Prolyl cis-trans isomerization as a molecular timer

Proline is unique in the realm of amino acids in its ability to adopt completely distinct cis and trans conformations, which allows it to act as a backbone switch that is controlled by prolyl cis-trans isomerization. This intrinsically slow interconversion can be catalyzed by the evolutionarily conserved group of peptidyl prolyl cis-trans isomerase enzymes. These enzymes include cyclophilins and FK506-binding proteins, which are well known for their isomerization-independent role as cellular targets for immunosuppressive drugs. The significance of enzyme-catalyzed prolyl cis-trans isomerization as an important regulatory mechanism in human physiology and pathology was not recognized until the discovery of the phosphorylation-specific prolyl isomerase Pin1. Recent studies indicate that both phosphorylation-dependent and phosphorylation-independent prolyl cis-trans isomerization can act as a novel molecular timer to help control the amplitude and duration of a cellular process, and prolyl cis-trans isomerization might be a new target for therapeutic interventions.     

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.     

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.     

FKBP51, a novel T-cell-specific immunophilin capable of calcineurin inhibition.

The immunosuppressive drugs FK506 and cyclosporin A block T-lymphocyte proliferation by inhibiting calcineurin, a critical signaling molecule for activation. Multiple intracellular receptors (immunophilins) for these drugs that specifically bind either FK506 and rapamycin (FK506-binding proteins [FKBPs]) or cyclosporin A (cyclophilins) have been identified. We report the cloning and characterization of a new 51-kDa member of the FKBP family from murine T cells. The novel immunophilin, FKBP51, is distinct from the previously isolated and sequenced 52-kDa murine FKBP, demonstrating 53% identity overall. Importantly, Western blot (immunoblot) analysis showed that unlike all other FKBPs characterized to date, FKBP51 expression was largely restricted to T cells. Drug binding to recombinant FKBP51 was demonstrated by inhibition of peptidyl prolyl isomerase activity. As judged from peptidyl prolyl isomerase activity, FKBP51 had a slightly higher affinity for rapamycin than for FK520, an FK506 analog. FKBP51, when complexed with FK520, was capable of inhibiting calcineurin phosphatase activity in an in vitro assay system. Inhibition of calcineurin phosphatase activity has been implicated both in the mechanism of immunosuppression and in the observed toxic side effects of FK506 in nonlymphoid cells. Identification of a new FKBP that can mediate calcineurin inhibition and is restricted in its expression to T cells suggests that new immunosuppressive drugs may be identified that, by virtue of their specific interaction with FKBP51, would be targeted in their site of action.     

Binding and regulation of the transcription factor NFAT by the peptidyl prolyl cis-trans isomerase Pin1

Nuclear factor of activated T cells (NFAT) plays a key role in T cell activation. The activation of NFAT involves calcium- and calcineurin-dependent dephosphorylation and nuclear translocation from the cytoplasm, a process that is opposed by protein kinases. We show here that the peptidyl prolyl cis-trans isomerase Pin1 interacts specifically with the phosphorylated form of NFAT. The NFAT-Pin1 interaction is mediated through the WW domain of Pin1 and the serine-proline-rich domains of NFAT. Furthermore, binding of Pin1 to NFAT inhibits the calcineurin-mediated dephosphorylation of NFAT in vitro, and overexpression of Pin1 in T cells inhibits calcium-dependent activation of NFAT in vivo. These results suggest a possible role for Pin1 in the regulation of NFAT in T cells.     

