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Joseph Heitman - One of the best experts on this subject based on the ideXlab platform.
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The Cyclophilins.
Genome biology, 2005Co-Authors: Ping Wang, Joseph HeitmanAbstract:Cyclophilins (Enzyme Commission (EC) number 5.1.2.8) belong to a group of proteins that have peptidyl-prolyl cis-trans isomerase activity; such proteins are collectively known as immunophilins and also include the FK-506-binding proteins and the parvulins. Cyclophilins are found in all cells of all organisms studied, in both prokaryotes and eukaryotes; humans have a total of 16 Cyclophilin proteins, Arabidopsis up to 29 and Saccharomyces 8. The first member of the Cyclophilins to be identified in mammals, Cyclophilin A, is the major cellular target for, and thus mediates the actions of, the immunosuppressive drug cyclosporin A. Cyclophilin A forms a ternary complex with cyclosporin A and the calcium-calmodulin-activated serine/threonine-specific protein phosphatase calcineurin; formation of this complex prevents calcineurin from regulating cytokine gene transcription. Recent studies have implicated a diverse array of additional cellular functions for Cyclophilins, including roles as chaperones and in cell signaling.
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Cyclophilin A and Ess1 interact with and regulate silencing by the Sin3-Rpd3 histone deacetylase.
The EMBO journal, 2000Co-Authors: Miguel Arévalo-rodríguez, Maria E Cardenas, Steven D. Hanes, Joseph HeitmanAbstract:Three families of prolyl isomerases have been identified: Cyclophilins, FK506-binding proteins (FKBPs) and parvulins. All 12 Cyclophilins and FKBPs are dispensable for growth in yeast, whereas the one parvulin homolog, Ess1, is essential. We report here that Cyclophilin A becomes essential when Ess1 function is compromised. We also show that overexpression of Cyclophilin A suppresses ess1 conditional and null mutations, and that Cyclophilin A enzymatic activity is required for suppression. These results indicate that Cyclophilin A and Ess1 function in parallel pathways and act on common targets by a mechanism that requires prolyl isomerization. Using genetic and biochemical approaches, we found that one of these targets is the Sin3-Rpd3 histone deacetylase complex, and that Cyclophilin A increases and Ess1 decreases disruption of gene silencing by this complex. We show that conditions that favor acetylation over deacetylation suppress ess1 mutations. Our findings support a model in which Ess1 and Cyclophilin A modulate the activity of the Sin3-Rpd3 complex, and excess histone deacetylation causes mitotic arrest in ess1 mutants.
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all Cyclophilins and fk506 binding proteins are individually and collectively dispensable for viability in saccharomyces cerevisiae
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Kara Dolinski, Scott Muir, Maria E Cardenas, Joseph HeitmanAbstract:The Cyclophilins and FK506 binding proteins (FKBPs) bind to cyclosporin A, FK506, and rapamycin and mediate their immunosuppressive and toxic effects, but the physiological functions of these proteins are largely unknown. Cyclophilins and FKBPs are ubiquitous and highly conserved enzymes that catalyze peptidyl-prolyl isomerization, a rate-limiting step during in vitro protein folding. We have addressed their functions by a genetic approach in the yeast Saccharomyces cerevisiae. Five Cyclophilins and three FKBPs previously were identified in yeast. We identified four additional enzymes: Cpr6 and Cpr7, which are homologs of mammalian Cyclophilin 40 that have also recently been independently isolated by others, Cpr8, a homolog of the secretory pathway Cyclophilin Cpr4, and Fpr4, a homolog of the nucleolar FKBP, Fpr3. None of the eight Cyclophilins or four FKBPs were essential. Surprisingly, yeast mutants lacking all 12 immunophilins were viable, and the phenotype of the dodecuplet mutant resulted from simple addition of the subtle phenotypes of each individual mutation. We conclude that Cyclophilins and FKBPs do not play an essential general role in protein folding and find little evidence of functional overlap between the different enzymes. We propose that each Cyclophilin and FKBP instead regulates a restricted number of unique partner proteins that remain to be identified.
