Cyclophilin A

Human cyclophilin A, or CypA, encoded by the gene peptidyl prolyl isomerase A (PPIA), is incorporated into the HIV type 1 (HIV-1) virion and promotes HIV-1 infectivity by facilitating virus uncoating. We examined the effect of single nucleotide polymorphisms (SNPs) and haplotypes within the PPIA gene on HIV-1 infection and disease progression in five HIV-1 longitudinal history cohorts. Kaplan-Meier survival statistics and Cox proportional hazards model were used to assess time to AIDS outcomes. Among eight SNPs tested, two promoter SNPs (SNP3 and SNP4) in perfect linkage disequilibrium were associated with more rapid CD4 þ T-cell loss (relative hazard ¼ 3.7, p ¼ 0.003) in African Americans. Among European Americans, these alleles were also associated with a significant trend to more rapid progression to AIDS in a multi-point categorical analysis (p ¼ 0.005). Both SNPs showed differential nuclear protein-binding efficiencies in a gel shift assay. In addition, one SNP (SNP5) located in the 59 UTR previously shown to be associated with higher ex vivo HIV-1 replication was found to be more frequent in HIV-1-positive individuals than in those highly exposed uninfected individuals. These results implicate regulatory PPIA polymorphisms as a component of genetic susceptibility to HIV-1 infection or disease progression, affirming the important role of PPIA in HIV-1 pathogenesis.

In humans, apolipoprotein E (apoE) has 3 isoforms: apoE2, apoE3, and apoE4. APOE4 is a major genetic risk factor for Alzheimer disease (AD). Apolipoprotein E4 has direct effects on the cerebrovascular system, resulting in microvascular lesions and blood-brain barrier (BBB) damage, as recently reviewed. 2 Neurovascular dysfunction is also present in cognitively normal APOE4 carriers and individuals with APOE4-associated disorders including AD. Moreover, postmortem brain tissue analysis has indicated that BBB breakdown in patients with AD is more pronounced in APOE4 carriers compared with APOE3 or APOE2. Our recent studies in transgenic mice have demonstrated that apoE4 leads to BBB breakdown by activating the proinflammatory cyclophilin A (CypA)-matrix metalloproteinase 9 (MMP-9) pathway in brain pericytes, which in turn results in degradation of the BBB tight junctions and basement membrane proteins. It has also been shown that apoE4-mediated BBB breakdown leads to secondary neuronal injury and cognitive decline in transgenic mice. Apolipoprotein E2 and apoE3 maintained normal BBB integrity in transgenic mice by suppressing the CypA-MMP-9 pathway. Here, we studied the cerebrospinal fluid (CSF)/plasma albumin quotient (Q Alb ), an established marker of BBB breakdown, 8 and CypA and active MMP-9 levels in the CSF of cognitively normal individuals with different APOE genotypes to determine whether apoE4dependent changes in BBB permeability and CypA-MMP-9 pathway as shown in APOE4, but not APOE3 and APOE2 transgenic mice, also occur in humans. Methods | Participants were volunteers who were recruited through advertisements or from the Memory Education and Research Initiative Program at the Nathan S. Kline Institute for

Human apolipoprotein E has three isoforms: APOE2, APOE3 and APOE4 1 . APOE4 is a major genetic risk factor for Alzheimer's disease 2, 3 and is associated with Down's syndrome dementia and poor neurological outcome after traumatic brain injury and haemorrhage 3 . Neurovascular dysfunction is present in normal APOE4 carriers and individuals withAPOE4-associated disorders . In mice, lack of Apoe leads to blood-brain barrier (BBB) breakdown , whereas APOE4 increases BBB susceptibility to injury 13 . How APOE genotype affects brain microcirculation remains elusive. Using different APOE transgenic mice, including mice with ablation and/or inhibition of cyclophilin A (CypA), here we show that expression of APOE4 and lack of murine Apoe, but not APOE2 and APOE3, leads to BBB breakdown by activating a proinflammatory CypA-nuclear factor-κB-matrix-metalloproteinase-9 pathway in pericytes. This, in turn, leads to neuronal uptake of multiple blood-derived neurotoxic proteins, and microvascular and cerebral blood flow reductions. We show that the vascular defects in Apoe-deficient and APOE4-expressing mice precede neuronal dysfunction and can initiate neurodegenerative changes. Astrocyte-secreted APOE3, but not APOE4, suppressed the CypA-nuclear factor-κBmatrix-metalloproteinase-9 pathway in pericytes through a lipoprotein receptor. Our data suggest