Activation of colicin M by the FkpA prolyl cis-trans isomerase/chaperone

Colicin M (Cma) is specifically imported into the periplasm of Escherichia coli and kills the cells. Killing depends on the periplasmic peptidyl prolyl cis-trans isomerase/chaperone FkpA. To identify the Cma prolyl bonds targeted by FkpA, we replaced the 15 proline residues individually with alanine. Seven mutant proteins were fully active; Cma(P129A), Cma(P176A), and Cma(P260A) displayed 1%, and Cma(P107A) displayed 10% of the wild-type activity. Cma(P107A), Cma(P129A), and Cma(P260A), but not Cma(P176A), killed cells after entering the periplasm via osmotic shock, indicating that the former mutants were translocation-deficient; Cma(P129A) did not bind to the FhuA outer membrane receptor. The crystal structures of Cma and Cma(P176A) were identical, excluding inactivation of the activity domain located far from Pro-176. In a new peptidyl prolyl cis-trans isomerase assay, FkpA isomerized the Cma prolyl bond in peptide Phe-Pro-176 at a high rate, but Lys-Pro-107 and Leu-Pro-260 isomerized at only <10% of that rate. The four mutant proteins secreted into the periplasm via a fused signal sequence were toxic but much less than wild-type Cma. Wild-type and mutant Cma proteins secreted or translocated across the outer membrane by energy-coupled import or unspecific osmotic shock were only active in the presence of FkpA. We propose that Cma unfolds during transfer across the outer or cytoplasmic membrane and refolds to the active form in the periplasm assisted by FkpA. Weak refolding of Cma(P176A) would explain its low activity in all assays. Of the four proline residues identified as being important for Cma activity, Phe-Pro-176 is most likely targeted by FkpA.     

Anti-Trypanosoma cruzi effects of cyclosporin A derivatives: possible role of a P-glycoprotein and parasite cyclophilins

Cyclophilins are target molecules for cyclosporin A (CsA), an immunosuppressive antimicrobial drug. We have previously reported the in vitro anti-Trypanosoma cruzi activity of H-7-94 and F-7-62 non-immunosuppressive CsA analogues. In this work, we continue the study of the parasiticidal effect of H-7-94 and F-7-62 CsA analogues in vitro and in vivo and we analyse 3 new CsA derivatives: MeIle-4-CsA (NIM 811), MeVal-4-CsA (MeVal-4) and D-MeAla-3-EtVal-4-CsA, (EtVal-4). The most efficient anti-T. cruzi effect was observed with H-7-94, F-7-62 and MeVal-4 CsA analogues evidenced as inhibition of epimastigote proliferation, trypomastigote penetration, intracellular amastigote development and in vivo T. cruzi infection. This trypanocidal activity could be due to inhibition of the peptidyl prolyl cis-trans isomerase activity on the T. cruzi recombinant cyclophilins tested. Furthermore, CsA and F-7-62 derivative inhibited the efflux of rhodamine 123 from T. cruzi epimastigotes, suggesting an interference with a P-glycoprotein activity. Moreover, H-7-94 and F-7-62 CsA analogues were not toxic as shown by cell viability and by aminopyrine-N-demethylase activity on mammalian cells. Our results show that H-7-94, F-7-62 and MeVal-4 CsA analogues expressed the highest inhibiting effects on T. cruzi, being promissory parasiticidal drugs worthy of further studies.     

FK506 binding protein from a thermophilic archaeon, Methanococcus thermolithotrophicus, has chaperone-like activity in vitro

The in vitro protein folding activity of an FKBP (FK506 binding protein, abbreviated to MTFK) from a thermophilic archaeon, Methanococcus thermolithotrophicus, was investigated. MTFK exhibited FK506 sensitive PPIase (peptidyl prolyl cis-trans isomerase) activity which accelerated the speed of ribonuclease T1 refolding, which is rate-limited by isomerization of two prolyl peptide bonds. In addition, MTFK suppressed the aggregation of folding intermediates and elevated the final yield of rhodanese refolding. We called this activity of MTFK the chaperone activity. The chaperone activity of MTFK was also inhibited by FK506. Alignment of the amino acid sequences of MTFK with human FKBP12 showed that MTFK has two insertion sequences, consisting of 13 and 44 amino acids, at the N- and C-termini, respectively [Furutani, M., Iida, T., Yamano, S., Kamino, K., and Maruyama, T. (1998) J. Bacteriol. 180, 388-394]. To study the relationship between chaperone and PPIase activities of MTFK, mutant MTFKs with deletions of these insertion sequences or with amino acid substitutions were created. Their PPIase and chaperone activities were measured using a synthetic oligopeptide and denatured rhodanese as the substrates, respectively. The far-UV circular dichroism spectra of the wild type and the mutants were also analyzed. The results suggested that (1) the PPIase activity did not correlate with chaperone activity, (2) both insertion sequences were required for MTFK to take a proper conformation, and (3) the insertion sequence (44 amino acids) in the C-terminus was important for the chaperone activity.     

