Identification and comparative analysis of the peptidyl-prolyl cis/trans isomerase repertoires of H. sapiens, D. melanogaster, C. elegans, S. cerevisiae and Sz. pombe

The peptidyl-prolyl cis/trans isomerase (PPIase) class of proteins comprises three member families that are found throughout nature and are present in all the major compartments of the cell. Their numbers appear to be linked to the number of genes in their respective genomes, although we have found the human repertoire to be smaller than expected due to a reduced cyclophilin repertoire. We show here that whilst the members of the cyclophilin family (which are predominantly found in the nucleus and cytoplasm) and the parvulin family (which are predominantly nuclear) are largely conserved between different repertoires, the FKBPs (which are predominantly found in the cytoplasm and endoplasmic reticulum) are not. It therefore appears that the cyclophilins and parvulins have evolved to perform conserved functions, while the FKBPs have evolved to fill ever-changing niches within the constantly evolving organisms. Many orthologous subgroups within the different PPIase families appear to have evolved from a distinct common ancestor, whereas others, such as the mitochondrial cyclophilins, appear to have evolved independently of one another. We have also identified a novel parvulin within Drosophila melanogaster that is unique to the fruit fly, indicating a recent evolutionary emergence. Interestingly, the fission yeast repertoire, which contains no unique cyclophilins and parvulins, shares no PPIases solely with the budding yeast but it does share a majority with the higher eukaryotes in this study, unlike the budding yeast. It therefore appears that, in comparison with Schizosaccharomyces pombe, Saccharomyces cerevisiae is a poor representation of the higher eukaryotes for the study of PPIases.     

Peptidylprolylisomerases,Protein Folders,or Scaffolders? The Example of FKBP51 and FKBP52

Peptidylprolyl-isomerases (PPIases) comprise of the protein families of FK506 binding proteins (FKBPs), cyclophilins, and parvulins. Their common feature is their ability to expedite the transition of peptidylprolyl bonds between the cis and the trans conformation. Thus, it seemed highly plausible that PPIase enzymatic activity is crucial for protein folding. However, this has been difficult to prove over the decades since their discovery. In parallel, more and more studies have discovered scaffolding functions of PPIases. This essay discusses the hypothesis that PPIase enzymatic activity might be the consequence of binding to peptidylprolyl protein motifs. The main focus of this paper is the large immunophilins FKBP51 and FKBP52, but other PPIases such as cyclophilin A and Pin1 are also described. From the hypothesis, it follows that the PPIase activity of these proteins might be less relevant, if at all, than the organization of protein complexes through versatile protein binding. Also see the video abstract here https://youtu.be/A33la0dx5LE .     

The binding of FKBP23 to BiP modulates BiP's ATPase activity with its PPIase activity

Peptidyl-prolyl cis-trans-isomerases (PPIases) are enzymes that can cis-trans-isomerize a Xaa-Pro peptide bond. Three families of PPIases are known: cyclophilins, FKBPs, and parvulins. The physiological functions of the PPIases are only poorly understood. In previous work, we reported that the mouse FK506-binding protein 23 (mFKBP23), which comprises an N-terminal PPIase domain and a C-terminal domain with Ca(2+)-binding sites, binds to mBiP in the endoplasmic reticulum (ER) and this binding is affected by the Ca(2+) concentration. In this study, we demonstrate the ability of mFKBP23 to modulate the ATPase activity of BiP, and that the bound mFKBP23, but not the free mFKBP23, can suppress the ATPase activity of mBiP through its PPIase activity.     

Molecular cloning and characterization of genes encoding FK506-binding proteins (FKBPs) in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

The family of FK506-binding proteins (FKBPs) consists of several members, which show peptidyl prolyl cis–trans isomerase (PPIase) activity. PPIases facilitate the conversion of peptidyl prolyl bonds from cis to trans conformation, a rate-limiting step in protein folding. In the present study, we carried out cloning of cDNAs encoding three different wheat FKBPs viz., TaFKBP20-1, TaFKBP16-1 and TaFKBP15-1. In silico analysis suggested their likely localization to nucleus, cytosol and endoplasmic reticulum, respectively. Biochemical analyses demonstrated that none of the three purified FKBP proteins possesses detectable PPIase activity. Several putative interacting partners of TaFKBP20-1, TaFKBP16-1 and TaFKBP15-1were identified using online software tools. The results of this study provide further evidence that PPIase activity in plant FKBPs is not conserved, and these proteins may be playing important roles in the cell through interaction with target proteins.     