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A yeast Cyclophilin gene essential for lactate metabolism at high temperature.
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: Edward S. Davis, Joseph Heitman, Andrea Becker, Michael N. Hall, Miles B. BrennanAbstract:The Cyclophilins are a family of ubiquitous eukaryotic proteins first identified by high affinity for cyclosporin A (CsA). The immunosuppressant and cytotoxic effects of CsA are thought to result from formation of a toxic complex between Cyclophilin and CsA rather than from inhibition of Cyclophilin function. The physiological role(s) of the Cyclophilins is unknown. Cyclophilins have in vitro peptidylprolyl cistrans isomerase (PPIase) activity, and thus may be involved in protein folding in vivo. We have isolated a yeast Cyclophilin gene, CPR3, which encodes a presumptive mitochondrial isoform. While CPR3 disruption mutants lack any phenotype at 30 degrees C, they are unable to grow on L-lactate at 37 degrees C. Disruptions of two other Cyclophilin genes (CPR1, CPR2) and of FPR1, the gene encoding an FK506 binding protein with PPIase activity, do not affect growth on L-lactate at 37 degrees C. L-Lactate metabolism requires transcriptional induction of CYB2, the gene encoding flavocytochrome b2; cpr3 mutants induce transcription of this gene normally. This result demonstrates a conditional lethal phenotype for a Cyclophilin mutation and presents a system for genetic and biochemical analysis of Cyclophilin function.
Peter Peichl - One of the best experts on this subject based on the ideXlab platform.
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Presence of Cyclophilin A in Synovial Fluids of Patients with Rheumatoid Arthritis
The Journal of experimental medicine, 1997Co-Authors: Andreas Billich, Gottfried Winkler, Heinrich Aschauer, Antal Rot, Peter PeichlAbstract:Cyclophilins have been suggested to act as leukocyte chemotactic factors produced in the course of inflammation. Therefore we looked for the presence of Cyclophilins in the synovial fluids (SF) from patients with rheumatoid arthritis (RA). Peptidyl prolyl cis-trans isomerase activity (PPIase) was measured in SF from knee punctures of 26 patients with RA and five patients with knee osteoarthritis (OA). PPIase was detected in SF from RA patients, but not in samples from OA patients. Enzyme activity was sensitive to inhibition by cyclosporin A (IC50 = 28-50 nM). Estimated concentrations of the SF-derived Cyclophilin based on the enzyme activity were in the range of 11 to 705 nM. The presence of Cyclophilin in the SF showed disease correlation; its concentration correlated with the number of cells in the SF (r = 0.91, P < 0.0001) and with the percentage of neutrophils in the cellular infiltrate and was higher in more acute cases of joint swelling. In immunoblots of partially purified preparations of SF from RA patients, an approximately 18-kD protein band reacted with polyclonal antibodies that recognize Cyclophilin A and B, but not with antibodies specific for Cyclophilin B. Sequencing of this protein revealed identity of the NH2-terminal amino acids with those of human Cyclophilin A. The finding is unexpected since Cyclophilin B rather than A is generally regarded as the secreted isoform, the presence of Cyclophilin A being confined to the cytoplasm. Our data support the hypothesis that Cyclophilins may contribute to the pathogenesis of inflammatory diseases, possibly by acting as cytokines. This may offer a possible explanation of the effectiveness of cyclosporin A in RA, in addition to the known immunosuppressive effects of the drug.