Determination of kinetic constants for peptidyl prolyl cis-trans isomerases by an improved spectrophotometric assay

The kinetic properties and substrate specificity of two well-characterized peptidyl prolyl cis-trans isomerases (PPIases), cyclophilin and the FK-506 binding protein (FKBP), have been previously examined [Fischer, G., Bang, H., Berger, E., & Schellenberger, A. (1984) Biochim. Biophys. Acta 791, 87-97; Harrison, R.K., & Stein, R.L. (1990) Biochemistry 29, 1684-1689; Albers, M.W., Walsh, C.T., & Schreiber, S. L. (1990) J. Org. Chem. 55, 4984-4986]. The chymotrypsin-coupled enzymatic assay employed in these studies suffers from two serious shortcomings. Due to the low equilibrium population of the X-cis-Pro-Phe-pNA isomer (the PPIase substrate), in conjunction with the low solubility of p-nitroaniline generated by chymotrypsin hydrolysis, substrate concentrations in the saturating region are not experimentally attainable. Secondly, the uncatalyzed cis-trans isomerization obscures the interpretation of the initial velocity. As a result of these limitations, the steady-state kinetic parameters (Km,Kcat) have not been determined. Here we introduce an improved version of the spectrophotometric assay and report for the first time the Michaelis constants and turnover numbers for both PPIases with established substrates. The improvements in the experimental conditions originate in a medium-induced increase in the equilibrium population of the cis X-Pro conformer and in conducting the assay at 0 degrees C to suppress the uncatalyzed thermal isomerization. In addition, we present a rigorous mathematical model of the spectrophotometric progress curves that accounts for the contributions of the residual background rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substrate specificities of the peptidyl prolyl cis-trans isomerase activities of cyclophilin and FK-506 binding protein: evidence for the existence of a family of distinct enzymes.

Substrate specificities, as reflected in kc/Km, were determined for the peptidyl prolyl cis-trans isomerase activities of cyclophilin and the FK-506 binding protein (FKBP). The substrates investigated were peptides of the general structure Suc-Ala-Xaa-Pro-Phe-p-nitroanilide, where Xaa = Gly, Ala, Val, Leu, Phe, His, Lys, on Glu. While kc/Km for cyclophilin-catalyzed isomerization shows little dependence on Xaa, kc/Km values for FKBP-catalyzed isomerization display a marked dependence on Xaa and vary over 3 orders of magnitude. An important outcome of this work is the discovery that Suc-Ala-Leu-Pro-Phe-pNA is a reactive substrate for FKBP (kc/Km = 640,000 M-1 s-1). This substrate can be used with FKBP concentrations that are low enough to allow, for the first time, accurate determinations of Ki values for tight-binding inhibitors of FKBP. Using this new assay, we found that FK-506 inhibits FKBP with Ki = 1.7 +/- 0.6 nM. The results of this work support the hypothesis that cyclophilin and FKBP are members of a family of peptidyl prolyl cis-trans isomerases and that the members of this family possess distinct substrate specificities that allow them to play diverse physiologic roles.     