Immunophilin AtFKBP13 sustains all peptidyl-prolyl isomerase activity in the thylakoid lumen from Arabidopsis thaliana deficient in AtCYP20-2

The physiological roles of immunophilins are unclear, but many possess peptidyl-prolyl isomerase (PPIase) activity, and they have been found in all organisms examined to date, implying that they are involved in fundamental, protein-folding processes. The chloroplast thylakoid lumen of the higher plant Arabidopsis thaliana contains up to 16 immunophilins (five cyclophilins and 11 FKBPs), but only two of them, AtCYP20-2 and AtFKBP13, have been found to be active PPIases, indicating that the other immunophilins in this cellular compartment may have lost their putative PPIase activities. To assess this possibility, we characterized two independent Arabidopsis knockout lines lacking AtCYP20-2 in enzymological and quantitative proteomic analyses. The PPIase activity in thylakoid lumen preparations of both mutants was equal to that of corresponding wild-type preparations, and comparative two-dimensional difference gel electrophoresis analyses of the lumenal proteins of the mutants and wild type showed that none of the potential PPIases was more abundant in the AtCYP20-2 deficient plants. Enzymatic analyses established that all PPIase activity in the mutant thylakoid lumen was attributable to AtFKBP13, and oxidative activation of this enzyme compensated for the lack of AtCYP20-2. Accordingly, sequence analyses of the potential catalytic domains of lumenal cyclophilins and FKBPs demonstrated that only AtCYP20-2 and AtFKBP13 possess all of the amino acid residues found to be essential for PPIase activity in earlier studies of human cyclophilin A and FKBP12. Thus, none of the immunophilins in the chloroplast thylakoid lumen of Arabidopsis except AtCYP20-2 and AtFKBP13 appear to possess prolyl isomerase activity toward peptide substrates.     

Transcriptional and biochemical characterization of two <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis> FKBP family members

Peptidyl-prolyl cis/trans isomerases (PPIases, EC: 5.2.1.8), a class of enzymes that catalyse the rate-limiting step of the cis/trans isomerization in protein folding, are divided into three structurally unrelated families: cyclophilins, FK506-binding proteins (FKBPs), and parvulins. Two recombinant FKBPs from the soil nitrogen-fixing bacterium Azotobacter vinelandii, designated as AvfkbX and AvfkbB, have been purified and their peptidyl-prolyl cis/trans isomerase activity against Suc-Ala-Xaa-Pro-Phe-pNA synthetic peptides characterised. The substrate specificity of both enzymes is typical for bacterial FKBPs, with Suc-Ala-Phe-Pro-Phe-pNA being the most rapidly catalysed substrate by AvfkbX and Suc-Ala-Leu-Pro-Phe-pNA by AvfkbB. Both FKBPs display chaperone activity as well in the citrate synthase thermal aggregation assay. Furthermore, using real-time RT-qPCR, we demonstrated that both genes were expressed during the exponential growth phase on glucose minimal medium, while their expression declined dramatically during the stationary growth phase as well as when the growth medium was supplied exogenously with ammonium.     