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Presence of Cyclophilin A in Synovial Fluids of Patients with Rheumatoid Arthritis
The Journal of experimental medicine, 1997Co-Authors: Andreas Billich, Gottfried Winkler, Heinrich Aschauer, Antal Rot, Peter PeichlAbstract:Cyclophilins have been suggested to act as leukocyte chemotactic factors produced in the course of inflammation. Therefore we looked for the presence of Cyclophilins in the synovial fluids (SF) from patients with rheumatoid arthritis (RA). Peptidyl prolyl cis–trans isomerase activity (PPIase) was measured in SF from knee punctures of 26 patients with RA and five patients with knee osteoarthritis (OA). PPIase was detected in SF from RA patients, but not in samples from OA patients. Enzyme activity was sensitive to inhibition by cyclosporin A (IC50 = 28–50 nM). Estimated concentrations of the SF-derived Cyclophilin based on the enzyme activity were in the range of 11 to 705 nM. The presence of Cyclophilin in the SF showed disease correlation; its concentration correlated with the number of cells in the SF ( r = 0.91, P
Nikolai V. Naoumov - One of the best experts on this subject based on the ideXlab platform.
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alisporivir inhibition of hepatocyte Cyclophilins reduces hbv replication and hepatitis b surface antigen production
Gastroenterology, 2015Co-Authors: Sandra Phillips, S Chokshi, Udayan Chatterji, A Riva, Michael Bobardt, Roger Williams, Philippe Gallay, Nikolai V. NaoumovAbstract:Background & Aims Cyclophilins are host factors required for hepatitis C virus replication. Cyclophilin inhibitors such as alisporivir have shown strong anti–hepatitis C virus activity in vitro and in clinical studies. However, little is known about whether hepatocyte Cyclophilins are involved in the hepatitis B virus (HBV) life cycle. We investigated the effects of 2 Cyclophilin inhibitors (alisporivir and NIM811) on HBV replication and hepatitis B surface antigen (HBsAg) production in cell lines. Methods Liver-derived cell lines producing full-length HBV and HBsAg particles, owing to stable (HepG2215) or transient (HuH-7) transfection, or infected with HBV (HepaRG cells; Invitrogen [Carlsbad, CA]), were incubated with alisporivir or NIM811 alone, or alisporivir in combination with a direct antiviral (telbivudine). The roles of individual Cyclophilins in drug response was evaluated by small interfering RNA knockdown of Cyclophilin (CYP)A, CYPC, or CYPD in HepG2215 cells, or CYPA knockdown in HuH-7 cells. The kinetics of antiviral activity were assessed based on levels of HBV DNA and HBsAg and Southern blot analysis. Results In HepG2215, HuH-7, and HepaRG cells, alisporivir reduced intracellular and secreted HBV DNA, in a dose-dependent manner. Knockdown of CYPA, CYPC, or CYPD (reduced by 80%) significantly reduced levels of HBV DNA and secreted HBsAg. Knockdown of CYPA significantly reduced secretion of HBsAg, leading to accumulation of intracellular HBsAg; the addition of alisporivir greatly reduced levels of HBsAg in these cells. The combination of alisporivir and telbivudine had greater antiviral effects than those of telbivudine or alisporivir alone. Conclusions Alisporivir inhibition of Cyclophilins in hepatocyte cell lines reduces replication of HBV DNA and HBsAg production and secretion. These effects are potentiated in combination with direct antiviral agents that target HBV-DNA polymerase.
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Cyclophilin inhibition as potential therapy for liver diseases.
Journal of hepatology, 2014Co-Authors: Nikolai V. NaoumovAbstract:The Cyclophilins are a group of proteins with peptidyl-prolyl isomerase enzymatic activity, localised in different cellular compartments and involved in a variety of functions related to cell metabolism and energy homeostasis, having enhanced expression in inflammation or malignancy. Cyclophilin A (CypA), the most abundantly expressed Cyclophilin, is present mainly in the cytoplasm and is a host factor involved in the life cycle of multiple viruses. The extracellular fractions of CypA and CypB are potent pro-inflammatory mediators. CypD, located in mitochondria, is a key regulator of mitochondrial permeability transition pores, and is critical for necrotic cell death. Cyclosporines are the prototype Cyclophilin inhibitors. Cyclic peptides, which bind and inhibit Cyclophilins without having immunosuppressive properties, have been generated by chemical modifications of cyclosporin A. In addition, Cyclophilin inhibitors that are structurally different from cyclosporines have been synthesized. The involvement of Cyclophilins in the pathogenesis of different liver diseases has been established using both in vitro and in vivo investigations, thus indicating that Cyclophilin inhibition may be of therapeutic benefit. This review summarises the evidence for potential therapeutic applications of non-immunosuppressive Cyclophilin inhibitors, alone or in combination with other agents, in virus-induced liver diseases like hepatitis C, B or Delta, liver inflammation and fibrosis, acetaminophen-induced liver toxicity and hepatocellular carcinoma.