Hind limb ischemia is a useful model to assess metabolic and cellular responses. Here, we present a protocol for evaluating post-natal angiogenesis in a mouse hind limb ischemia model. We describe steps to induce a severe restriction of blood supply of the femoral artery and vein that mimics the real-life scenario observed in clinical settings. We then detail procedures for follow-up laser Doppler imaging to compare post-ischemic responses of four different mouse strains in their capacity to trigger compensatory arteriogenesis.

The viral protein Nef and the cellular factor cyclophilin A are both required for full infectivity of human immunodeficiency virus type 1 (HIV-1) virions. In contrast, HIV-2 and simian immunodeficiency virus (SIV) do not incorporate cyclophilin A into virions or need it for full infectivity. Since Nef and cyclophilin A appear to act in similar ways on postentry events, we determined whether chimeric HIV-1 virions that contained either HIV-2 or SIV Nef would have a direct effect on cyclophilin A dependence. Our results show that chimeric HIV-1 virions containing either HIV-2 or SIV Nef are resistant to treatment by cyclosporine and enhance the infectivity of virions with mutations in the cyclophilin A binding loop of Gag. Amino acids at the C terminus of HIV-2 and SIV are necessary for inducing cyclosporine resistance. However, transferring these amino acids to the C terminus of HIV-1 Nef is insufficient to induce cyclosporine resistance in HIV-1. These results suggest that HIV-2 and...

Gains of 1q21-q23 have been associated with metastasis and chemotherapy response, particularly in bladder cancer, hepatocellular carcinomas and sarcomas. By positional cloning of ampli®ed genes by yeast arti®cial chromosome-mediated cDNA capture using magnetic beads, we have identi®ed three candidate genes (COAS1, -2 and -3) in the ampli®ed region in sarcomas. COAS1 and -2 showed higher ampli®cation levels than COAS3. Most notably, ampli®cation was very common in osteosarcomas, where in particular COAS2 was highly expressed. COAS1 has multiple repeats and shows no homology to previously described genes, whereas COAS2 is a novel member of the cyclosporin-binding peptidylprolyl isomerase family, very similar to cyclophilin A. COAS2 was overexpressed almost exclusively in aggressive metastatic or chemotherapy resistant tumours. Although COAS2 was generally more ampli®ed than COAS1, it was not expressed in well-dierentiated liposarcomas, where ampli®cation of this region is very common. All three genes were found to be ampli®ed and over-expressed also in breast carcinomas. The complex nature of the 1q21-23 amplicons and close proximity of the genes make unequivocal determination of the gene responsible dicult. Quite likely, the dierent genes may give selective advantages to dierent subsets of tumours.

This protocol details the uorescence assay for Enterovirus A71 (EV-A71) 3C protease activity measurement. This method is intended to measure the activity of viral proteases by using a speci c labelled-peptide that allows the detection of the cleaved product. The substrate contains the cleavage-sequence speci c to the tested protease and is labeled in Cterminal by the uorophore Edans (ex 336 nm; em: 455 nm) and in N-ternimal by the quencher Dabcyl (ex 472 nm). In the case of a non-cleaved substrate, the proximity of Dabcyl to Edans prevents the emission and the detection of the uorescence at 455 nm. The cleavage of the peptide by the protease allows Edans' uorescence emission and detection.

