Streptomycetes possess peptidyl-prolyl cis-trans isomerases that strongly resemble cyclophilins from eukaryotic organisms

A functionally active 17.5 kDa peptidyl-prolyl cis-trans isomerase was purified to homogeneity from Streptomyces chrysomallus, a Gram-positive filamentous bacterium. Characterization of the enzyme revealed inhibition and binding characteristics, against the immunsuppressive drug cyclosporin A, which were similar to cyclophilins from eukaryotes such as mammals, plants, fungi and yeasts, but different from those of cyclophilins from enterobacteria such as Escherichia coli. The amino acid sequence of the S. chrysomallus cyclophilin, as deduced from the gene sequence, revealed a striking degree of amino acid sequence identity with the corresponding 17 kDa proteins of humans (66%), Neurospora (70%) and yeast (69%). Comparison with cyclophilin sequences from the Gram-negative enterobacteria revealed much less homology (25% identity with E. coli b, 23% identity with E. coli a). Cyclophilin was detected in each of the four other Streptomyces species tested. The cyclophilins from the various streptomycetes differed in size, varying between 17 and 20.5 kDa. The cyclophilins were abundant in the Streptomyces cells, and present throughout growth.     

Cloning and characterization of a Streptomyces antibioticus ATCC11891 cyclophilin related to Gram negative bacteria cyclophilins

Cyclophilins are folding helper enzymes and represent a family of the enzyme class of peptidyl-prolyl cis-trans isomerases. Here, we report the molecular cloning and biochemical characterization of SanCyp18, an 18-kDa cyclophilin from Streptomyces antibioticus ATCC11891 located in the cytoplasm and constitutively expressed during development. Amino acid sequence analysis revealed a much higher homology to cyclophilins from Gram negative bacteria than to known cyclophilins from Streptomyces or other Gram positive bacteria. SanCyp18 is inhibited weakly by CsA, with a K(i) value of 21 microM, similar to cyclophilins from Gram negative bacteria. However, this value is more than 20-fold higher than the K(i) values reported for cyclophilins from other Gram positive bacteria, which makes SanCyp18 unique within this group. The presence of SanCyp18 in Streptomyces is likely due to horizontal gene transmission from Gram-negative bacteria to Streptomyces.     

Characterization of the cyclophilin of Trichophyton mentagrophytes

A genetic approach to cyclophilins in a dermatophyte, Trichophyton mentagrophytes, was carried out. The nucleotide and deduced amino acid sequences of the cyclophilin of T. mentagrophytes shared about 70% sequence similarity with those of Schizosaccharomyces pombe, Saccharomyces cerevisiae and Candida albicans. However, the first 21 amino acid and the C-terminal amino acid regions of 188 to 226 of the T. mentagrophytes cyclophilin were distinct from those of the other fungal cyclophilins. The recombinant glutathione S-transferase (GST)-T. mentagrophytes cyclophilin fusion protein produced by Escherichia coli was purified. The protease digest of the fusion protein had a molecular weight of about 13 kDa and peptidyl-prolyl cis-trans isomerase (PPI) activity. This digest protein from T. mentagrophytes was confirmed to be cyclophilin by proving PPI activity.     

A multicopy suppressor of a cell cycle defect in S. pombe encodes a heat shock-inducible 40 kDa cyclophilin-like protein.

Cyclophilins are peptidyl-prolyl cis-trans isomerases (PPIases) which have been implicated in intracellular protein folding, transport and assembly. Cyclophilins are also known as the intracellular receptors for the immunosuppressive drug cyclosporin A (CsA). The most common type of cyclophilins are the 18 kDa cytosolic proteins containing only the highly conserved core domain for PPIase and CsA binding activities. The wis2+ gene of the fission yeast Schizosaccharomyces pombe was isolated as a multicopy suppressor of wee1-50 cdc25-22 win1-1, a triple mutant strain which exhibits a cell cycle defect phenotype. Sequence analysis of wis2+ reveals that it encodes a 40 kDa cyclophilin-like protein, homologous to the mammalian cyclophilin 40. The 18 kDa cyclophilin domain (CyP-18) of wis2 is followed by a C-terminal region of 188 amino acids. The C-terminal region of wis2 is essential for suppression of the triple mutant defect. Furthermore this region of the protein is able to confer suppression activity on the 18 kDa S.pombe cyclophilin, cyp1, since a hybrid protein consisting of an 18 kDa S.pombe cyclophilin (cyp1) fused to the C-terminus of wis2 shows suppression activity. We also demonstrate that the level of wis2+ mRNA increases 10- to 20-fold upon heat shock of S.pombe cells suggesting a role for wis2+ in the heat-shock response.     