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oc 025 alisporivir inhibition of cellular Cyclophilins disrupts hepatitis b virus hbv replication and this effect is further enhanced in combination with direct antiviral targeting hbv dna polymerase in vitro
Gut, 2012Co-Authors: S Chokshi, A Riva, S Phillips, Nikolai V. NaoumovAbstract:Introduction Cyclophilins are intracellular proteins with enzymatic activity—peptidyl-prolyl-isomerase that plays a major role in the life cycle of Hepatitis C virus. By targeting host Cyclophilins Alisporivir (DEB025) exerts potent anti-HCV activity in vitro and in clinical studies. We have recently shown in vitro that Cyclophilin inhibition with Alisporivir or NIM811 also interferes with HBV replication, with Alisporivir having a greater effect than NIM811. To elucidate the underlying mechanisms, in the present study we compared in vitro the effects on HBV replication of Alisporivir alone, Alisporivir in combination with a potent antiviral targeting HBV-DNA polymerase, and in cells after selective knockdown of individual Cyclophilins. Methods Stably (HepG2215) and transiently (HUH-7) transfected cells, producing full HBV virions and HBsAg particles, were treated with different Alisporivir concentrations (0.25/1.0/5.0/20 ug/ml) alone, Telbivudine alone, or combinations of Alisporivir and Telbivudine. To determine the involvement of individual Cyclophilins, HepG2215 cells were transfected with siRNA-specific for Cyclophilin (Cyp) A, C or D and additionally treated with Alisporivir. Cytoplasmic extracts and supernatants were harvested at baseline; 24, 48 and 72 h post-treatment. The kinetics of antiviral activity was assessed by quantitation of intracellular and secreted HBV-DNA (real-time qPCR) and HBsAg levels (ELISA). Results Alisporivir treatment resulted in dose-dependent reduction of intracellular and secreted HBV-DNA from HepG2215 and HUH-7 cells at all time points, by 70% (p=0.004) and 63% (p 3-fold reduction of HBsAg vs either Alisporivir or Telbivudine alone. CypA, C or D expression was markedly reduced after transfection with corresponding siRNA, which was associated with significant decrease of HBV-DNA and HBsAg levels (p Conclusion These results suggest that Alisporivir interferes with multiple sites of HBV replication and has synergistic antiviral activity with direct antiviral targeting viral DNA polymerase, such as Telbivudine. Competing interests None declared.
