Chaperone-like activity of peptidyl-prolyl cis-trans isomerase during creatine kinase refolding

Porcine kidney 18 kD peptidyl-prolyl cis-trans isomerase (PPIase) belongs to the cyclophilin family that is inhibited by the immunosuppressive drug cyclosporin A. The chaperone activity of PPIase was studied using inactive, active, and alkylated PPIase during rabbit muscle creatine kinase (CK) refolding. The results showed that low concentration inactive or active PPIase was able to improve the refolding yields, while high concentration PPIase decreased the CK reactivation yields. Aggregation was inhibited by inactive or active PPIase, and completely suppressed at 32 or 80 times the CK concentration (2.7 microM). However, alkylated PPIase was not able to prevent CK aggregation. In addition, the ability of inactive PPIase to affect CK reactivation and prevent CK aggregation was weaker than that of active PPIase. These results indicate that PPIase interacted with the early folding intermediates of CK, thus preventing their aggregation in a concentration-dependent manner. PPIase exhibited chaperone-like activity during CK refolding. The results also suggest that the isomerase activity of PPIase was independent of the chaperone activity, and that the proper molar ratio was important for the chaperone activity of PPIase. The cysteine residues of PPIase may be a peptide binding site, and may be an essential group for the chaperone function.     

X-ray structure of peptidyl-prolyl cis-trans isomerase A from Mycobacterium tuberculosis.

Peptidyl-prolyl cis-trans isomerases (EC 5.2.1.8) catalyse the interconversion of cis and trans peptide bonds and are therefore considered to be important for protein folding. They are also thought to participate in processes such as signalling, cell surface recognition, chaperoning and heat-shock response. Here we report the soluble expression of recombinant Mycobacterium tuberculosis peptidyl-prolyl cis-trans isomerase PpiA in Escherichia coli, together with an investigation of its structure and biochemical properties. The protein was shown to be active in a spectrophotometric assay, with an estimated kcat/Km of 2.0 x 10(6) m(-1).s(-1). The X-ray structure of PpiA was solved by molecular replacement, and refined to a resolution of 2.6 A with R and Rfree values of 21.3% and 22.9%, respectively. Comparisons to known structures show that the PpiA represents a slight variation on the peptidyl-prolyl cis-trans isomerase fold, previously not represented in the Protein Data Bank. Inspection of the active site suggests that specificity for substrates and cyclosporin A will be similar to that found for most other enzymes of this structural family. Comparison to the sequence of the second M. tuberculosis enzyme, PpiB, suggests that binding of peptide substrates as well as cyclosporin A may differ in that case.     

A Candida albicans homolog of a human cyclophilin gene encodes a peptidyl-prolyl cis-trans isomerase

A Candida albicans cDNA and its genomic counterpart were isolated from lambda phage libraries using a human T-cell cyclophilin (Cyp) cDNA as a hybridization probe. The clones contain a 486-bp open reading frame predicting a 162-amino acid, approx. 18 kDa protein which is similar in size to, and which shares 68 and 81% homology with, human T-cell Cyp and cytosolic Saccharomyces cerevisiae Cyp, respectively. Northern blots show the presence of a single mRNA species of about 800 bp. However, genomic Southern blots suggest the presence of at least one other Cyp-related gene in C. albicans. The cDNA was engineered for expression in Escherichia coli, and the resulting recombinant protein, like mammalian Cyps, exhibited a peptidyl-prolyl cis-trans isomerase (PPIase) activity which was sensitive to inhibition by cyclosporin A in vitro. These results indicate that the gene which we have cloned encodes a C. albicans Cyp. We designate this gene CYP1 (cyclophilin). Interestingly, the predicted C. albicans protein contains only two cysteine residues which do not align with any of the four cysteines conserved among mammalian Cyps. This suggests that the PPIase catalytic mechanism may not involve an enzyme-bound hemithioorthoamide, as previously reported for porcine Cyp.     

Secretion by Trypanosoma cruzi of a peptidyl-prolyl cis-trans isomerase involved in cell infection.

