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Brucella spp. are the etiological agent of animal and human brucellosis. We have reported previously that cyclophilins of Brucella (CypA and CypB) are upregulated within the intraphagosomal replicative niche and required for stress adaptation and host intracellular survival and virulence. Here, we characterize B. abortus cyclophilins, CypA, and CypB from a biochemical standpoint by studying their PPIase activity, chaperone activity, and oligomer formation. Even though CypA and CypB are very similar in sequence and share identical chaperone and PPIase activities, we were able to identify outstanding differential features between them. A series of differential peptide loops were predicted when comparing CypA and CypB, differences that might explain why specific antibodies (anti-CypA or anti-CypB) were able to discriminate between both cyclophilins without cross-reactivity. In addition, we identified the presence of critical amino acids in CypB, such as the Trp134 which is responsible for the cyclosporin A inhibition, and the Cys128 that leads to CypB homodimer formation by establishing a disulfide bond. Here, we demonstrated that CypB dimer formation was fully required for stress adaptation, survival within HeLa cells, and mouse infection in B. abortus. The presence of Trp134 and the Cys128 in CypB, which are not present in CypA, suggested that two different kinds of cyclophilins have evolved in Brucella, one with eukaryotic features (CypB), another (CypA) with similar features to Gramnegative cyclophilins. [ABSTRACT FROM AUTHOR]

Brucella spp. are the etiological agent of animal and human brucellosis. We have reported previously that cyclophilins of Brucella (CypA and CypB) are upregulated within the intraphagosomal replicative niche and required for stress adaptation and host intracellular survival and virulence. Here, we characterize B. abortus cyclophilins, CypA, and CypB from a biochemical standpoint by studying their PPIase activity, chaperone activity, and oligomer formation. Even though CypA and CypB are very similar in sequence and share identical chaperone and PPIase activities, we were able to identify outstanding differential features between them. A series of differential peptide loops were predicted when comparing CypA and CypB, differences that might explain why specific antibodies (anti-CypA or anti-CypB) were able to discriminate between both cyclophilins without cross-reactivity. In addition, we identified the presence of critical amino acids in CypB, such as the Trp134 which is responsible for the cyclosporin A inhibition, and the Cys128 that leads to CypB homodimer formation by establishing a disulfide bond. Here, we demonstrated that CypB dimer formation was fully required for stress adaptation, survival within HeLa cells, and mouse infection in B. abortus. The presence of Trp134 and the Cys128 in CypB, which are not present in CypA, suggested that two different kinds of cyclophilins have evolved in Brucella, one with eukaryotic features (CypB), another (CypA) with similar features to Gram-negative cyclophilins.

We recently reported that the PPIase Par14 and Par17 encoded by PIN4 upregulate HBV replication in an HBx-dependent manner by binding to conserved arginine–proline (RP) motifs of HBx. HBV core protein (HBc) has a conserved 133RP134 motif; therefore, we investigated whether Par14/Par17 bind to HBc and/or core particles. Native agarose gel electrophoresis (NAGE) and immunoblotting and co-immunoprecipitation were used. Chromatin immunoprecipitation from HBV-infected HepG2-hNTCP-C9 cells was performed. NAGE and immunoblotting revealed that Par14/Par17 bound to core particles and co-immunoprecipitation revealed that Par14/Par17 interacted with core particle assembly-defective, and dimer-positive HBc-Y132A. Thus, core particles and HBc interact with Par14/Par17. Par14/Par17 interacted with the HBc 133RP134 motif possibly via substrate-binding E46/D74 and E71/D99 motifs. Although Par14/Par17 dissociated from core particles upon heat treatment, they were detected in 0.2 N NaOH-treated opened-up core particles, demonstrating that Par14/Par17 bind outside and inside core particles. Furthermore, these interactions enhanced the stabilities of HBc and core particles. Like HBc-Y132A, HBc-R133D and HBc-R133E were core particle assembly-defective and dimer-positive, demonstrating that a negatively charged residue at position 133 cannot be tolerated for particle assembly. Although positively charged R133 is solely important for Par14/17 interactions, the 133RP134 motif is important for efficient HBV replication. Chromatin immunoprecipitation from HBV-infected cells revealed that the S19 and E46/D74 residues of Par14 and S44 and E71/D99 residues of Par17 were involved in recruitment of 133RP134 motif-containing HBc into cccDNA. Our results demonstrate that interactions of HBc, Par14/Par17, and cccDNA in the nucleus and core particle–Par14/Par17 interactions in the cytoplasm are important for HBV replication. [ABSTRACT FROM AUTHOR]