Three-dimensional solution structure of an archaeal FKBP with a dual function of peptidyl prolyl cis-trans isomerase and chaperone-like activities

Here we report the solution structure of an archaeal FK506-binding protein (FKBP) from a thermophilic archaeum, Methanococcus thermolithotrophicus (MtFKBP17), which has peptidyl prolyl cis-trans isomerase (PPIase) and chaperone-like activities, to reveal the structural basis for the dual function. In addition to a typical PPIase domain, a newly identified domain is formed in the flap loop by a 48-residue insert that is required for the chaperone-like activity. The new domain, called IF domain (the Insert in the Flap), is a novel-folding motif and exposes a hydrophobic surface, which we consider to play an important role in the chaperone-like activity.     

Cyclophilin D links programmed cell death and organismal aging in Podospora anserina

Cyclophilin D (CYPD) is a mitochondrial peptidyl prolyl‐cis,trans‐isomerase involved in opening of the mitochondrial permeability transition pore (mPTP). CYPD abundance increases during aging in mammalian tissues and in the aging model organism Podospora anserina. Here, we show that treatment of the P. anserina wild‐type with low concentrations of the cyclophilin inhibitor cyclosporin A (CSA) extends lifespan. Transgenic strains overexpressing PaCypD are characterized by reduced stress tolerance, suffer from pronounced mitochondrial dysfunction and are characterized by accelerated aging and induction of cell death. Treatment with CSA leads to correction of mitochondrial function and lifespan to that of the wild‐type. In contrast, PaCypD deletion strains are not affected by CSA within the investigated concentration range and show increased resistance against inducers of oxidative stress and cell death. Our data provide a mechanistic link between programmed cell death (PCD) and organismal aging and bear implications for the potential use of CSA to intervene into biologic aging.     

Structural Basis of Cyclophilin B Binding by the Calnexin/Calreticulin P-domain

Little is known about how chaperones in the endoplasmic reticulum are organized into complexes to assist in the proper folding of secreted proteins. One notable exception is the complex of ERp57 and calnexin that functions as part the calnexin cycle to direct disulfide bond formation in N-glycoproteins. Here, we report three new complexes composed of the peptidyl prolyl cis-trans-isomerase cyclophilin B and any of the lectin chaperones: calnexin, calreticulin, or calmegin. The 1.7 Å crystal structure of cyclophilin with the proline-rich P-domain of calmegin reveals that binding is mediated by the same surface that binds ERp57. We used NMR titrations and mutagenesis to measure low micromolar binding of cyclophilin to all three lectin chaperones and identify essential interfacial residues. The immunosuppressant cyclosporin A did not affect complex formation, confirming the functional independence of the P-domain binding and proline isomerization sites of cyclophilin. Our results reveal the P-domain functions as a unique protein-protein interaction domain and implicate a peptidyl prolyl isomerase as a new element in the calnexin cycle.     

Human and Escherichia coli cyclophilins: sensitivity to inhibition by the immunosuppressant cyclosporin A correlates with a specific tryptophan residue

The human T-cell protein cyclophilin shows high affinity for and is the proposed target of the major immunosuppressant drug cyclosporin A (CsA). Cyclophilin also has peptidyl prolyl cis-trans isomerase activity that is inhibited by CsA with an IC50 of 6 nM, while by contrast a homologous PPIase from Escherichia coli has been found to be much less sensitive to CsA, shown here to be 500-fold less potent at an IC50 of 3000 nM. This E. coli rotamase lacks the single highly conserved tryptophan residue of eukaryotic cyclophilins, and we show here that mutation of the natural F112 to W112 enhances E. coli rotamase susceptibility to CsA inhibition by 23-fold. Correspondingly, the human W121 mutations to F121 or A121 yield cyclophilins with 75- and 200-fold decreased sensitivity to CsA, while kcat/Km values of rotamase activity in a tetrapeptide assay drop only 2- and 13-fold, respectively. This complementary gain and loss of CsA sensitivity to mutation to or from tryptophan validate the indole side chain as a major determinant in immunosuppressant drug recognition and the separation of PPIase catalytic efficiency from CsA affinity.     