Macrophage infectivity potentiators are membrane proteins described as virulence factors in bacterial intracellular parasites, such as Legionella and Chlamydia. These factors share amino acid homology to eukaryotic peptidyl-prolyl cis-trans isomerases that are inhibited by FK506, an inhibitor of signal transduction in mammalian cells with potent immunosuppressor activity. We report here the characterization of a protein released into the culture medium by the infective stage of the protozoan intracellular parasite Trypanosoma cruzi. The protein possesses a peptidyl-prolyl cis-trans isomerase activity that is inhibited by FK506 and its non-immunosuppressing derivative L-685,818. The corresponding gene presents sequence homology with bacterial macrophage infectivity potentiators. The addition of the protein, produced heterologously in Escherichia coli, to cultures of trypomastigotes and simian epithelial or HeLa cells enhances invasion of the mammalian cells by the parasites. Antibodies raised in mice against the T.cruzi isomerase greatly reduce infectivity. A similar reduction of infectivity is obtained by addition to the cultures of FK506 and L-685,818. We concluded that the T.cruzi isomerase is involved in cell invasion.     

Cyclosporin A binding to Mycobacterium tuberculosis peptidyl-prolyl cis-trans isomerase A--investigation by CD, FTIR and fluorescence spectroscopy

Circular dichroism and resolution-enhanced Fourier transform infrared reveal induction of secondary structural elements on peptidyl-prolyl cis–trans isomerase A (PpiA) from Mycobacterium tuberculosis upon binding cyclosporin A (CsA). Thermal denaturation shows aggregation of PpiA at higher temperatures (>70 °C) and CsA fails to impart stabilization in protein structure. However, CsA stabilizes PpiA structure in urea denaturation. In presence/absence of CsA, urea-induced reversible unfolding of secondary and tertiary structures follows two-state and three-state transition, respectively. The chemical unfolding results also demonstrate that loss in the tertiary structure precedes the loss in secondary structure both in presence and absence of CsA at the initial stages. Fluorescence quenching suggests presence of a positive barrier around tryptophan microenvironment of PpiA.     

Chloroplast cyclophilin is a target protein of thioredoxin. Thiol modulation of the peptidyl-prolyl cis-trans isomerase activity

Chloroplast cyclophilin has been identified as a potential candidate of enzymes in chloroplasts that are regulated by thioredoxin (Motohashi, K., Kondoh, A., Stumpp, M. T., and Hisabori, T. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 11224-11229). In the present study we found that the peptidyl-prolyl cis-trans isomerase activity of cyclophilin is fully inactivated in the oxidized form. Reduction of cyclophilin by thioredoxin-m recovered the isomerase activity. Two crucial disulfide bonds were determined by disulfide-linked peptide mapping. The relevance of these cysteines for isomerase activity was confirmed by the mutagenesis studies. Because four cysteine residues in Arabidopsis thaliana cyclophilin were conserved in the isoforms from several organisms, it appears that this redox regulation must be one of the common regulation systems of cyclophilin.     

The influence of peptidyl-prolyl cis-trans isomerase on the in vitro folding of type III collagen

Peptidyl-prolyl cis-trans isomerase was extracted from pig kidney cortex and partially purified. Enzyme activity was monitored against the cis-trans isomerization of succinyl-Ala-Ala-Pro-Phe-methylcoumaryl amide by means of a two-step process using chymotrypsin as the trans cleaving activity. The in vitro refolding of denatured type III collagen, which is rate-limited by the cis-trans isomerization of peptide bonds, was studied in the presence of peptidyl-prolyl cis-trans isomerase by optical rotatory dispersion and by resistance to tryptic digestion. A 3-fold increase in the initial rate of folding was observed compared to the uncatalyzed refolding. This rate increase is comparable to the rate increase found for the CT-phase in the refolding of urea-denatured ribonuclease A, but it is smaller than the increase in the rate of isomerization of succinyl-Ala-Ala-Pro-Phe-methylcoumarylamide.     

The cytosolic-binding protein for the immunosuppressant FK-506 is both a ubiquitous and highly conserved peptidyl-prolyl cis-trans isomerase.