We recently reported that the PPIase Par14 and Par17 encoded by PIN4 upregulate HBV replication in an HBx-dependent manner by binding to conserved arginine–proline (RP) motifs of HBx. HBV core protein (HBc) has a conserved 133RP134 motif; therefore, we investigated whether Par14/Par17 bind to HBc and/or core particles. Native agarose gel electrophoresis (NAGE) and immunoblotting and co-immunoprecipitation were used. Chromatin immunoprecipitation from HBV-infected HepG2-hNTCP-C9 cells was performed. NAGE and immunoblotting revealed that Par14/Par17 bound to core particles and co-immunoprecipitation revealed that Par14/Par17 interacted with core particle assembly-defective, and dimer-positive HBc-Y132A. Thus, core particles and HBc interact with Par14/Par17. Par14/Par17 interacted with the HBc 133RP134 motif possibly via substrate-binding E46/D74 and E71/D99 motifs. Although Par14/Par17 dissociated from core particles upon heat treatment, they were detected in 0.2 N NaOH-treated opened-up core particles, demonstrating that Par14/Par17 bind outside and inside core particles. Furthermore, these interactions enhanced the stabilities of HBc and core particles. Like HBc-Y132A, HBc-R133D and HBc-R133E were core particle assembly-defective and dimer-positive, demonstrating that a negatively charged residue at position 133 cannot be tolerated for particle assembly. Although positively charged R133 is solely important for Par14/17 interactions, the 133RP134 motif is important for efficient HBV replication. Chromatin immunoprecipitation from HBV-infected cells revealed that the S19 and E46/D74 residues of Par14 and S44 and E71/D99 residues of Par17 were involved in recruitment of 133RP134 motif-containing HBc into cccDNA. Our results demonstrate that interactions of HBc, Par14/Par17, and cccDNA in the nucleus and core particle–Par14/Par17 interactions in the cytoplasm are important for HBV replication.

Hepatic ischemia/reperfusion injury is a complication of liver surgery that involves mitochondrial dysfunction resulting from mitochondrial permeability transition pore (mPTP) opening. Cyclophilin D (PPIF or CypD) is a peptidyl-prolyl cis - trans isomerase that regulates mPTP opening in the inner mitochondrial membrane. We investigated whether and how recently created small-molecule inhibitors of CypD prevent opening of the mPTP in hepatocytes and the resulting effects in cell models and livers of mice undergoing ischemia/reperfusion injury. We measured the activity of 9 small-molecule inhibitors of cyclophilins in an assay of CypD activity. The effects of the small-molecule CypD inhibitors or vehicle on mPTP opening were assessed by measuring mitochondrial swelling and calcium retention in isolated liver mitochondria from C57BL/6J (wild-type) and Ppif –/– (CypD knockout) mice and in primary mouse and human hepatocytes by fluorescence microscopy. We induced ischemia/reperfusion injury in livers of mice given a small-molecule CypD inhibitor or vehicle before and during reperfusion and collected samples of blood and liver for histologic analysis. The compounds inhibited peptidyl-prolyl isomerase activity (half maximal inhibitory concentration values, 0.2–16.2 μmol/L) and, as a result, calcium-induced mitochondrial swelling, by preventing mPTP opening (half maximal inhibitory concentration values, 1.4–132 μmol/L) in a concentration-dependent manner. The most potent inhibitor (C31) bound CypD with high affinity and inhibited swelling in mitochondria from livers of wild-type and Ppif –/– mice (indicating an additional, CypD-independent effect on mPTP opening) and in primary human and mouse hepatocytes. Administration of C31 in mice with ischemia/reperfusion injury before and during reperfusion restored hepatic calcium retention capacity and oxidative phosphorylation parameters and reduced liver damage compared with vehicle. Recently created small-molecule inhibitors of CypD reduced calcium-induced swelling in mitochondria from mouse and human liver tissues. Administration of these compounds to mice during ischemia/reperfusion restored hepatic calcium retention capacity and oxidative phosphorylation parameters and reduced liver damage. These compounds might be developed to protect patients from ischemia/reperfusion injury after liver surgery or for other hepatic or nonhepatic disorders related to abnormal mPTP opening. [ABSTRACT FROM AUTHOR]

