Presence Of Cyclophilin Research Articles

Effective reduction of Tau amyloid aggregates in the presence of Cyclophilin from Platanus orientalis pollens; An alternative mechanism of action of the allergen.

A hallmark pathology of Alzheimer's disease (AD) is the construction of neurofibrillary tangles, which are made of hyperphosphorylated Tau. The cis-proline isomer of the pThr/Ser-Pro sequence has been suggested to act as an aggregation precursor according to the 'Cistauosis' hypothesis; however, this aggregation scheme is not yet completely approved. Various peptidyl-prolyl isomerases (PPIases) may specifically isomerize cis/trans-proline bonds and restitute Tau's ability to attach microtubules and may control Tau amyloid aggregation in AD. In this study, we provided experimental evidence for indicating the effects of the plant Cyclophilin (P-Cyp) from Platanus orientalis pollens on the Tau aggregation by various spectroscopic techniques. Our findings disclosed that the rate/extent of amyloid formation in the Tau sample which is incubated with P-Cyp decreased and these observations do not seem to be due to the macromolecular crowding effect. Also, as proven that 80% of the prolines in the unfolded protein are in the trans conformation, urea-induced unfolding analyses confirmed this conclusion and showed that the aggregation rate/extent of urea-treated Tau samples decreased compared with those of the native protein. Also, XRD analysis indicated the reduction of scattering intensities and beta structures of amyloid fibrils in the presence of P-Cyp. Therefore, the ability of P-Cyp to suppress Tau aggregation probably depends on cis to trans isomerization of proline peptide bonds (X-Pro) and decreasing cis isomers in vitro. The findings of the current study may inspire possible protective/detrimental effects of various types of cyclophilins on AD onset/progression through direct regulation of intracellular Tau molecules and provide evidence that a protein from a plant source is able to enter the cell cytoplasm and may affect the behavior of cytoplasmic proteins.

Metformin attenuates effects of cyclophilin A on macrophages, reduces lipid uptake and secretion of cytokines by repressing decreased AMPK activity.

Growing evidence implicates cyclophilin A secreted by vascular wall cells and monocytes as a key mediator in atherosclerosis. Cyclophilin A in addition to its proliferative effects, during hyperglycemic conditions, increases lipid uptake in macrophages by increasing scavenger receptors on the cell's surface. It also promotes macrophage migration across endothelial cells and conversion of macrophages into foam cells. Given the known effects of metformin in reducing vascular complications of diabetes, we investigated the effect of metformin on cyclophilin A action in macrophages. Using an ex vivo model of cultured macrophages isolated from patients with type 2 diabetes with and without coronary artery disease (CAD), we measured the effect of metformin on cyclophilin A expression, lipid accumulation, expression of scavenger receptors, plasma cytokine levels and AMP-activated protein kinase (AMPK) activity in macrophages. In addition, the effects of metformin on migration of monocytes, reactive oxygen species (ROS) formation, lipid uptake in the presence of cyclophilin A inhibitors and comparison with pioglitazone were studied using THP-1 monocytes. Metformin reduced cyclophilin A expression in human monocyte-derived macrophages. Metformin also decreased the effects of cyclophilin A on macrophages such as oxidized low-density lipoprotein (oxLDL) uptake, scavenger receptor expression, ROS formation and secretion of inflammatory cytokines in high-glucose conditions. Metformin reversed cyclophilin A-induced decrease in AMPK-1α activity in macrophages. These effects of metformin were similar to those of cyclophilin A inhibitors. Metformin can thus function as a suppressor of pro-inflammatory effects of cyclophilin A in high-glucose conditions by attenuating its expression and repressing cyclophilin A-induced decrease in AMPK-1α activity in macrophages.

Microbial cyclophilins: specialized functions in virulence and beyond.