Background: Immunoglobulin E (IgE) plays a central role in allergic disease. The B-cell receptor CD23 is pivotal in regulating IgE synthesis. Results: Ca 2ϩ -dependent structural changes in CD23 enable additional interactions with IgE. Conclusion: Mechanism for Ca 2ϩ -induced increase in affinity is revealed. Significance: Ca 2ϩ binding brings an extra degree of modulation to CD23 function. Ig receptors in that it belongs to the C-type (calcium-dependent) lectin-like superfamily. Although the interaction of CD23 with IgE is carbohydrate-independent, calcium has been reported to increase the affinity for IgE, but the structural basis for this activity has previously been unknown. We have determined the crystal structures of the human lectin-like head domain of CD23 in its Ca 2؉ -free and Ca 2؉ -bound forms, as well as the crystal structure of the Ca 2؉ -bound head domain of CD23 in complex with a subfragment of IgE-Fc consisting of the dimer of C⑀3 and C⑀4 domains (Fc⑀3-4). Together with site-directed mutagenesis, the crystal structures of four Ca 2؉ ligand mutants, isothermal titration calorimetry, surface plasmon resonance, and stopped-flow analysis, we demonstrate that Ca 2؉ binds at the principal and evolutionarily conserved binding site in CD23. Ca 2؉ binding drives Pro-250, at the base of an IgE-binding loop (loop 4), from the trans to the cis configuration with a concom-itant conformational change and ordering of residues in the loop. These Ca 2؉ -induced structural changes in CD23 lead to additional interactions with IgE, a more entropically favorable interaction, and a 30-fold increase in affinity of a single head domain of CD23 for IgE. Taken together, these results suggest that binding of Ca 2؉ brings an extra degree of modulation to CD23 function.

Background: Immunoglobulin E (IgE) plays a central role in allergic disease. The B-cell receptor CD23 is pivotal in regulating IgE synthesis. Results: Ca 2ϩ -dependent structural changes in CD23 enable additional interactions with IgE. Conclusion: Mechanism for Ca 2ϩ -induced increase in affinity is revealed. Significance: Ca 2ϩ binding brings an extra degree of modulation to CD23 function. Ig receptors in that it belongs to the C-type (calcium-dependent) lectin-like superfamily. Although the interaction of CD23 with IgE is carbohydrate-independent, calcium has been reported to increase the affinity for IgE, but the structural basis for this activity has previously been unknown. We have determined the crystal structures of the human lectin-like head domain of CD23 in its Ca 2؉ -free and Ca 2؉ -bound forms, as well as the crystal structure of the Ca 2؉ -bound head domain of CD23 in complex with a subfragment of IgE-Fc consisting of the dimer of C⑀3 and C⑀4 domains (Fc⑀3-4). Together with site-directed mutagenesis, the crystal structures of four Ca 2؉ ligand mutants, isothermal titration calorimetry, surface plasmon resonance, and stopped-flow analysis, we demonstrate that Ca 2؉ binds at the principal and evolutionarily conserved binding site in CD23. Ca 2؉ binding drives Pro-250, at the base of an IgE-binding loop (loop 4), from the trans to the cis configuration with a concom-itant conformational change and ordering of residues in the loop. These Ca 2؉ -induced structural changes in CD23 lead to additional interactions with IgE, a more entropically favorable interaction, and a 30-fold increase in affinity of a single head domain of CD23 for IgE. Taken together, these results suggest that binding of Ca 2؉ brings an extra degree of modulation to CD23 function.

Huntington's disease (HD) is a fatal neurodegenerative disease in which an early and selective vulnerability of striatal Spiny Projection Neurons is observed. However, several studies have highlighted the implication of glial cells, and in particular astrocytes, in the pathophysiological mechanisms of this disease. A better understanding of the respective contributions of neurons and astrocytes in HD is needed and would be important for the development of new therapeutic approaches. Today, no comparable in vivo models expressing the mutant HTT selectively in astrocytes or in neurons are available. In this study, we developed comparable cell‐type specific mouse models expressing a fragment of Huntingtin specifically in neurons, astrocytes, or in both cell populations of the adult mouse basal ganglia circuit. This approach allowed us to characterize behavioral alterations occurring as soon as 4 weeks postinjection. Interestingly, less severe but significant behavioral alterations ...

Computational methods have been devised in the past to predict the interface residues using amino acid sequence information but have been majorly applied to predict for prokaryotic protein complexes. Since the composition and rate of evolution of the primary sequence are different between prokaryotes and eukaryotes, it is important to develop a method specifically for eukaryotic complexes. Here we report a new hybrid pipeline for the prediction of protein-protein interaction interfaces from the amino acid sequence information alone based on the framework of Co-evolution, machine learning (Random forest) and Network Analysis named CoRNeA trained specifically on eukaryotic protein complexes. We incorporate the intra contact information of the individual proteins to eliminate false positives from the predictions as the amino acid sequence also holds information for its own folding along with the interface propensities. Our prediction on various case studies shows that CoRNeA can succes...