FKBP22, an Escherichia coli -made peptidyl-prolyl cis - trans isomerase, has shown considerable homology with Mip-like virulence factors. While the C-terminal domain of this enzyme is used for executing catalytic function and binding inhibitor, the N-terminal domain is employed for its dimerization. To precisely determine the underlying factors of FKBP22 dimerization, its structural model, developed using a suitable template, was carefully inspected. The data show that the dimeric FKBP22, like dimeric Mip proteins, has a V-like shape. Further, it dimerizes using 40 amino acid residues including Ile 9, Ile 17, Ile 42, and Ile 65. All of the above Ile residues except Ile 9 are partly conserved in the Mip-like proteins. To confirm the roles of the partly conserved Ile residues, three FKBP22 mutants, constructed by substituting them with an Ala residue, were studied as well. The results together indicate that Ile 65 has little role in maintaining the dimeric state or enzymatic activity of FKBP22. Conversely, both Ile 17 and Ile 42 are essential for preserving the structure, enzymatic activity, and dimerization ability of FKBP22. Ile 42 in particular looks more essential to FKBP22. However, none of these two Ile residues is required for binding the cognate inhibitor. Additional computational studies also indicated the change of V-shape and the dimeric state of FKBP22 due to the Ala substitution at position 42. The ways Ile 17 and Ile 42 protect the structure, function, and dimerization of FKBP22 have been discussed at length.Communicated by Ramaswamy H. Sarma.

Alternative splicing (AS) is an important molecular mechanism by which single genes can generate multiple mRNA isoforms. We reported previously that, in Oryza sativa, the cyclophilin 19-4 (OsCYP19-4.1) transcript was significantly upregulated in response to cold stress, and that transgenic plants were cold tolerant. Here we show that, under cold stress, OsCYP19-4 produces eight transcript variants by intron retention and exon skipping, resulting in production of four distinct protein isoforms. The OsCYP19-4 AS isoforms exhibited different cellular localizations in the epidermal cells: in contrast to OsCYP19-4.1, the OsCYP19-4.2 and OsCYP19-4.3 proteins were primarily targeted to guard and subsidiary cells, whereas OsCYP19-4.5, which consists largely of an endoplasmic reticulum (ER) targeting signal, was co-localized with the RFP-BiP marker in the ER. In OsCYP19-4.2, the key residues of the PPIase domain are altered; consistent with this, recombinant OsCYP19-4.2 had significantly lower PPIase activity than OsCYP19-4.1 in vitro. Specific protein-protein interactions between OsCYP19-4.2/3 and AtRCN1 were verified in yeast two-hybrid (Y2H) and bimolecular fluoresence complementation (BiFC assays), although the OsCYP19-4 isoforms could not bind each other. Based on these results, we propose that two OsCYP19-4 AS isoforms, OsCYP19-4.2 and OsCYP19-4.3, play roles linking auxin transport and cold stress via interactions with RCN1. [ABSTRACT FROM AUTHOR]

Background: Cyclophilin A (CypA), a receptor for the immunosuppressive agent cyclosporin A (CsA), is a cis-trans peptidyl-prolyl isomerase (PPIase) which accelerates the cis-trans isomerization of prolyl-peptide bonds and interacts with a variety of proteins to regulate their activities. Results: The full-length cDNA of crab Eriocheir sinensis CypA (EsCypA) was cloned by EST and RACE technique. The complete sequence of EsCypA cDNA contained a 5' untranslated region (UTR) of 50 bp, a 3' UTR of 233 bp with a polyA tail, and an open reading frame (ORF) of 495 bp encoding a polypeptide of 164 amino acids with the predicted molecular weight of 17.36 kDa. The deduced amino acid sequence of EsCypA contained two highly conserved signature sequences of peptidyl-prolyl cis-trans isomerase and a pro-isomerase domain. The mRNA transcripts of EsCypA were detectable in all the examined tissues, including haemocytes, gill, hepatopancreas, gonad, muscle and heart, with higher expression level in hepatopancreas and gonad. No significant difference in the relative mRNA expression level of EsCypA was observed during the whole course of bacteria challenge, whereas it was up-regulated during fungi challenge. The purified recombinant protein rEsCypA exhibited a significant PPIase activity and an antifungal activity. Conclusions: All these results indicated that it was a typical CypA member and potentially involved in the innate immune responses of crab. [ABSTRACT FROM AUTHOR]