Cyclophilins belong to the superfamily of peptidyl-prolyl cis/trans isomerases (PPIases, EC: 5.2.1.8), the enzymes that catalyze the cis/trans isomerization of peptidyl-prolyl peptide bonds in unfolded and partially folded polypeptide chains and native state proteins. Cyclophilins have been extensively studied, since they are involved in multiple cellular processes related to human pathologies, such as neurodegenerative disorders, infectious diseases, and cancer. However, the presence of cyclophilins in all domains of life indicates a broader biological importance. In this mini-review, we summarize current advances in the study of microbial cyclophilins. Apart from their anticipated role in protein folding and chaperoning, cyclophilins are involved in several other biological processes, such as cellular signal transduction, adaptation to stress, control of pathogens virulence, and modulation of host immune response. Since many existing family members do not have well-defined functions and novel ones are being characterized, the requirement for further studies on their biological role and molecular mechanism of action is apparent.

Cyclophilin A enhances macrophage differentiation and lipid uptake in high glucose conditions: a cellular mechanism for accelerated macro vascular disease in diabetes mellitus.

BackgroundVascular disease in diabetes is initiated by monocyte adhesion to vascular endothelium, transmigration and formation of foam cells. Increasing clinical evidence supports a role for the secretory protein, cyclophilin A in diabetic vascular disease. The means by which cyclophilin A contributes to vascular lesion development in diabetes is however largely unknown.MethodsIn this study we investigated using THP1 cells and human monocytes whether cyclophilin A under hyperglycemic conditions, functions in the inflammatory cascade as a chemoattractant and increases lipid uptake by formation of foam cells invitro. We developed an invitro model of monocytes cultured in 20 mm glucose (high glucose) equivalent to 360 mg/dL of plasma glucose levels. These monocytes were then differentiated into macrophages using PMA and subsequently transformed to lipid laden foam cells using oxidized low density lipoproteins in the presence and absence of cyclophilin A. This cellular model was used to study monocyte to macrophage differentiation, transmigration and foam cell formation. A similar cellular model using siRNA mediated transient elimination of the cyclophilin A gene as well as chemical inhibitors were used to further confirm the role of cyclophilin A in the differentiation and foam cell formation process.ResultsCyclophilin A effectively increased migration of high glucose treated monocytes to the endothelial cell monolayer (p < 0.0001). In the presence of cyclophilin A, differentiated macrophages, when treated with oxLDL had a 36 percent increase in intracellular lipid accumulation (p = 0.01) when compared to cells treated with oxLDL alone. An increased flux of reactive oxygen species was also observed (p = 0.01). Inflammatory cytokines such as TNF-α, MCP-1 and cyclophilin A were significantly increased. Silencing cyclophilin A in THP-1 cells and human monocytes using siRNA or chemical inhibitor, TMN355 resulted in decrease in lipid uptake by 65–75% even after exposure to oxidized LDL. The expression of scavenger receptors expressed during differentiation process, CD36 and LOX-1 were decreased (p < 0.0001). Levels of extracellular cyclophilin A and other inflammatory cytokines such as TNF-α and MCP-1also significantly reduced.ConclusionsTaken together, we describe here a possible cellular basis by which cyclophilin A may accelerate atherogenesis in diabetes mellitus.Electronic supplementary materialThe online version of this article (doi:10.1186/s12933-016-0467-5) contains supplementary material, which is available to authorized users.

Mitochondrial Calcium and Reactive Oxygen Species Regulate Agonist-Initiated Platelet Phosphatidylserine Exposure