We have recently isolated an abundant cytosolic protein from human T-cells which specifically binds the immunosuppressive agent, FK-506. The FK-506-binding protein (FKBP) is a member of a novel class of proteins possessing peptidyl-prolyl cis-trans isomerase activity. These proteins are believed to play an important role in accelerating the rate at which proteins fold into their native conformations. In the present study, we demonstrate that FKBP is not a lymphoid-specific protein, but is widely distributed and phylogenically conserved. FKBP, purified from three sources (a human T-lymphocyte cell line JURKAT, bovine calf thymus, and Saccharomyces cerevisiae) exhibit identical molecular weights, immunological cross-reactivities, and a high degree of NH2-terminal amino acid sequence homology. In addition, FKBP from all sources possesses peptidyl-prolyl cis-trans isomerase activity which can be specifically inhibited by FK-506. We conclude that FKBP may serve an important biological function in all eukaryotic cells.     

Transgenic mice overexpressing cyclophilin A are resistant to cyclosporin A-induced nephrotoxicity via peptidyl-prolyl cis-trans isomerase activity

Cyclosporin A (CsA) suppresses immune reaction by inhibiting calcineurin activity after forming complex with cyclophilins and is currently widely used as an immunosuppressive drug. Cyclophilin A (CypA) is the most abundantly and ubiquitously expressed family member of cyclophilins. We previously showed that CsA toxicity is mediated by ROS generation as well as by inhibition of peptidyl-prolyl cis-trans isomerase (PPIase) activity of CypA in CsA-treated myoblasts [FASEB J. 16 (2002) 1633]. Since CsA-induced nephrotoxicity is the most significant adverse effect in its clinical utilization, we here investigated the role of CsA inhibition of CypA PPIase activity in its nephrotoxicity using transgenic mouse models. Transgenic mice of either wild type (CypA/wt) or R55A PPIase mutant type (CypA/R55A), a dominant negative mutant of CypA PPIase activity, showed normal growth without any apparent abnormalities. However, CsA-induced nephrotoxicity was virtually suppressed in CypA/wt mice, but exacerbated in CypA/R55A mice, compared to that of littermates. Also, life expectancy was extended in CypA/wt mice and shortened in CypA/R55A mice during CsA administration. Besides, CsA-induced nephrotoxicity was inversely related to the levels of catalase expression and activity. In conclusion, our data provide in vivo evidence that supplement of CypA PPIase activity allows animal's resistance toward CsA-induced nephrotoxicity.     

Domain structure and denaturation of a dimeric Mip-like peptidyl-prolyl cis-trans isomerase from Escherichia coli

FKBP22, a protein expressed by Escherichia coli, possesses PPIase (peptidyl-prolyl cis-trans isomerase) activity, binds FK506 (an immunosuppressive drug), and shares homology with Legionella Mip (a virulence factor) and its related proteins. To understand the domain structure and the folding-unfolding mechanism of Mip-like proteins, we investigated a recombinant E. coli FKBP22 (His-FKBP22) as a model protein. Limited proteolysis indicated that His-FKBP22 harbors an N-terminal domain (NTD), a C-terminal domain (CTD), and a long flexible region linking the two domains. His-FKBP22, NTD(+) (NTD with the entire flexible region), and CTD(+) (CTD with a truncated flexible region) were unfolded by a two-state mechanism in the presence of urea. Urea induced the swelling of dimeric His-FKBP22 molecules at the pretransition state but dissociated it at the early transition state. In contrast, guanidine hydrochloride (GdnCl)-induced equilibrium unfolding of His-FKBP22 or NTD(+) and CTD(+) seemed to follow three-step and two-step mechanisms, respectively. Interestingly, the intermediate formed during the unfolding of His-FKBP22 with GdnCl was not a molten globule but was thought to be composed of the partially unfolded dimeric as well as various multimeric His-FKBP22 molecules. Dimeric His-FKBP22 did not dissociate gradually with increasing concentrations of GdnCl. Very low GdnCl concentrations also had little effect on the molecular dimensions of His-FKBP22. Unfolding with either denaturant was found to be reversible, as refolding of the unfolded His-FKBP22 completely, or nearly completely, restored the structure and function of the protein. Additionally, denaturation of His-FKBP22 appeared to begin at the CTD(+).     

