡核苷酸均明显减轻缺氧／复氧诱导的ERK1/2激活,显著减少ECV-304细胞与中性粒细胞柑附。ERK112信号通路特异性阻断剂PD98059亦显著抑制ECV-304细胞与中性粒细胞的粘附。上述结果提示,CsA抑制缺氧气／复氧诱导的ECV-304细胞与中性粒细胞粘附,并可能通过抑制ROS-Cyclophilin A-ERK112的信号转导途径实现。     

Interaction of the retinoblastoma gene product,RB, with cyclophilin A negatively affects cyclosporin-inhibited NFAT signaling

The retinoblastoma susceptibility gene product, p105Rb (RB), is generally believed to be an important regulator in the control of cell growth, differentiation, and apoptosis. Several cellular factors that form complexes with RB and exert their cellular regulatory functions have been identified, such as the newly identified RB:cyclophilin A (CypA) complex. The physical interactions between RB and CypA were demonstrated by glutathione S-transferase affinity matrix binding assays and immunoprecipitation, followed by Western blot analyses. The N-terminal region of CypA mediated the interaction with RB, whereas the region upstream of the A-pocket of RB was required for binding to CypA. Ectopic expression of RB into Jurkat cells partially blocks the function of cyclosporin (CsA) to inhibit nuclear factor for activation of T cell (NFAT) activation by phorbol ester (PMA) plus ionomycin A (IA), suggesting that RB may prevent CsA inhibition of T lymphocyte activation. These results are further evidenced by the effect of RB on both calcineurin (CN) and NFAT binding activity in vitro, suggesting that the interaction of RB with CypA interferes with the CsA:CypA complex and blocks CsA-inhibited CN activity. These data reveal the functional link between RB and CypA and their involvement in T cell activation signaling.     

Kaempferol: a new immunosuppressant of calcineurin

Calcineurin (CN), the Ca(2+)/calmodulin (CaM)-dependant protein phosphatase, is the target for immunosuppressive drugs cyclosporine A (CsA) and FK506. These immunosuppressants can inhibit CN activity after binding with respective immunophilins. Based on the model of screening by using p-nitrophenyl phosphate as a substrate for preliminary screening and (32)P-labeled 19-residue phosphopeptide as a specific substrate for final determination, we found Kaempferol, a natural flavonol, could inhibit CN activity in purified enzyme and Jurkat T-cells. Unlike CsA and FK506, CN inhibition by kaempferol is independent of matchmaker protein and the inhibitory manner is noncompetitive. Through investigation of inhibitions for CNA and a series of its truncated mutants, we suggested that Kaempferol could directly act on the catalytic domain. Data also indicated that the CN inhibition by kaempferol could be enhanced when the enzyme was activated in the presence of CaM and CNB. CNB is necessary for mediating inhibition of enzyme by kaempferol. The result of RT-PCR also indicated that kaempferol had an inhibitory activity against IL-2 gene expression in activated Jurkat cells. All data suggested that kaempferol could be a new immunosuppressant of CN.     

Parasite cyclophilins and antiparasite activity of cyclosporin A

Cyclosporin A (CsA) was initially developed as an immunosuppressive drug. In the past several years, it has been shown to possess antiparasite activity independent of the immune system. It is not known how the drug exerts these antiparasite effects, or why it is stage and/or species specific. The answers may lie in the enzymatic function of cyclophilins. The cyclophilins are a growing family of proteins that exhibit peptidyl-prolyl cis-trans isomerase (PPiase) activity and bid CsA to varying degrees. PPiases have been shown to play a role in the folding of many essential proteins. Antony Page, Sanjay Kumar and Clotilde Carlow here review parasite cyclophilins and their association with CsA. The possible biological function of parasite cyclophilins and their potential role in future drug discovery are also discussed.     

Sanglifehrin A acts as a potent inhibitor of the mitochondrial permeability transition and reperfusion injury of the heart by binding to cyclophilin-D at a different site from cyclosporin A

Cyclosporin A (CsA) inhibits opening of the mitochondrial permeability transition pore (MPTP), a critical event in some forms of necrotic and apoptotic cell death, by binding to cyclophilin D (CyP-D) and inhibiting its peptidyl-prolyl cis-trans isomerase (PPIase) activity. Sanglifehrin A (SfA), like CsA, exerts its immunosuppressive action by binding to cyclophilin A but at a different site from CsA, and unlike the latter, SfA does not inhibit calcineurin activity. Here we demonstrate that SfA inhibits the PPIase activity of CyP-D (K(0.5) 2 nm) and acts as a potent inhibitor of MPTP opening under both energized and de-energized conditions. However, unlike CsA, the dose-response curve for inhibition by SfA is sigmoidal rather than hyperbolic, suggesting a multimeric structure for the MPTP with cooperativity between subunits. Furthermore, SfA does not prevent CyP-D binding to submitochondrial particles or detergent-solubilized adenine nucleotide translocase (ANT), implying that CyP-D binding to the ANT does not require PPIase activity but pore opening does. Once bound to the MPTP, SfA is not readily dissociated, and inhibition of pore opening is maintained following extensive washing. To investigate the potential of SfA as an inhibitor of cell death in vivo, we used the Langendorff perfused rat heart. SfA caused a time-dependent inhibition of the MPTP that was maintained on mitochondrial isolation to a greater extent than was CsA inhibition. We demonstrate that SfA, like CsA, improves the recovery of left ventricular developed pressure during reperfusion after 30 min of global ischemia and greatly reduces lactate dehydrogenase release, implying inhibition of necrotic damage. Because SfA does not inhibit calcineurin activity, our data suggest that it may be more desirable than CsA for protecting tissues recovering from ischemic episodes and for studying the role of the MPTP in cell death.     