Biochemical characterization of two Azotobacter vinelandii FKBPs and analysis of their interaction with the small subunit of carbamoyl phosphate synthetase

FK-506 binding proteins (FKBPs) belong to the peptidyl-prolyl cis/trans isomerase superfamily (PPIases, EC: 5.2.1.8) which catalyzes the interconversion of peptidyl-prolyl bonds while they can also act on polypeptides, as folding helper enzymes. Here, we biochemically characterize two recombinant FKBPs, AvfkbA1 and AvfkbA2, from the soil nitrogen-fixing bacterium Azotobacter vinelandii and show that both possess PPIase activity while AvfkbA2 possesses chaperone activity as well. Further, we demonstrate their physical interaction with AvcarA, the small subunit of carbamoyl phosphate synthetase. Using RT-qPCR, we show that AvfkbA1 and AvfkbA2 are co-expressed with AvcarA under the same growth conditions. A decrease in AvfkbA1 or AvfkbA2 PPIase activity, in the presence of AvcarA, further confirms each interaction. However, PPIase activity does not seem to be essential for these interactions since PPIase active site mutations of both FKBPs do not abolish the AvcarA binding. The P³⁵⁸ residue of AvcarA, possibly retaining a cis configuration, is critical only for the interaction with AvfkbA1. The presence of either of the two FKBPs did not influence the measured glutamine hydrolyzing activity of AvcarA. Taken together, these data indicate that although the two FKBPs have a common biological substrate they probably have differing physiological roles.     

Classification of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>l. japonica nipponbare) immunophilins (FKBPs,CYPs) and expression patterns under water stress

Background  

FK506 binding proteins (FKBPs) and cyclophilins (CYPs) are abundant and ubiquitous proteins belonging to the peptidyl-prolyl cis/trans isomerase (PPIase) superfamily, which regulate much of metabolism through a chaperone or an isomerization of proline residues during protein folding. They are collectively referred to as immunophilin (IMM), being present in almost all cellular organs. In particular, a number of IMMs relate to environmental stresses.     

The peptidyl-prolyl isomerase and chaperone Par27 of Bordetella pertussis as the prototype for a new group of parvulins

Proteins that pass through the periplasm in an unfolded state are highly sensitive to proteolysis and aggregation and, therefore, often require protection by chaperone-like proteins. The periplasm of Gram-negative bacteria is well equipped with ATP-independent chaperones and folding catalysts, including peptidyl-prolyl isomerases (PPIases). The filamentous hemagglutinin of Bordetella pertussis, which is secreted by the two-partner secretion pathway, crosses the periplasm in an unfolded conformation. By affinity chromatography, we identified a new periplasmic PPIase of the parvulin family, Par27, which binds to an unfolded filamentous hemagglutinin fragment. Par27 differs from previously characterized bacterial and eukaryotic parvulins. Its central parvulin-like domain is flanked by atypical N- and C-terminal extensions that are found in a number of putative PPIases present mostly in β proteobacteria. Par27 displays both PPIase and chaperone activities in vitro. In vivo, Par27 might function as a general periplasmic chaperone in B. pertussis.     

Functional characterization of two novel parvulins in Trypanosoma brucei

Parvulins belong to a family of peptidyl-prolyl cis/trans isomerases (PPIases) that catalyze the cis/trans conformations of prolyl-peptidyl bonds. Herein, we characterized two novel parvulins, TbPIN1 and TbPAR42, in Trypanosoma brucei. TbPIN1, a 115 amino-acid protein, contains a single PPIase domain but lacks the N-terminal WW domain. Using NMR spectroscopy, TbPIN1 was found to exhibit PPIase activity toward a phosphorylated substrate. Overexpression of TbPIN1 can rescue the impaired temperature-sensitive phenotype in a mutant yeast strain. TbPAR42, containing 383 amino acids, comprises a novel FHA domain at its N terminus and a C-terminal PPIase domain but is a non-Pin1-type PPIase. Functionally, a knockdown of TbPAR42 in its procyclic form results in reduced proliferation rates suggesting an important role in cell growth.     