Abstract: Cyclophilin A (CypA) plays an important role in many physiology processes and its overexpression has been involved in many diseases including immune disease, viral infection, neuro-degenerative disease, and cancer. However, the actual role of CypA in the diseases is still far from clear, and a complete understanding of CypA is necessary in order to direct more specific and effective therapeutic strategies. Based on the screening of our in-house library through the isomer-specific proteolysis method, we find a CypA activator (1-(2,6-Dibenzyloxybenzoyl)-3-(9H-fluoren-9-yl)-urea), compound 1a, which can increase CypA’s PPIase activity and give allosteric behavior. The binding affinity of compound 1a to CypA has been confirmed by Fortebio’s Octet RED system and the increased phosphorylation of ERK in H446 cells is observed by treatment with both compound 1a and CsA. In order to further evaluate the binding mode between the activator and CypA, the allosteric binding site and allosteric mechanism of CypA are investigated by molecular dynamics (MD) simulations in combination with mutagenesis experiments. The results show that the allosteric binding site of CypA is 7Å away from its catalytic site and is composed of Cys52, His70, His54, Lys151, Thr152 and Lys155. Compound 1a binds to the allosteric site of CypA, stabilizing the active conformation of catalytic residues, and finally promotes the catalytic efficiency of CypA. We believe our finding of the CypA allosteric activator will be used as an effective chemical tool for further studies of CypA mechanisms in diseases. [Copyright &y& Elsevier]

Cyclophilin, a cytosolic receptor for the immunosuppressive drug cyclosporin A, plays a role in diverse pathophysiologies along with its receptor, CD147. Although the interaction between cyclophilin A and CD147 is well established in inflammatory disease, that of cyclophilin B (CypB) with CD147 has not been fully explored, especially in cancer cell biology, and the exact molecular mechanism underlying such an association is poorly understood. In this study, we first identified high expression levels of CypB in 54 % of hepatocellular carcinoma patient tissues but in only 12.5 % of normal liver tissues. Then, we demonstrated that CypB overexpression protects human hepatoma cells against oxidative stress through its binding to CD147; this protective effect depends on the peptidyl prolyl isomerase activity of CypB. siRNA-mediated knockdown of CypB expression rendered hepatoma cells more vulnerable to ROS-mediated apoptosis. Furthermore, we also determined that a direct interaction between secreted CypB and CD147 regulates the extracellular signal-regulated kinase intracellular signaling pathway and is indispensible for the protective functions of CypB. For the first time, we demonstrated that CypB has an essential function in protecting hepatoma cells against oxidative stress through binding to CD147 and regulating the ERK pathway. [ABSTRACT FROM AUTHOR]

Abstract: Through virtual screening of a rationally built database consisting of 40 peptides, we identified three short peptides. After testing these three synthetic peptides, we found that the peptide Trp-Gly-Pro (WGP) showed comparable inhibitory ability as positive control cyclosporine A (CsA) on CypA-mediated PPIase activity with IC50 values of 33.11nM and 10.25nM, respectively. The peptide WGP had same order of CypA-binding affinity as CsA with dissociation equilibrium constant K D of 3.41×10−6 and 6.42×10−6 M, respectively. This peptide could also inhibit HIV-1IIIB infection. This study provides a novel strategy for rational design and development of peptidic drugs. [Copyright &y& Elsevier]

Abstract: We describe a new type of protein-imprinted polymer for separation/enrichment of active natural protein present at a relatively low level in cell extracts, with a cloned bacterial protein as template. In this work, cloned pig cyclophilin 18 (pCyP18) was used as template. The template protein was selectively assembled with assistant recognition polymer chains (ARPCs) from their library, which consists of numerous limited length polymer chains with randomly distributed recognition and immobilizing sites. These assemblies of protein and ARPCs were adsorbed by porous polymeric beads and immobilized by cross-linking polymerization. After removing the template, the synthesized imprinted polymer was used to adsorb authentic pCyP18 from cell extract, and its proportional content was enriched 200 times. The assay of peptidyl-prolyl cis–trans-isomerase (PPIase) activity showed that natural pCyP18 is more active than cloned pCyP18 and, in particular, it is much more sensitive to the suppressant cyclosporine A (CsA). [Copyright &y& Elsevier]