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.     

Identification and characterization of Moca-cyp. A Drosophila melanogaster nuclear cyclophilin

Cyclophilins are enzymes catalyzing the cis-trans isomerization of peptidyl-prolyl bonds and belong to the enzyme class of peptidyl-prolyl cis-trans isomerases (PPIases), which includes two more families (FK506 binding proteins and parvulins). We report the characterization of a novel cyclophilin (Moca-cyp) isolated from Drosophila melanogaster. The single-copy Moca-cyp gene, which is localized on chromosome 3R, was cloned and sequenced. The sequence alignment of the gene against Moca-cyp cDNA allowed us to define its intron/exon structure and to identify a variant cDNA corresponding to an alternatively spliced mRNA. By embryo in situ RNA hybridization and immunostaining, we show that the expression of Moca-cyp is regulated during embryogenesis of Drosophila. The 120-kDa nuclear Moca-cyp protein belongs to a subfamily of large cyclophilins sharing structural and enzymatic features: their highly conserved N-terminal PPIase domain is extended by a positively charged and divergent C-terminal tail. Compared with cyclophilin 18, the enzymatic activity carried by the PPIase domain of Moca-cyp is low, exhibits characteristic substrate specificity, and shows a reduced sensitivity to the drug cyclosporin A (CsA). The reduced affinity for CsA is one of the typical features linking members of this subfamily and is probably the consequence of two amino acid substitutions within their active site. Another structural feature shared by members of this subfamily is a conserved polypeptidic segment ("moca" domain) that we report for the first time. The moca domain is located within the C-terminal tail and is the exclusive hallmark of a group of large cyclophilins found in multicellular organisms of the animal kingdom.     

1H, 13C,and 15N backbone and side chain resonance assignments of thermophilic Geobacillus kaustophilus cyclophilin-A

Cyclophilins catalyze the reversible peptidyl-prolyl isomerization of their substrates and are present across all kingdoms of life from humans to bacteria. Although numerous biological roles have now been discovered for cyclophilins, their function was initially ascribed to their chaperone-like activity in protein folding where they catalyze the often rate-limiting step of proline isomerization. This chaperone-like activity may be especially important under extreme conditions where cyclophilins are often over expressed, such as in tumors for human cyclophilins (Lee Archiv Pharm Res 33(2): 181–187, 2010), but also in organisms that thrive under extreme conditions, such as theromophilic bacteria. Moreover, the reversible nature of the peptidyl-prolyl isomerization reaction catalyzed by cyclophilins has allowed these enzymes to serve as model systems for probing the role of conformational changes during catalytic turnover (Eisenmesser et al. Science 295(5559): 1520–1523, 2002; Eisenmesser et al. Nature 438(7064): 117–121, 2005). Thus, we present here the resonance assignments of a thermophilic cyclophilin from Geobacillus kaustophilus derived from deep-sea sediment (Takami et al. Extremophiles 8(5): 351–356, 2004). This thermophilic cyclophilin may now be studied at a variety of temperatures to provide insight into the comparative structure, dynamics, and catalytic mechanism of cyclophilins.     

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.     

Ricinus communis cyclophilin: functional characterisation of a sieve tube protein involved in protein folding

The phloem translocation stream of the angiosperms contains a special population of proteins and RNA molecules which appear to be produced in the companion cells prior to being transported into the sieve tube system through the interconnecting plasmodesmata. During this process, these non-cell-autonomous proteins are thought to undergo partial unfolding. Recent mass spectroscopy studies identified peptidyl-prolyl cis-trans isomerase (PPIases) as potential molecular chaperones functioning in the phloem translocation stream (Giavalisco et al. 2006). In the present study, we describe the cloning and characterisation of a castor bean phloem cyclophilin, RcCYP1 that has high peptidyl-prolyl cis-trans isomerase activity. Equivalent enzymatic activity was detected with phloem sap or purified recombinant (His)(6)-tagged RcCYP1. Mass spectrometry analysis of proteolytic peptides, derived from a 22 kDa band in HPLC-fractionated phloem sap, immunolocalisation studies and Western analysis of proteins extracted from castor bean tissues/organs indicated that RcCYP1 is an abundant protein in the companion cell-sieve element complex. Microinjection experiments established that purified recombinant (His)(6)-RcCYP1 can interact with plasmodesmata to both induce an increase in size exclusion limit and mediate its own cell-to-cell trafficking. Collectively, these findings support the hypothesis that RcCYP1 plays a role in the refolding of non-cell-autonomous proteins after their entry into the phloem translocation stream.     