Human immunodeficiency virus type 1 (HIV-1) specifically incorporates the host cell peptidyl-prolyl isomerase cyclophilin A into virions via contacts with the capsid (CA) domain of the Gag polyprotein Pr55gag. The immunosuppressant drug cyclosporin A and the nonimmunosuppressive cyclosporin A analog SDZ NIM 811 bind to cyclophilin A and inhibit its incorporation into HIV-1 virions. Both drugs inhibit the virion association of cyclophilin A and the replication of HIV-1 with a similar dose dependence. In contrast, these compounds are inactive against other primate lentiviruses which do not incorporate cyclophilin A, such as simian immunodeficiency virus (SIV). To locate determinants which confer sensitivity to SDZ NIM 811, we generated chimeric proviruses between HIV-1 and SIVmac. A hybrid SIVmac which has the CA-p2 domain of the Gag polyprotein replaced by the corresponding domain from HIV-1 replicated in an established CD4+ cell line and in human but not macaque peripheral blood mon...

HIV-1 specifically incorporates the peptidyl prolyl isomerase cyclophilin A (CyPA), the cytosolic receptor for the immunosuppressant cyclosporin A (CsA). HIV-1 replication is inhibited by CsA as well as by nonimmunosuppressive CsA analogues that bind to CyPA and interfere with its virion association. In contrast, the related simian immunodeficiency virus SIV mac , which does not interact with CyPA, is resistant to these compounds. The incorporation of CyPA into HIV-1 virions is mediated by a specific interaction between the active site of the enzyme and the capsid (CA) domain of the HIV-1 Gag polyprotein. We report here that the transfer of HIV-1 CA residues 86–93, which form part of an exposed loop, to the corresponding position in SIV mac resulted in the efficient incorporation of CyPA and conferred an HIV-1-like sensitivity to a nonimmunosuppressive cyclosporin. HIV-1 CA residues 86–90 were also sufficient to transfer the ability to efficiently incorporate CyPA, provided that the...

Background: Traditional cardiovascular risk factors and a large number of biomarkers are well known in their association with the diagnosis and prognosis of coronary artery disease (CAD). Cyclophilin C (CypC) is a subfamily of immunophilins that modulates macrophage activation and redox homeostasis. Purpose: This study is aimed at investigating the changes in serum CypC levels and their relationship with cardiovascular events at 12 months of follow-up in CAD patients. Methods: The study included a total of 125 subjects (40 patients with acute CAD, 40 patients with chronic CAD and 45 control volunteers). We analyzed plasma CypC levels from baseline to 6 and 12 months for a better understanding of its behaviour in atherosclerosis. Results: Serum CypC levels were shown to be gradually increased in CAD patients [(30.

Mass spectrometry-based tissue profiling and imaging are technologies that allow identification and visualization of protein signals directly on thin sections cut from fresh frozen tissue specimens. These technologies were utilized to evaluate protein expression profiles in the normal mouse prostate during development (1-5 weeks of age), at sexual maturation (6 weeks of age), and in adult prostate (at 10, 15, or 40 weeks of age). The evolution of protein expression during normal prostate development and maturation were subsequently compared with 15week prostate tumors derived from genetically engineered mice carrying the Large T antigen gene under regulation of the prostate-specific probasin promoter (LPB-Tag mouse model for prostate cancer). This approach identified proteins differentially expressed at specific time points during prostate development. Furthermore expression of some of these proteins, for example probasin and spermine-binding protein, were associated with prostate maturation, and prostate tumor formation resulted in their loss of expression. Cyclophilin A, a protein found in other cancers, was differentially ␣-acetylated on the N terminus, and both isoforms appeared during normal prostate and prostate tumor development. Imaging mass spectrometry localized the protein signals to specific prostatic lobes or regions. Thus, tissue profiling and imaging can be utilized to analyze the ontogeny of protein expression during prostate morphogenesis and tumorigenesis and identify proteins that could potentially serve as biomarkers for prostate cancer. Molecular & Cellular Proteomics 7:411-423, 2008.