To study the interactions of cytoplasmic calcium elevation, mitochondrial permeability transition pore (mPTP) formation, and reactive oxygen species formation in the regulation of phosphatidylserine (PS) exposure in platelets. mPTP formation, but not the degree of cytoplasmic calcium elevation, was associated with PS exposure in wild-type, cyclophilin D-null, ionomycin-treated, and reactive oxygen species-treated platelets. In the absence of the mPTP regulator cyclophilin D, agonist-initiated mPTP formation and high-level PS exposure were markedly blunted, but cytoplasmic calcium transients were unchanged. Mitochondrial calcium (Ca(2+)(mit)) transients and reactive oxygen species, key regulators of mPTP formation, were examined in strongly stimulated platelets. Increased reactive oxygen species production occurred in strongly stimulated platelets and was dependent on extracellular calcium entry, but not the presence of cyclophilin D. Ca(2+)(mit) increased significantly in strongly stimulated platelets. Abrogation of Ca(2+)(mit) entry, either by inhibition of the Ca(2+)(mit) uniporter or mitochondrial depolarization, prevented mPTP formation and exposure but not platelet aggregation or granule release. Sustained cytoplasmic calcium levels are necessary, but not sufficient, for high-level PS exposure in response to agonists. Increased Ca(2+)(mit) levels are a key signal initiating mPTP formation and PS exposure. Blockade of Ca(2+)(mit) entry allows the specific inhibition of platelet procoagulant activity.

Functional interaction of Azotobacter vinelandii cytoplasmic cyclophilin with the biotin carboxylase subunit of acetyl-CoA carboxylase

Cyclophilins (E.C. 5.1.2.8) are protein chaperones with peptidyl-prolyl cis/trans isomerase activity (PPIase). In the present study, we demonstrate a physical interaction among AvppiB, encoding the cytoplasmic cyclophilin from the soil nitrogen-fixing bacterium Azotobacter vinelandii, and AvaccC, encoding the biotin carboxylase subunit of acetyl-CoA carboxylase, which catalyzes the committed step in long-chain fatty acid synthesis. A decrease in AvppiB PPIase activity, in the presence of AvaccC, further confirms the interaction. However, PPIase activity seems not to be essential for these interactions since a PPIase active site mutant of cyclophilin does not abolish the AvaccC binding. We further show that the presence of cyclophilin largely influences the measured ATP hydrolyzing activity of AvaccA in a way that is negatively regulated by the PPIase activity. Taken together, our data support a novel role for cyclophilin in regulating biotin carboxylase activity.

An optimised system for refolding of human glucose 6-phosphate dehydrogenase.

BackgroundHuman glucose 6-phosphate dehydrogenase (G6PD), active in both dimer and tetramer forms, is the key entry enzyme in the pentose phosphate pathway (PPP), providing NADPH for biosynthesis and various other purposes, including protection against oxidative stress in erythrocytes. Accordingly haemolytic disease is a major consequence of G6PD deficiency mutations in man, and many severe disease phenotypes are attributed to G6PD folding problems. Therefore, a robust refolding method with high recovery yield and reproducibility is of particular importance to study those clinical mutant enzymes as well as to shed light generally on the refolding process of large multi-domain proteins.ResultsThe effects of different chemical and physical variables on the refolding of human recombinant G6PD have been extensively investigated. L-Arg, NADP+ and DTT are all major positive influences on refolding, and temperature, protein concentration, salt types and other additives also have significant impacts. With the method described here, ~70% enzyme activity could be regained, with good reproducibility, after denaturation with Gdn-HCl, by rapid dilution of the protein, and the refolded enzyme displays kinetic and CD properties indistinguishable from those of the native protein. Refolding under these conditions is relatively slow, taking about 7 days to complete at room temperature even in the presence of cyclophilin A, a peptidylprolyl isomerase reported to increase refolding rates. The refolded protein intermediates shift from dominant monomer to dimer during this process, the gradual emergence of dimer correlating well with the regain of enzyme activity.ConclusionL-Arg is the key player in the refolding of human G6PD, preventing the aggregation of folding intermediate, and NADP+ is essential for the folding intermediate to adopt native structure. The refolding protocol can be applied to produce high recovery yield of folded protein with unaltered properties, paving the way for future studies on clinical G6PD mutants with folding defects and providing a useful model system to study the folding process of oligomeric proteins.