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p43 cellular protein Cyclophilin a is involved in hepatitis b virus replication and its inhibition with deb025 alisporivir or nim811 demonstrates antiviral activity in vitro
Gut, 2011Co-Authors: S Chokshi, A Riva, S Phillips, Nikolai V. NaoumovAbstract:Introduction Cyclophilins are ubiquitously expressed intracellular proteins that have enzymatic activity—peptidyl-prolyl cis-trans isomerase, and are involved in regulation of mitochondrial/cytosolic transport in the cells. DEB025 (Alisporivir) and NIM811 are non-immunosuppressive Cyclophilin inhibitors that efficiently block hepatitis C virus replication by targeting host rather than viral proteins, however the potential impact of cyclophillin inhibition on hepatitis B virus (HBV) life cycle is poorly understood. Aim In the present study we employed a panel of liver cell lines to investigate the ability of Cyclophilin inhibitors DEB025 (Alisporivir) and NIM811 to affect HBV replication and viral particle secretion from the cells. Method HepG2215 cells, stably transfected with the full HBV genome and supporting the production of both infectious virions and HBsAg particles, and the parent cells (HepG2) were cultured for 7 days (baseline), prior to treatment with DEB025 or NIM811 at 0.25; 1.0 and 5.0 mg/ml. The cells and supernatants were harvested separately at baseline; 6, 24, 48 and 72 h after addition of Cyclophilin inhibitors. HBV DNA levels - both intracellular and in culture supernatants—were quantitated by Taqman qPCR (ABI7500). Western blot and ELISA were used to assess intracellular and secreted HBsAg, respectively. PLC/PRF/5 cells, expressing only HBsAg, were also tested. Cyclophilin expression in the cells was silenced by transfection separately with siRNA for Cyclophilins A, B, C or D to determine the role of individual Cyclophilins in HBV replication. Results Cyclophilin inhibition with either DEB025 or NIM811 significantly reduced cytoplasmic core-particle associated HBV DNA levels in the cells, between 2 and 10-fold as compared with the control cells. The most pronounced reduction of intracellular HBV DNA (by 10-fold at 72 h) was observed with DEB025 5 mg/ml, which was greater than the reduction observed with NIM811. Similarly, DEB025 (at 1 and 5 mg/ml) showed a greater impact in reducing HBV virion secretion in the supernatants, compared with NIM811. HBsAg secretion from the cells was also reduced by up to 50% when compared to controls. Cyclophilin-A expression was markedly reduced after transfection with corresponding siRNA, which led to a rapid decrease of intracellular HBV DNA by 2 logs. HBV-DNA was reduced further when the cyclophillin-A silenced cultures were treated with NIM811 or DEB025. Conclusion These results demonstrate that Cyclophilin A is directly involved in HBV replication. Cyclophilin inhibition by DEB025 or NIM811 interferes with HBV replication within liver cells and reduces the secretion of infectious virions and HBsAg particles from the cells, with DEB025 having a greater antiviral activity than NIM811.
Mrinal Bhave - One of the best experts on this subject based on the ideXlab platform.
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Characterisation and physical mapping of Cyclophilin A genes and identification of new classes of Cyclophilins in wheat
Journal of Cereal Science, 2004Co-Authors: Joshua Johnson, Mrinal BhaveAbstract:The Cyclophilins, a class of ‘foldase’ enzymes that catalyse the rate-limiting step of peptidyl-prolyl cis–trans isomerization, are up-regulated during wheat endosperm development and suggested to be important for folding of the storage proteins, thus influencing wheat quality. However, little information exists on the types of Cyclophilins expressed, the genes encoding these, and their possible functions in wheat endosperm. We have characterised three isoforms of genes encoding Cyclophilin A from eight wheat cultivars, using previously isolated cDNAs. The genes are small, intronless, comprise a small multi-gene family, lack any inter-cultivar polymorphisms and localise to chromosomal arms 6AS, 6BS and 6DS, in a region where genes for other quality traits are localised, the locus at 6AS possibly having duplicated genes. Further, cDNAs encoding two novel, endosperm-expressed classes of Cyclophilins have been isolated, one being potentially plastid-localised and the other, a nuclear protein with Cyclophilin-like domains. In-silico analyses have further led to identification of another form of Cyclophilin A and a potentially ER-localised, endosperm-expressed Cyclophilin B. The plastid and ER-localised forms are of particular relevance to events occurring during endosperm maturation. The results thus provide valuable data and molecular tools for isolation and analysis of these genes, to address their roles and any association with wheat quality traits
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isolation and characterisation of cdnas encoding protein disulphide isomerases and Cyclophilins in wheat
Journal of Cereal Science, 2001Co-Authors: Joshua Johnson, Mrinal Bhave, B C ClarkeAbstract:The enzyme families of protein disulphide isomerases (PDI) and Cyclophilins catalyse the isomerisation of disulphide bonds and the rotation of Xaa-Pro peptide bonds in nascent proteins, respectively, making them excellent candidates for regulating the deposition of storage proteins in wheat. This study aimed to isolate and characterise clones encoding Cyclophilins and PDIs from a wheat endosperm cDNA library. A number of cDNA clones were isolated, of which three different cDNAs each of PDI and Cyclophilin were selected for complete sequencing. The three PDI cDNAs encoded putative protein products of 513 or 516 amino acids, all exhibiting conserved sequences for the N-terminal signal peptide, the two thioredoxin-like domains at the catalytic sites and the C-terminal ER retention signal. The three Cyclophilin cDNAs exhibited 68–87% identity to other reported Cyclophilins and encoded putative protein products of 171 amino acids containing the conserved tryptophan residue and a 7 amino acid sequence unique to certain plant Cyclophilins. The sequence variations within and outside the coding regions of all the cDNAs suggest that both enzymes are encoded by multiple genes. This information will allow further investigations into the roles of these enzymes in storage protein deposition in the developing endosperm and thus in wheat quality.