Analysis of cyclosporin A and a set of analogs as inhibitors of a T. cruzi cyclophilin by docking and molecular dynamics

Cyclophilins (CyPs) are enzymes involved in protein folding. In Trypanosoma cruzi (T. cruzi), the most abundantly expressed CyP is the isoform TcCyP19. It has been shown that TcCyP19 is inhibited by the immunosuppressive drug cyclosporin A (CsA) and analogs, which also proved to have potent trypanosomicidal activity in vitro. In this work, we continue and expand a previous study on the molecular interactions of CsA, and a set of analogs modeled in complexes with TcCyP19. The modeled complexes were used to evaluate binding free energies by molecular dynamics (MD), applying the Linear Interaction Energy (LIE) method. In addition, putative binding sites were identified by molecular docking. In our analysis, the binding free energy calculations did not correlate with experimental data. The heterogeneity of the non-bonded energies and the variation in the pattern of hydrogen bonds suggest that the systems may not be suitable for the application of the LIE method. Further, the docking calculations identified two other putative binding sites with comparable scoring energies to the active site, a fact that may also explain the lack of correlation found. Kinetic experiments are needed to confirm or reject the multiple binding sites hypothesis. In the meantime, MD simulations at the alternative sites, employing other methods to compute binding free energies, might be successful at finding good correlations with the experimental data.     

Isomerase-independent chaperone function of cyclophilin ensures aggregation prevention of adenosine kinase both in vitro and under in vivo conditions

Using inactive aggregates of adenosine kinase (AdK) from Leishmania donovani as the model substrate, we recently demonstrated that a cyclophilin (LdCyP) from the same source in an isomerase-independent fashion reactivated the enzyme in vitro by disaggregating its inactive oligomers [Chakraborty et al. (2002) J. Biol. Chem. 277, 47451-47460]. Besides disrupting preformed aggregates, LdCyP also prevents reaggregation of the newly formed active protein that is generated after productive refolding from its urea-denatured state. To investigate possible physiological implications of such phenomena, a unique expression system that simultaneously induces both AdK and LdCyP in naturally AdK-deficient Escherichia coli, was developed. Both in vitro and in vivo experiments revealed that oligomerization is an inherent property of this particular enzyme. In vivo protein cross-linking studies, activity determination analysis and Ado phosphorylation experiments carried out in cells coexpressing both the proteins unequivocally demonstrated that, similar to the phenomena observed in vitro, aggregates of the enzyme formed in vivo are able to interact with both LdCyP and its N-terminal truncated form (N(22-88)DEL LdCyP) in a crowded intracellular environment, resulting in aggregation prevention and reactivation of the enzyme. Our results indicate that the isomerase-independent chaperone function of LdCyP, detected in vitro, participates in vivo as well to keep aggregation-prone proteins in a monomeric state. Furthermore, analogous to yeast/bacterial two-hybrid systems, development of this simple coexpression system may help in the confirmation of interaction of two proteins under simulated in vivo conditions.     

A mitochondrial-targeted cyclosporin A with high binding affinity for cyclophilin D yields improved cytoprotection of cardiomyocytes

Mitochondrial CyP-D (cyclophilin-D) catalyses formation of the PT (permeability transition) pore, a key lesion in the pathogenesis of I/R (ischaemia/reperfusion) injury. There is evidence [Malouitre, Dube, Selwood and Crompton (2010) Biochem. J. 425, 137-148] that cytoprotection by the CyP inhibitor CsA (cyclosporin A) is improved by selective targeting to mitochondria. To investigate this further, we have developed an improved mtCsA (mitochondrial-targeted CsA) by modifying the spacer linking the CsA to the TPP+ (triphenylphosphonium) (mitochondrial-targeting) cation. The new mtCsA exhibits an 18-fold increase in binding affinity for CyP-D over the prototype and a 12-fold increase in potency of inhibition of the PT in isolated mitochondria, owing to a marked decrease in non-specific binding. The cytoprotective capacity was assessed in isolated rat cardiomyocytes subjected to transient glucose and oxygen deprivation (pseudo-I/R). The new mtCsA was maximally effective at lower concentrations than CsA (3-15 nM compared with 50-100 nM) and yielded improved cytoprotection for up to 3 h following the pseudo-ischaemic insult (near complete compared with 40%). These data indicate the potential value of selective CyP-D inhibition in cytoprotection.