A novel cyclophilin from parasitic and free-living nematodes with a unique substrate- and drug-binding domain

A highly diversified member of the cyclophilin family of peptidyl-prolyl cis-trans isomerases has been isolated from the human parasite Onchocerca volvulus (OvCYP-16). This 25-kDa cyclophilin shares 43-46% similarity to other filarial cyclophilins but does not belong to any of the groups previously defined in invertebrates or vertebrates. A homolog was also isolated from Caenorhabditis elegans (CeCYP-16). Both recombinant O. volvulus and C. elegans cyclophilins were found to possess an enzyme activity with similar substrate preference and insensitivity to cyclosporin A. They represent novel cyclophilins with important differences in the composition of the drug-binding site in particular, namely, a Glu(124) (C. elegans) or Asp(123) (O. volvulus) residue present in a critical position. Site-directed mutagenesis studies and kinetic characterization demonstrated that the single residue dictates the degree of binding to substrate and cyclosporin A. CeCYP-16::GFP-expressing lines were generated with expression in the anterior and posterior distal portions of the intestine, in all larval stages and adults. An exception was found in the dauer stage, where fluorescence was observed in both the cell bodies and processes of the ventral chord motor neurons but was absent from the intestine. These studies highlight the extensive diversification of cyclophilins in an important human parasite and a closely related model organism.     

Overexpression, purification and assessment of cyclosporin binding of a family of cyclophilins and cyclophilin-like proteins of the human malarial parasite Plasmodium falciparum

Malaria represents a global health, economic and social burden of enormous magnitude. Chemotherapy is at the moment a largely effective weapon against the disease, but the appearance of drug-resistant parasites is reducing the effectiveness of most drugs. Finding new drug-target candidates is one approach to the development of new drugs. The family of cyclophilins may represent a group of potential targets. They are involved in protein folding and regulation due to their peptidyl-prolyl cis-trans isomerase and/or chaperone activities. They also mediate the action of the immunosuppressive drug cyclosporin A, which additionally has strong antimalarial activity. In the genome database of the most lethal human malarial parasite Plasmodium falciparum, 11 genes apparently encoding cyclophilin or cyclophilin-like proteins were found, but most of these have not yet been characterized. Previously a pET vector conferring a C-terminal His? tag was used for recombinant expression and purification of one member of the P. falciparum cyclophilin family in Escherichia coli. The approach here was to use an identical method to produce all of the other members of this family and thereby allow the most consistent functional comparisons. We were successful in generating all but three of the family, plus a single amino-acid mutant, in the same recombinant form as either full-length proteins or isolated cyclophilin-like domains. The recombinant proteins were assessed by thermal melt assay for correct folding and cyclosporin A binding.     

S-cyclophilin. New member of the cyclophilin family associated with the secretory pathway

Cyclophilin is an abundant and ubiquitous cytosolic protein that is conserved throughout evolution from man to bacteria. It is the target of the immunosuppressive drug cyclosporin A. Cyclophilin has peptidyl-prolyl cis/trans-isomerase activity, and it accelerates protein folding in vitro, suggesting that it might be involved in the folding of cytosolic proteins. We describe a novel cyclophilin-like protein, S-cyclophilin, in the chick. Analysis of S-cyclophilin cDNA revealed the presence of a signal sequence followed by an open reading frame coding for a protein very similar to cytosolic cyclophilin, except for the presence of unique additional short amino acid segments at the N and C termini of the protein. S-Cyclophilin mRNA was abundant and present in all embryonic chick tissues tested. Cyclophilin and S-cyclophilin are coded by separate genes in the chick genome. Recombinant S-cyclophilin was expressed in insect cells by means of the baculovirus system. Pulse-chase experiments revealed that a significant fraction of newly synthesized recombinant S-cyclophilin was rapidly secreted into the culture medium. Our findings indicate that cyclophilins are associated with most if not all intra- and extracellular compartments and suggest that enzyme-assisted conformational conversions in proteins might also take place in post-endoplasmic reticulum compartments, possibly including the extracellular space.     