Dynamic Combinatorial Self‐Assembly of Cyclophilin hCyp‐18 Ligands through Oxorhenium Coordination

The dynamic combinatorial assembly of independent modules A and B through oxorhenium(V) coordination by a NS2+S motif in the presence of cyclophilin hCyp-18-an important peptidyl-prolyl isomerase-was investigated. Increasing glutathione (GSH) concentrations were used to dissociate [ARe(V)OB] complexes that displayed low affinity for hCyp-18. Conversely, coordinates that displayed submicromolar affinities for hCyp-18 were protected against thiol exchange and could be detected by LC-MS. Determination of the GSH concentration that decreased the extracted ionic current of the complex by 50 % (CC(50)) enabled the selection of three oxorhenium coordinates that were shown to bind to the active site of hCyp-18 and to inhibit its peptidyl-prolyl isomerase activity in the micromolar to submicromolar range.

Thrombin-Cleaved COOH-Terminal Osteopontin Peptide Binds with Cyclophilin C to CD147 in Murine Breast Cancer Cells

Osteopontin is a glycoprotein that has been linked to metastatic function in breast, lung, and prostate cancers. However, the mechanism by which osteopontin acts to induce metastatic properties is largely unknown. One intriguing feature of osteopontin is the presence of a conserved thrombin cleavage site that is COOH-terminal from a well-characterized RGD domain. Although the COOH-terminal fragment may bind to cell surface CD44 receptors, little is known about the COOH-terminal osteopontin fragment. In the current study, we use the murine mammary epithelial tumor cell lines 4T1 and 4T07; these cells are thioguanine-resistant sublines derived from the parental population of 410.4 cells from Balb/cfC3H mice. Using flow cytometry and Forster resonance energy transfer, we show that the COOH-terminal fragment of osteopontin binds with another marker of metastatic function (cyclophilin C or rotamase) to the CD147 cell surface glycoprotein (also known as extracellular matrix metalloproteinase inducer), to activate Akt1/2 and matrix metalloproteinase-2. In in vitro assays, thrombin cleavage of osteopontin to generate short COOH-terminal osteopontin in the presence of cyclophilin C increases migration and invasion of both 4T07 and 4T1 cells. This interaction between osteopontin peptide and cyclophilin C has not been previously described but assigns a heretofore unknown function for the thrombin-cleaved osteopontin COOH-terminal fragment.

Aha1 competes with Hop, p50 and p23 for binding to the molecular chaperone Hsp90 and contributes to kinase and hormone receptor activation

The ATP-dependent molecular chaperone Hsp90 (heat-shock protein 90) is essential for the maturation of hormone receptors and protein kinases. During the process of client protein activation, Hsp90 co-operates with cofactors/co-chaperones of unique sequence, e.g. Aha1 (activator of Hsp90 ATPase 1), p23 or p50, and with cofactors containing TPR (tetratricopeptide repeat) domains, e.g. Hop, immunophilins or cyclophilins. Although the binding sites for these different types of cofactors are distributed along the three domains of Hsp90, sterical overlap and competition for binding sites restrict the combinations of cofactors that can bind to Hsp90 at the same time. The recently discovered cofactor Aha1 associates with the middle domain of Hsp90, but its relationship to other cofactors of the molecular chaperone is poorly understood. Therefore we analysed whether complexes of Aha1, p23, p50, Hop and a cyclophilin with Hsp90 are disrupted by the other four cofactors by gel permeation chromatography using purified proteins. It turned out that Aha1 competes with the early cofactors Hop and p50, but can bind to Hsp90 in the presence of cyclophilins, suggesting that Aha1 acts as a late cofactor of Hsp90. In contrast with p50, which can bind to Hop, Aha1 does not interact directly with any of the other four cofactors. In vivo studies in yeast and in mammalian cells revealed that Aha1 is not specific for kinase activation, but also contributes to maturation of hormone receptors, proposing a general role for this cofactor in the activation of Hsp90-dependent client proteins.