Cyclophilins: Proteins in search of function

Cyclophilins constitute a subgroup of large family of proteins called immunophilins, which also include FKBPs and Parvulins. They are remarkably conserved in all genera, highlighting their pivotal role in important cellular processes. Most cyclophilins display PPIase enzymatic activity, multiplicity, diverse cellular locations and active role in protein folding which render them to be included in the class of diverse set of proteins called molecular chaperones. Due to their distinct PPIase function, besides protein disulfide isomerases and protein foldases, cyclophilins have been deemed necessary for in vivo chaperoning activity. Unlike other cellular chaperones, these proteins are specific in their respective targets. Not all cyclophilin proteins possess PPIase activity, indicating a loss of their PPIase activity during the course of evolution and gain of function independent of their PPIase activity. The PPIase function of cyclophilins is also compensated by their functional homologs, like FKBPs. Multiple cyclophilin members in plants like Arabidopsis and rice have been reported to be associated with diverse functions and regulatory pathways through their foldase, scaffolding, chaperoning or other unknown activities. Although many functions of plant cyclophilins were reported or suggested, the physiological relevance and molecular basis of stress-responsive expression of plant cyclophilins is still largely unknown. However, their wide distribution and ubiquitous nature signifies their fundamental importance in plant survival. Several of these members have also been directly linked to multiple stresses. This review attempts to deal with plant cyclophilins with respect to their role in stress response.     

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.     

PPIase activities and interaction partners of FK506-binding proteins in the wheat thylakoid

FK506-binding proteins (FKBPs) and cyclophilins, collectively called immunophilins, conserve peptidyl-prolyl cis/trans isomerase (PPIase) active sites, although many lack PPIase activity. The chloroplast thylakoid contains a large proportion of the plant immunophilin family, but their functions within this compartment are unclear. Some lumenal immunophilins are important for assembly of photosynthetic complexes, implicating them in the maintenance and turnover of the photosynthetic apparatus during acclimation processes. In this investigation into the functions of three FKBPs localized to the thylakoid of Triticum aestivum (wheat), we present the first evidence of PPIase activity in the thylakoid of a cereal plant, and also show that PPIase activity is not conserved in all lumenal FKBPs. Using yeast two-hybrid analysis we found that the PPIase-active FKBP13 interacts with the globular domain of the wheat Rieske protein, with potential impact on photosynthetic electron transfer. Specific interaction partners for PPIase-deficient FKBP16-1 and FKBP16-3 link these isoforms to photosystem assembly.     

Immunophilins and parvulins. Superfamily of peptidyl prolyl isomerases in Arabidopsis

Immunophilins are defined as receptors for immunosuppressive drugs including cyclosporin A, FK506, and rapamycin. The cyclosporin A receptors are referred to as cyclophilins (CYPs) and FK506- and rapamycin-binding proteins are abbreviated as FKBPs. These two groups of proteins (collectively called immunophilins) share little sequence homology, but both have peptidyl prolyl cis/trans isomerase (PPIase) activity that is involved in protein folding processes. Studies have identified immunophilins in all organisms examined including bacteria, fungi, animals, and plants. Nevertheless, the physiological function of immunophilins is poorly understood in any organism. In this study, we have surveyed the genes encoding immunophilins in Arabidopsis genome. A total of 52 genes have been found to encode putative immunophilins, among which 23 are putative FKBPs and 29 are putative CYPs. This is by far the largest immunophilin family identified in any organism. Both FKBPs and CYPs can be classified into single domain and multiple domain members. The single domain members contain a basic catalytic domain and some of them have signal sequences for targeting to a specific organelle. The multiple domain members contain not only the catalytic domain but also defined modules that are involved in protein-protein interaction or other functions. A striking feature of immunophilins in Arabidopsis is that a large fraction of FKBPs and CYPs are localized in the chloroplast, a possible explanation for why plants have a larger immunophilin family than animals. Parvulins represent another family of PPIases that are unrelated to immunophilins in protein sequences and drug binding properties. Three parvulin genes were found in Arabidopsis genome. The expression of many immunophilin and parvulin genes is ubiquitous except for those encoding chloroplast members that are often detected only in the green tissues. The large number of genes and diversity of structure domains and cellular localization make PPIases a versatile superfamily of proteins that clearly function in many cellular processes in plants.     

A ribosome-associated peptidyl-prolyl cis/trans isomerase identified as the trigger factor.