Specific cross-linking of the proline isomerase cyclophilin to a non-proline-containing peptide.

A peptide corresponding to an efficient peroxisomal targeting sequence, the carboxy terminal 12 amino acids of PMP20 from Candida boidinii, was employed as an affinity ligand to search for a peroxisomal targeting receptor. Two proteins from yeast extracts with apparent molecular masses of 20 and 80 kDa were detected by chemical cross-linking to radioiodinated peptide. Both proteins were present in cytosolic supernatants. The 20-kDa species did not cross-link to a control peptide with reversed sequence, whereas the 80-kDa protein cross-linked to both peptides. The cross-linking assay was used to purify the 20-kDa protein from Saccharomyces cerevisiae. Partial protein sequencing identified this protein as cyclophilin, the product of the CYP1 gene. This protein, a peptidyl-prolyl cis-trans isomerase, is the yeast homologue of the protein that mediates the immunosuppressant effects of the drug cyclosporin A (CsA). Cross-linking of peptide to cyclophilin was inhibited by CsA. The cross-linking of cyclophilin to the PMP20-derived peptide was unanticipated because the peptide contains no prolines. The CYP1-encoded protein was not required to target proteins to peroxisomes because this organelle appeared to be assembled normally in a CYP1-disrupted strain. Furthermore, the final three amino acids of the peptide, which are critical for peroxisomal sorting, were not required for cross-linking to cyclophilin. We conclude that either cyclophilin is playing a nonessential facilitating role in peroxisomal targeting or that the interaction of the targeting peptide to cyclophilin is mimicking an interaction with an unidentified substrate or effector of cyclophilin.     

Depletion of Cyclophilins B and C Leads to Dysregulation of Endoplasmic Reticulum Redox Homeostasis

Protein folding within the endoplasmic reticulum is assisted by molecular chaperones and folding catalysts that include members of the protein-disulfide isomerase and peptidyl-prolyl isomerase families. In this report, we examined the contributions of the cyclophilin subset of peptidyl-prolyl isomerases to protein folding and identified cyclophilin C as an endoplasmic reticulum (ER) cyclophilin in addition to cyclophilin B. Using albumin and transferrin as models of cis-proline-containing proteins in human hepatoma cells, we found that combined knockdown of cyclophilins B and C delayed transferrin secretion but surprisingly resulted in more efficient oxidative folding and secretion of albumin. Examination of the oxidation status of ER protein-disulfide isomerase family members revealed a shift to a more oxidized state. This was accompanied by a >5-fold elevation in the ratio of oxidized to total glutathione. This “hyperoxidation” phenotype could be duplicated by incubating cells with the cyclophilin inhibitor cyclosporine A, a treatment that triggered efficient ER depletion of cyclophilins B and C by inducing their secretion to the medium. To identify the pathway responsible for ER hyperoxidation, we individually depleted several enzymes that are known or suspected to deliver oxidizing equivalents to the ER: Ero1αβ, VKOR, PRDX4, or QSOX1. Remarkably, none of these enzymes contributed to the elevated oxidized to total glutathione ratio induced by cyclosporine A treatment. These findings establish cyclophilin C as an ER cyclophilin, demonstrate the novel involvement of cyclophilins B and C in ER redox homeostasis, and suggest the existence of an additional ER oxidative pathway that is modulated by ER cyclophilins.     

Crystal structure of the cyclophilin-like domain from the parasitic nematode Brugia malayi.

Cyclophilins are a family of proteins that exhibit peptidyl-prolyl cis-trans isomerase activity and bind the immunosuppressive agent cyclosporin A (CsA). Brugia malayi is a filarial nematode parasite of humans, for which a cyclophilin-like domain was identified at the N-terminal of a protein containing 843 amino acid residues. There are two differences in sequence in the highly conserved CsA binding site: A histidine and a lysine replace a tryptophan and an alanine, respectively. The crystal structure of this domain has been determined by the molecular replacement method and refined to an R-factor of 16.9% at 2.15 A resolution. The overall structure is similar to other cyclophilins; however, major differences occur in two loops. Comparison of the CsA binding site of this domain with members of the cyclophilin family shows significant structural differences, which can account for the reduced sensitivity of the Brugia malayi protein to inhibition by CsA.     

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.