The Nature of the Rate-limiting Steps in the Refolding of the Cofactor-dependent Protein Aspartate Aminotransferase

The refolding of mitochondrial aspartate aminotransferase (mAAT; EC 2.6.1.1) has been studied following unfolding in 6 m guanidine hydrochloride for different periods of time. Whereas reactivation of equilibrium-unfolded mAAT is sigmoidal, reactivation of the short term unfolded protein displays a double exponential behavior consistent with the presence of fast and slow refolding species. The amplitude of the fast phase decreases with increasing unfolding times (k approximately 0.75 min(-1) at 20 degrees C) and becomes undetectable at equilibrium unfolding. According to hydrogen exchange and stopped-flow intrinsic fluorescence data, unfolding of mAAT appears to be complete in less than 10 s, but hydrolysis of the Schiff base linking the coenzyme pyridoxal 5'-phosphate (PLP) to the polypeptide is much slower (k approximately 0.08 min(-1)). This implies the existence in short term unfolded samples of unfolded species with PLP still attached. However, since the disappearance of the fast refolding phase is about 10-fold faster than the release of PLP, the fast refolding phase does not correspond to folding of the coenzyme-containing molecules. The fast refolding phase disappears more rapidly in the pyridoxamine and apoenzyme forms of mAAT, both of which lack covalently attached cofactor. Thus, bound PLP increases the kinetic stability of the fast refolding unfolding intermediates. Conversion between fast and slow folding forms also takes place in an early folding intermediate. The presence of cyclophilin has no effect on the reactivation of either equilibrium or short term unfolded mAAT. These results suggest that proline isomerization may not be the only factor determining the slow refolding of this cofactor-dependent protein.

Human immunodeficiency virus type 1 capsid protein is a substrate of the retroviral proteinase while integrase is resistant toward proteolysis

The capsid protein of human immunodeficiency virus type 1 was observed to undergo proteolytic cleavage in vitro when viral lysate was incubated in the presence of dithiothreitol at acidic pH. Purified HIV-1 capsid protein was also found to be a substrate of the viral proteinase in a pH-dependent manner; acidic pH (<7) was necessary for cleavage, and decreasing the pH toward 4 increased the degree of processing. Based on N-terminal sequencing of the cleavage products, the capsid protein was found to be cleaved at two sites, between residues 77 and 78 as well as between residues 189 and 190. Oligopeptides representing these cleavage sites were also cleaved at the expected peptide bonds. The presence of cyclophilin A decreased the degree of capsid protein processing. Unlike the capsid protein, integrase was found to be resistant toward proteolysis in good agreement with its presence in the preintegration complex.

Prolactin in the Nucleus

Prolactin is a growth factor and hormone involved in stimulating milk production. Prolactin binds to a cytokine type I receptor to initiate a signaling cascade that includes the activation of the transcription factor STAT5. Ligand-occupied prolactin receptor is also internalized, and a small fraction of the internalized prolactin can be found in the nucleus. Rycyzyn and Clevenger investigated the interaction between prolactin and cyclophilin b, a peptidyl-prolyl isomerase known for its role in the endoplasmic reticulum as a protein-folding chaperone and also known to potentiate the prolactin to stimulate proliferation. In transfected cells, the ability of cyclophilin B to potentiate prolactin-stimulated proliferation or transcription was blocked if cyclophilin B was rendered catalytically inactive by mutation. STAT5 was coprecipitated from the nucleus in cells expressing catalytically active cyclophilin B after stimulation of cells with epitope-tagged prolactin (the complex was precipitated with an antibody against the tagged prolactin). The presence of cyclophilin B inhibited the prolactin-stimulated interaction between STAT5 and an inhibitor of STAT5 called PIAS3. Thus, cyclophilin B may promote the entry of prolactin into the nucleus and then, through a mechanism that requires the catalytic activity of cyclophin B, promote the dissociation of a prolactin-stimulated STAT5-PIAS3 interaction, enhancing DNA binding and transcriptional activation.M. A. Rycyzyn, C. V. Clevenger, The intranuclear prolactin/cyclophilin B complex as a transcriptional inducer. Proc. Natl. Acad. Sci. U.S.A. 99, 6790-6795 (2002). [Abstract][Full Text]