John A. Cidlowski - One of the best experts on this subject based on the ideXlab platform.
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Native Recombinant Cyclophilins A, B, and C Degrade DNA Independently of Peptidylprolyl cis-trans-Isomerase Activity POTENTIAL ROLES OF CyclophilinS IN APOPTOSIS
The Journal of biological chemistry, 1997Co-Authors: Jennifer W. Montague, Francis M. Hughes, John A. CidlowskiAbstract:Previous work in our laboratory (Montague, J., Gaido, M., Frye, C., and Cidlowski, J. (1994) J. Biol. Chem. 269, 18877-18880) has shown that human recombinant Cyclophilins A, B, and C have sequence homology with the apoptotic nuclease NUC18 and that denatured Cyclophilins can degrade DNA. We have now evaluated the nucleolytic activity of recombinant Cyclophilins under native conditions. We show that nuclease activity inherent to Cyclophilins is distinct from cis-trans-peptidylprolyl isomerase activity and is similar to that described for apoptotic nucleases. Cyclophilin nucleolytic activity is stimulated by Ca2+ and/or Mg2+, with a combination of the two being optimal for Cyclophilins A and B. Mg2+ alone is sufficient for Cyclophilin C nuclease activity. pH optimums are in the range of pH 7.5-9.5. Cyclophilins can degrade both single-stranded and double-stranded DNA. Additionally, Cyclophilins produce 3'-OH termini in linear double-stranded substrates, suggesting the cuts produced are similar to those of apoptotic cells. Cyclophilins also display endonucleolytic activity, demonstrated by their ability to degrade supercoiled DNA. In the absence of ions, Cyclophilins bind linearized DNA. When added to nuclei from nonapoptotic cells, Cyclophilin C induces 50-kilobase pair DNA fragmentation but not internucleosomal fragmentation. Together, these data suggest that Cyclophilins are involved in degradation of the genome during apoptosis.
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a calcium dependent nuclease from apoptotic rat thymocytes is homologous with Cyclophilin recombinant Cyclophilins a b and c have nuclease activity
Journal of Biological Chemistry, 1994Co-Authors: Jennifer W. Montague, M L Gaido, C Frye, John A. CidlowskiAbstract:Apoptosis is an important physiological process that involves the deletion of specific cells in a controlled and timely manner. A biochemical hallmark typifying apoptosis in normal lymphocytes is DNA cleavage caused by a calcium-dependent nuclease. We have previously identified and purified an 18-kDa nuclease (NUC18) from glucocorticoid-treated rat thymocytes whose activity is associated with this apoptotic DNA fragmentation. Partial protein sequencing of pure NUC18 has generated two peptide sequences that have a remarkable similarity to rat Cyclophilin A and other members of the Cyclophilin family. We report here that recombinant Cyclophilins A, B, and C have a calcium/magnesium-dependent nuclease activity with biochemical and pharmacological properties similar to those of NUC18. Our results raise the intriguing possibility that Cyclophilin or a Cyclophilin-related protein may play a role in lymphocyte apoptosis.