Abstract: We have shown here the identification and characterization of two cyclophilin, cyclophilin A (CyPA) and B (CyPB), from the ixodid tick, Haemaphysalis longicornis. Both CyPA and CyPB contain the conserved peptidyl-prolyl isomerase (PPIase) domain, with CyPA consisting of 188 amino acids and CyPB of 216 amino acids. CyPA and CyPB share 50–67% amino acid sequence identity with the cyclophilins of other organisms, and are found in multiple organs throughout the developmental stages of ixodid ticks. In addition, recombinant CyPA and CyPB exhibited PPIase activity that could be inhibited by the cyclic peptides cyclosporin A (CsA). Silencing of CyPA through RNA interference has led to a significant reduction in the body weight of engorged ticks and their failure to lay eggs, in contrast to CyPB whose silencing did not result in any detectable phenotypic changes. Our results indicate that CyPA represents the major cyclophilin protein in H. longicornis involved in blood ingestion, viability, and oocyte development. This is the first report of cyclophilin proteins from ixodid and argasid ticks. [Copyright &y& Elsevier]

Abstract: Human nuclear cyclophilin 33 (hCyP33) was the first protein which was found to contain an RNA-binding motif and a PPIase domain. It was not known what cellular and physiological roles are played by the RNA-binding activity as well as the PPIase activity of hCyP33. In this paper, we investigated the binding specificity of hCyP33 to different cellular RNA using ion-exchange chromatography and affinity adsorption. Furthermore, the influence of different cellular RNAs to the PPIase activity of hCyP33 was investigated using a protease-coupled method. The results show that hCyP33 binds specifically to mRNA, namely poly(A)+RNA, and that binding stimulates the PPIase activity of hCyP33. [Copyright &y& Elsevier]

Abstract: 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 Ca2+-binding sites, binds to mBiP in the endoplasmic reticulum (ER) and this binding is affected by the Ca2+ 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. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

Alternative splicing (AS) is an important molecular mechanism by which single genes can generate multiple mRNA isoforms. We reported previously that, in Oryza sativa, the cyclophilin 19-4 (OsCYP19-4.1) transcript was significantly upregulated in response to cold stress, and that transgenic plants were cold tolerant. Here we show that, under cold stress, OsCYP19-4 produces eight transcript variants by intron retention and exon skipping, resulting in production of four distinct protein isoforms. The OsCYP19-4 AS isoforms exhibited different cellular localizations in the epidermal cells: in contrast to OsCYP19-4.1, the OsCYP19-4.2 and OsCYP19-4.3 proteins were primarily targeted to guard and subsidiary cells, whereas OsCYP19-4.5, which consists largely of an endoplasmic reticulum (ER) targeting signal, was co-localized with the RFP-BiP marker in the ER. In OsCYP19-4.2, the key residues of the PPIase domain are altered; consistent with this, recombinant OsCYP19-4.2 had significantly lower PPIase activity than OsCYP19-4.1 in vitro. Specific protein-protein interactions between OsCYP19-4.2/3 and AtRCN1 were verified in yeast two-hybrid (Y2H) and bimolecular fluoresence complementation (BiFC assays), although the OsCYP19-4 isoforms could not bind each other. Based on these results, we propose that two OsCYP19-4 AS isoforms, OsCYP19-4.2 and OsCYP19-4.3, play roles linking auxin transport and cold stress via interactions with RCN1.

Aim: To characterize the biochemical features of the newest member of cyclophilin family of peptidyl-prolyl cis/trans-isomerases (PPIases), cyclophilin J (CYPJ)., Materials and Methods: PPIase assays were performed on purified hCYPJ and its mutated variants. The substrate specificity, half-maximal inhibitory concentration (IC50) of cyclosporin A (CsA) inhibition and circular dichroism (CD) spectrum of CYPJ were measured. Mercury pathway profiling luciferase assays were also performed., Results: The catalytic number/Michaelis constant (kcat/KM) value of CYPJ was 9.5×10(4) s(-1)M(-1). CYPJ additionally catalyzed norleucine-proline, isoleucine-proline and glutamine-proline peptides compared to CYPA and Escherichia coli PPIases. CYPJ was inhibited by CsA in a dose-dependent manner with IC50 of 12.1±0.9 μM. The CD spectrum of CYPJ was similar to CYPA. CYPJ significantly up-regulated the transcription of E-box, E2F, retinoblastoma (Rb), p53, activator protein 1 (AP1), NF-κB and phospho-cAMP response element (CRE) cis-response element in 293T cells., Conclusion: CYPJ structurally resembles CYPA. It is sensitive to inhibition by CsA and plays a role in regulating cell growth, proliferation, and apoptosis., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)