Hensin is a rabbit ortholog of DMBT1, a multifunctional, multidomain protein implicated in the regulation of epithelial differentiation, innate immunity, and tumorigenesis. Hensin in the extracellular matrix (ECM) induced morphological changes characteristic of terminal differentiation in a clonal cell line (clone C) of rabbit kidney intercalated cells. Although hensin is secreted in monomeric and various oligomeric forms, only the polymerized ECM form is able to induce these phenotypic changes. Here we report that hensin secretion and matrix assembly were inhibited by the peptidylprolyl cis-trans isomerase (PPIase) inhibitors cyclosporin A (CsA) and a derivative of cyclosporin A with modifications in the D-Ser side chain (Cs9) but not by the calcineurin pathway inhibitor FK506. PPIase inhibition led to failure of hensin polymerization in the medium and ECM, plus the loss of apical cytoskeleton, apical microvilli, and the columnar epithelial shape of clone C cells. Cyclophilin A was produced and secreted into the media to a much greater extent than cyclophilins B and C. Our results also identified the direct CsAsensitive interaction of cyclophilin A with hensin, suggesting that cyclophilin A is the PPIase that mediates the polymerization and matrix assembly of hensin. These results are significant because this is the first time a direct role of peptidylprolyl cis-trans isomerase activity has been implicated in the process of epithelial differentiation.

Background and Aims: It is expected that a cure for HBV will require drug combinations that interact at more than one stage of viral replication and propagation. Our lead drug, CMX157, a tenofovir (TFV) prodrug, is a novel lipid acyclic nucleoside (NUC) phosphonate designed to deliver high intrahepatic concentrations of TFV, while minimizing off-target effects caused by high levels of circulating TFV. CRV431, our earlier-stage molecule, is a host targeting antiviral that inhibits cyclophilins, namely cyclophilin A (cypA), a peptidyl prolyl isomerase. As a cypA inhibitor, CRV431 reduces HBV DNA, suppresses HBsAg, inhibits viral uptake via NTCP and, more recently, has been shown to impede HBx-cypA binding. The aim of the current study was to investigate the combination of CMX157 and CRV431, measuring HBV DNA. Methods: The current study measured inhibition of intracellular HBV DNA at concentrations of CRV431 ranging from 0 to 320 nM alone, and in combination with CMX157 ranging from 0 to 640 nM. Both drugs were tested in vitro in AD38, DE19, and DES19 cells. Studies were each conducted twice, in triplicate, using DMSO as control. Drug concentration versus effect was evaluated using Prichard-Shipman MacSynergy. Additionally, CRV431 cytotoxicity was examined in a number of primary human cells and cell lines, utilizing DMSO and 0.5% saponin as negative and positive controls, respectively, comparing cyclosporine, alisporivir, and sanglifehrin A, to confirm cell viability in the assays. Results: Intracellular HBV DNA inhibition with CRV431 and CMX157, tested in combination with all three cell types, exhibited IC50 values in the low nanomolar range. Synergy scores for CRV431 with CMX157 indicated moderate to strong synergy. Further, cells were viable across all CRV431 concentrations tested, exceeding the viability of alisporivir, cyclosporine, and far exceeding sanglifehrin A. CRV431 exhibited CC50 values of approximately 24 µM. The selective index (SI) was widest for CRV431 compared with that of alisporivir, cyclosporine, and sanglifehrin A. Conclusions: CRV431 and CMX157, tested in combination, represents a viable therapeutic drug strategy for the cure of HBV. This strategy exploits the complementary modes of action of the two drugs, which allows for suppression of HBV DNA, HBsAg, HBeAg, inhibition of viral entry, and blocking of cypclophilin A binding to HBx. The complementary actions of CRV431 with CMX157 may reasonably extend to drugs with other modes of activity including, for example, core inhibitors.