FK506-binding protein-type peptidyl-prolyl cis-trans isomerase from a halophilic archaeum, Halobacterium cutirubrum

The halophilic archaeum, Halobacterium cutirubrum, has been shown to have a cyclophilin-type peptidyl-prolyl cis-trans isomerase (PPIase). Because most archaeal genomes studied only have genes for FK506-binding proteins (FKBPs) as a PPIase, it has been unclear whether H. cutirubrum has an FKBP-type PPIase or not. In the present study, a gene encoding an FKBP-type PPIase was cloned from genomic DNA of H. cutirubrum and then sequenced. This FKBP was deduced to be composed of 303 amino acid residues with a molecular mass of 33.3kDa. Alignment of its amino acid sequence with those of other reported FKBPs showed that it contained two insertion sequences in the regions corresponding to the bulge and flap of human FKBP12, which are common to archaeal FKBPs. Its C-terminal amino acid sequence was approximately 130 amino acids longer than the FKBPs of Methanococcus thermolithotrophicus and Thermococcus sp. KS-1. Among the 14 conserved amino acid residues that form the FK506 binding pocket, only three were found in this FKBP. This gene was expressed as a fusion protein with glutathione S-transferase (GST) in Escherichia coli, and the N-terminal GST portion was removed by protease digestion. The purified recombinant FKBP showed a weak PPIase activity with a low sensitivity to FK506. This FKBP suppressed aggregation of the unfolded protein.     

1.88 A crystal structure of the C domain of hCyP33: a novel domain of peptidyl-prolyl cis-trans isomerase

Cyclophilins (CyPs) are a widespreading protein family in living organisms and possess the activity of peptidyl-prolyl cis-trans isomerase (PPIase), which is inhibited by cyclosporin A (CsA). The human nuclear cyclophilin (hCyP33) is the first protein which was found to contain two RNA binding domains at the amino-terminus and a PPIase domain at the carboxyl-terminus. We isolated the hCyP33 gene from the human hematopoietic stem/progenitor cells and expressed it in Escherichia coli, and determined the crystal structure of the C domain of hCyP33 at 1.88 A resolution. The core structure is a beta-barrel covered by two alpha-helices. Superposition of the structure of the C domain of hCyP33 with the structure of CypA suggests that the C domain contains PPIase active site which binds to CsA. Furthermore, C domain seems to be able to bind with the Gag-encoded capsid (CA) of HIV-1 and may affect the viral replication of HIV-1. A key residue of the active site is changed from Ala-103-CypA to Ser-239-hCyP33, which may affect the PPIase domain/substrates interactions.     

The three-dimensional structure of two redox states of cyclophilin A from Schistosoma mansoni. Evidence for redox regulation of peptidyl-prolyl cis-trans isomerase activity

Treatment of schistosomiasis, a widespread human parasitic disease caused by the helminth parasites of the genus Schistosoma, relies mainly on one chemotherapeutic agent, praziquantel, although several other compounds exert anti-parasitic effects. One such compound is the immunosuppressant cyclosporin A, which has been shown to significantly diminish worm burden in mice infected with Schistosoma mansoni. Given the well established interaction between cyclosporin A and the cyclophilin superfamily of peptidylprolyl cis-trans isomerases, we solved the structure of cyclophilin A from S. mansoni (SmCypA) by x-ray crystallography in the reduced and oxidized states at 1.5 and 1.8 A of resolution, respectively. Oxidized SmCypA contains a disulfide bridge between two C-terminal cysteines (Cys-122 and Cys-126). This is the first example of a cyclophilin containing this disulfide bridge. Parallel functional studies suggest a mechanism for regulation of SmCypA activity via oxidation of its thiol groups; in fact, whereas oxidized SmCypA is inactive, reduced SmCypA is an efficient isomerase active at nanomolar levels with a k(cat)/K(m) of 1.1 x 10(7) M(-1) s(-1), and it is inhibited by cyclosporin A (IC(50) of 14 +/- 4 nM). The lack of conservation of this cysteine couple within the CypA superfamily, their close proximity to the active site, and the importance of thiol groups for peptidyl-prolyl cis-trans isomerase activity render this structural feature a challenge for the development of alternative and more effective anti-schistosomiasis inhibitors and may in addition imply an alternative function of SmCypA in the schistosome.