Peptidyl-prolyl cis/trans isomerases (PPIases) are enzymes that catalyse protein folding both in vitro and in vivo. We isolated a peptidyl-prolyl cis/trans isomerase (PPIase) which is specifically associated with the 50S subunit of the Escherichia coli ribosome. This association was abolished by adding at least 1.5 M LiCl. Sequencing the N-terminal amino acids in addition to three proteolytic fragments totalling 62 amino acids revealed that this PPIase is identical to the E.coli trigger factor. A comparison of the amino acid sequence of trigger factor with those of other PPIase families shows little similarities, suggesting that trigger factor may represent an additional family of PPIases. Trigger factor was purified to homogeneity on a preparative scale from E.coli and its enzymatic properties were studied. In its activity towards oligopeptide substrates, the trigger factor resembles the FK506-binding proteins (FKBPs). Additionally, the pattern of subsite specificities with respect to the amino acid preceding proline in Suc-Ala-Xaa-Pro-Phe-4-nitroanilides is reminiscent of FKBPs. However, the PPIase activity of the trigger factor was not inhibited by either FK506 or by cyclosporin A at concentrations up to 100 microM. In vitro, the trigger factor catalysed the proline-limited refolding of a variant of RNase T1 much better than all other PPIases that have been examined so far.     

Stress-Induced Changes in Peptidyl-Prolyl cis-trans Isomerase Activity of Sorghum bicolor Seedlings

Developmental changes and effects of various abiotic stresses on peptidyl prolyl cis-trans isomerase (PPIase) activity were studied in the seedlings of sorghum [Sorghum bicolor (L.) Moench cv. CSH-6]. The PPIase activity of sorghum seedlings markedly decreased after two days of germination. Up to 90 % of the PPIase activity was inhibited by cyclosporin-A. Maximal increase in specific PPIase activity in the 3-d-old seedlings was observed in response to osmotic stress and it was transient in nature. The stress-induced enhancement in PPIase activity, depending upon tissue and stress treatment, was due to induction of cyclophilins as well as other PPIases. Osmotic stress-induced enhancement in PPIase activity in the drought susceptible cv. SPRU-94008B was maximal in roots, as compared to shoots in the drought tolerant cv. ICSV-272. This revised version was published online in July 2006 with corrections to the Cover Date.     

Prolyl isomerases in a minimal cell. Catalysis of protein folding by trigger factor from Mycoplasma genitalium.

Peptidyl-prolyl cis/trans isomerases (PPIases) catalyze the isomerization of prolyl peptide bonds. Distinct families of this class of enzymes are involved in protein folding in vitro, whereas their significance in free living organisms is not known. Previously, we inspected the smallest known genome of a self-replicating organism and found that Mycoplasma genitalium is devoid of all known PPIases except the trigger factor. Despite the extensive sequence information becoming available, most genes remain hypothetical and enzyme activities in many species have not been assigned to an open reading frame. Therefore, we studied the PPIase activity in crude extracts of M. genitalium. We showed that this is solely attributed to a single enzyme activity, the trigger factor. Characterization of this enzyme revealed that its PPIase activity resides in a central 12-kDa domain. Only the complete trigger factor is able to cis/trans isomerize extended peptide substrates, while the PPIase domain alone can not. The N- and the C-terminal domains of the trigger factor seem to function in binding of proteins as substrates, as demonstrated by protein refolding experiments, in which the complete trigger factor catalyzed protein refolding towards a model protein 500-fold more efficiently than the isolated central PPIase domain. Protein modeling studies suggest that the PPIase domain can fold in a similar way as the PPIase domain of FK506 binding proteins (FKBPs), one class of PPIases, despite only very limited sequence homology. Differences at the active site explain why this enzyme is not inhibited by FK506 in contrast with FKBPs. Trigger factor expressed in Escherichia coli confirms its additional chaperone functions, as shown by its association with chaperones GroEL and GroES after induction of misfolding. In contrast, the isolated PPIase-domain lacks any association with chaperones from E. coli. In summary, trigger factor of M. genitalium is the single folding isomerase of this organism, which harbors an enzymatically active PPIase domain with structural homology to FKBPs. Its additional domains confer its ability to be an efficient catalyst of protein folding. The protein folding machinery is conserved and shows a dual function as a chaperone and a prolyl isomerase.     

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.