Binding analysis of a psychrotrophic FKBP22 to a folding intermediate of protein using surface plasmon resonance

SIB1 FKBP22 is a homodimer, with each subunit consisting of the C-terminal catalytic domain and N-terminal dimerization domain. This protein exhibits peptidyl prolyl cis-trans isomerase activity for both peptide and protein substrates. However, truncation of the N-terminal domain greatly reduces the activity only for a protein substrate. Using surface plasmon resonance, we showed that SIB1 FKBP22 loses the binding ability to a folding intermediate of protein upon truncation of the N-terminal domain but does not lose it upon truncation of the C-terminal domain. We propose that the binding site of SIB1 FKBP22 to a protein substrate of PPIase is located at the N-terminal domain.     

Genome wide analysis of Cyclophilin gene family from rice and Arabidopsis and its comparison with yeast

Cyclophilin proteins are the members of immunophillin group of proteins, known for their property of binding to the immune-suppressant drug cyclosporin A, hence named as cyclophilins. These proteins are characterized by the presence of peptidyl prolyl isomerase (PPIase) domain which catalyzes the cis-trans isomerisation process of proline residues. In the present study, an in-silico based approach was followed to identify and characterize the cyclophilin family from rice, Arabidopsis and yeast. We were able to identify 28 rice, 35 Arabidopsis and 8 yeast cyclophilin genes from their respective genomes on the basis of their annotation as well as the presence of highly conserved PPIase domain. The evolutionary relationship of the cyclophilin genes from the three genomes was analyzed using the phylogenetic tree. We have also classified the rice cyclophilin genes on the basis of localization of the protein in cell. The structural similarity of the cyclophilins was also analyzed on the basis of their homology model. The expression analysis performed using Genevestigator revealed a very strong stress responsive behavior of the gene family which was more prominent in later stages of stress. The study indicates the importance of the gene family in stress response as well as several developmental stages thus opening up many avenues for future study on the cyclophilin proteins.     

An endoplasmic reticulum-specific cyclophilin.

Cyclophilin is a ubiquitously expressed cytosolic peptidyl-prolyl cis-trans isomerase that is inhibited by the immunosuppressive drug cyclosporin A. A degenerate oligonucleotide based on a conserved cyclophilin sequence was used to isolate cDNA clones representing a ubiquitously expressed mRNA from mice and humans. This mRNA encodes a novel 20-kDa protein, CPH2, that shares 64% sequence identity with cyclophilin. Bacterially expressed CPH2 binds cyclosporin A and is a cyclosporin A-inhibitable peptidyl-prolyl cis-trans isomerase. Cell fractionation of rat liver followed by Western blot (immunoblot) analysis indicated that CPH2 is not cytosolic but rather is located exclusively in the endoplasmic reticulum. These results suggest that cyclosporin A mediates its effect on cells through more than one cyclophilin and that cyclosporin A-induced misfolding of T-cell membrane proteins normally mediated by CPH2 plays a role in immunosuppression.     

A nuclear FK506-binding protein is a histone chaperone regulating rDNA silencing

We report a novel chromatin-modulating factor, nuclear FK506-binding protein (FKBP). It is a member of the peptidyl prolyl cis-trans isomerase (PPIase) family, whose members were originally identified as enzymes that assist in the proper folding of polypeptides. The endogenous FKBP gene is required for the in vivo silencing of gene expression at the rDNA locus and FKBP has histone chaperone activity in vitro. Both of these properties depend on the N-terminal non-PPIase domain of the protein. The C-terminal PPIase domain is not essential for the histone chaperone activity in vitro, but it regulates rDNA silencing in vivo. Chromatin immunoprecipitation showed that nuclear FKBP associates with chromatin at rDNA loci in vivo. These in vivo and in vitro findings in nuclear FKBPs reveal a hitherto unsuspected link between PPIases and the alteration of chromatin structure.