Prolactin in the Nucleus

Prolactin is a growth factor and hormone involved in stimulating milk production. Prolactin binds to a cytokine type I receptor to initiate a signaling cascade that includes the activation of the transcription factor STAT5. Ligand-occupied prolactin receptor is also internalized, and a small fraction of the internalized prolactin can be found in the nucleus. Rycyzyn and Clevenger investigated the interaction between prolactin and cyclophilin b, a peptidyl-prolyl isomerase known for its role in the endoplasmic reticulum as a protein-folding chaperone and also known to potentiate the prolactin to stimulate proliferation. In transfected cells, the ability of cyclophilin B to potentiate prolactin-stimulated proliferation or transcription was blocked if cyclophilin B was rendered catalytically inactive by mutation. STAT5 was coprecipitated from the nucleus in cells expressing catalytically active cyclophilin B after stimulation of cells with epitope-tagged prolactin (the complex was precipitated with an antibody against the tagged prolactin). The presence of cyclophilin B inhibited the prolactin-stimulated interaction between STAT5 and an inhibitor of STAT5 called PIAS3. Thus, cyclophilin B may promote the entry of prolactin into the nucleus and then, through a mechanism that requires the catalytic activity of cyclophin B, promote the dissociation of a prolactin-stimulated STAT5-PIAS3 interaction, enhancing DNA binding and transcriptional activation. M. A. Rycyzyn, C. V. Clevenger, The intranuclear prolactin/cyclophilin B complex as a transcriptional inducer. Proc. Natl. Acad. Sci. U.S.A. 99 , 6790-6795 (2002). [Abstract] [Full Text]

The two slow refolding processes of creatine kinase are catalyzed by cyclophilin.

A burst phase occurs in the refolding kinetics of guanidine-denatured creatine kinase due to formation of an intermediate within the mixing dead time, with further refolding to the native state after the burst phase along a path following biphasic kinetics. In the presence of cyclophilin, the refolding rates of the two slow processes are accelerated and the values are proportional to the cyclophilin concentration. The activity of cyclophilin in accelerating the slow refolding processes of creatine kinase is totally inhibited by cyclosporin A, indicating that the cis-trans isomerization of the peptidyl-prolyl bonds is involved in the two slow refolding processes.

Crystal structure of the complex of brugia malayi cyclophilin and cyclosporin A.

The resistance of the human parasite Brugia malayi to the antiparasitic activity of cyclosporin A (CsA) may arise from the presence of cyclophilins with relatively low affinity for the drug. The structure of the complex of B. malayi cyclophilin (BmCYP-1) and CsA, with eight independent copies in the asymmetric unit, has been determined at a resolution of 2.7 A. The low affinity of BmCYP-1 for CsA arises from incomplete preorganization of the binding site so that the formation of a hydrogen bond between His132 of BmCYP-1 and N-methylleucine 9 of CsA is associated with a shift in the backbone of approximately 1 A in this region.

Characterization of cytosolic cyclophilin from guard cells of Vicia faba L.

The effect of immunosuppressant cyclosporin A (CsA) on inward-rectifying K(+)-channels and biochemical analysis have indicated the presence of cyclophilin in guard cells of Vicia faba. In this study, we identified a full-length cDNA sequence, vcCyP, encoding cyclophilin (CyP), a peptidyl-prolyl cis-trans isomerase of guard cell protoplasts (GCPs) from Vicia faba L. The deduced amino acid sequence revealed that vcCyP contained 171 amino acid residues and exhibited a strong similarity to previously described cytosolic CyP isoforms from other plants. vcCyP had seven extra amino acid residues, which is a characteristic of the cytosolic form of plant CyPs. A complex of recombinant vcCyP and CsA inhibited the phosphatase activity of bovine calcineurin, a type 2B protein phosphatase, with a half-inhibitory concentration of 0.2 microM. Protein phosphatase activity was measured in the cytosolic fraction of GCPs using a 32P-labeled myelin basic protein (32P-MBP) and the activity was increased by a physiological concentration of Ca2+ (1 microM). This Ca(2+)-stimulated phosphatase activity was inhibited by CsA, suggesting the presence of both cytosolic CyP and calcineurin-like protein phosphatase in guard cells. Northern blot analysis showed that the transcription level of vcCyP was much higher in GCPs than in root and leaf tissues of Vicia.

N-Terminal extension of human immunodeficiency virus capsid protein converts the in vitro assembly phenotype from tubular to spherical particles.

Expression of retroviral Gag polyproteins is sufficient for morphogenesis of virus-like particles with a spherical immature protein shell. Proteolytic cleavage of Gag into the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains (in the case of human immunodeficiency virus [HIV]) leads to condensation to the mature cone-shaped core. We have analyzed the formation of spherical or cylindrical particles on in vitro assembly of purified HIV proteins or inside Escherichia coli cells. CA protein alone yielded cylindrical particles, while all N-terminal extensions of CA abolished cylinder formation. Spherical particles with heterogeneous diameters or amorphous protein aggregates were observed instead. Extending CA by 5 amino acids was sufficient to convert the assembly phenotype to spherical particles. Sequences C-terminal of CA were not required for sphere formation. Proteolytic cleavage of N-terminally extended CA proteins prior to in vitro assembly led to the formation of cylindrical particles, while proteolysis of in vitro assembly products caused disruption of spheres but not formation of cylinders. In vitro assembly of CA and extended CA proteins in the presence of cyclophilin A (CypA) at a CA-to-CypA molar ratio of 10:1 yielded significantly longer cylinders and heterogeneous spheres, while higher concentrations of CypA completely disrupted particle formation. We conclude that the spherical shape of immature HIV particles is determined by the presence of an N-terminal extension on the CA domain and that core condensation during virion maturation requires the liberation of the N terminus of CA.

Parallel pathways in the folding of a short-term denatured scFv fragment of an antibody

Antibodies are prototypes of multimeric proteins and consist of structurally similar domains. The two variable domains of an antibody (VH and VL) interact through a large hydrophobic interface and can be expressed as covalently linked single-chain Fv (scFv) fragments. The in vitro folding of scFv fragments after long-term denaturation in guanidinium chloride is known to be slow. In order to delineate the nature of the rate-limiting step, the folding of the scFv fragment of an antibody after short-term denaturation has been investigated. Secondary structure formation, measured by H/D-exchange protection, of a mutant scFv fragment of an antibody after short incubation in 6 M guanidinium chloride was shown to be multiphasic. NMR analysis shows that an intermediate with significant proton protection is observed within the dead time of the manual mixing experiments. Subsequently, the folding reaction proceeds via a biphasic reaction and mass spectrometry analyses of the exchange experiments confirm the existence of two parallel pathways. In the presence of cyclophilin, however, the faster of the two phases vanishes (when followed by intrinsic tryptophan fluorescence), while the slower phase is not significantly enhanced by equimolar cyclophilin. The formation of an early intermediate, which shows amide-proton exchange protection, is independent of proline isomerization. Subsequently, a proline cis-trans isomerization reaction in the rapidly formed intermediate, producing 'non-native' isomers, competes with the fast formation of native species. Interface formation in a folding intermediate of the scFv fragment is proposed to prevent the back-isomerization of these prolines from being efficiently catalyzed by cyclophilin.

Presence of cyclophilin A in synovial fluids of patients with rheumatoid arthritis.

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.