HCV-related liver disease is the main cause of morbidity and mortality of HCV/HIV-1 coinfected patients. Despite the recent advent of anti-HCV direct acting antivirals (DAAs), the treatment of HCV/HIV-1 co-infected patients remains a challenge, as these patients are refractory to most therapies and develop liver fibrosis, cirrhosis and liver cancer more often than HCV mono-infected patients. Until the present study, there was no suitable in vitro assay to test the inhibitory activity of drugs on HCV/HIV-1 co-infection. Here we developed a novel in vitro "co-infection" model where HCV and HIV-1 concurrently replicate in their respective main host target cells-human hepatocytes and CD4+ T-lymphocytes. Using this co-culture model, we demonstrate that cyclophilin inhibitors (CypI), including a novel cyclosporin A (CsA) analog, CPI-431-32, simultaneously inhibits replication of both HCV and HIV-1 when added pre-and post-infection. In contrast, the HIV-1 protease inhibitor nelfinavir or the HCV NS5A inhibitor daclatasvir only blocks the replication of a single virus in the "co-infection" system. CPI-431-32 efficiently inhibits HCV and HIV-1 variants, which are normally resistant to DAAs. CPI-431-32 is slightly, but consistently more efficacious than the most advanced clinically tested CypI-alisporivir (ALV)-at interrupting an established HCV/HIV-1 co-infection. The superior antiviral efficacy of CPI-431-32 over ALV correlates with its higher potency inhibition of cyclophilin A (CypA) isomerase activity and at preventing HCV NS5A-CypA and HIV-1 capsid-CypA interactions known to be vital for replication of the respective viruses. Moreover, we obtained evidence that CPI-431-32 prevents the cloaking of both the HIV-1 and HCV genomes from cellular sensors. Based on these results, CPI-431-32 has the potential, as a single agent or in combination with DAAs, to inhibit both HCV and HIV-1 infections. a11111

Aplidin (plitidepsin) is an antitumoral agent that induces apoptosis via Rac1-JNK activation. A proteomic approach using 2D-DIGE technology found 52 cytosolic and 39 membrane proteins differentially expressed in wild-type and Aplidin-resistant HeLa cells, of which 39 and 27 were identified by MALDI-TOF mass spectrometry and database interrogation. A number of proteins involved in apoptosis pathways were found to be deregulated. Alterations in Rab geranylgeranyltransferase, protein disulfide isomerase (PDI), cystathionine γ-lyase, ezrin, and cyclophilin A (CypA) were confirmed by immunoblotting. Moreover, the role of PDI and CypA in Aplidin resistance was functionally confirmed by using the inhibitor bacitracin and overexpression, respectively. These deregulated proteins are candidates to mediate, at least partially, Aplidin action and might provide a route to the cells to escape the induction of apoptosis by this drug.

Naturally occurring histocompatibility responses, following tissue-to-tissue allogeneic contacts, are common among numerous colonial marine invertebrate taxa, including sponges, cnidarians, bryozoans and ascidians. These responses, often culminating in either tissue fusions or rejections, activate a wide array of innate immune components. By comparing two allorejection EST libraries, developed from alloincompatible challenged colonies of the stony coral Stylophora pistillata and the ascidian Botryllus schlosseri, we revealed a common basis for innate immunity in these two evolutionary distant species. Two prominent genes within this common basis were the immunophilins, Cyclophilin A (CypA) and FK506-binding protein (FKBP). In situ hybridizations revealed that mRNA expression of the coral and ascidian immunophilins was restricted to specific allorecognition effector cell populations (nematoblasts and nematocytes in the coral and morula cells in the ascidian). The expressions were limited to only some of the effector cells within a population, disclosing disparities in numbers and location between naïve colonies and their immune challenged counterparts. Administration of the immunosuppression drug Cyclosporine-A during ascidian's allogeneic assays inhibited both fusion and rejection reactions, probably through the inhibition of ascidian's immunocytes (morula cells) movement and activation. Our results, together with previous published data, depict an immunophilins-based immune mechanism, which is similarly activated in allogeneic responses of distantly related animals from sponges to humans.

Background: Marfan syndrome (MFS) is a genetic disease, characterized by thoracic aortic aneurysm (TAA), which treatment is to date purely surgical. Understanding of novel molecular targets is mandatory to unveil effective pharmacological approaches. Cyclophilin A (CyPA) and its receptor EMMPRIN are associated with several cardiovascular diseases, including abdominal aortic aneurysm. Here, we envisioned the contribution of CyPA/EMMPRIN axis in MFS-related TAA. Methods: We obtained thoracic aortic samples from healthy controls (HC) and MFS patients’ aortas and then isolated vascular smooth muscle cells (VSMC) from the aortic wall. Results: our findings revealed that MFS aortic tissue samples isolated from the dilated zone of aorta showed higher expression levels of EMMPRIN vs. MFS non-dilated aorta and HC. Interestingly, angiotensin II significantly stimulated CyPA secretion in MFS-derived VSMC (MFS-VSMC). CyPA treatment on MFS-VSMC led to increased levels of EMMPRIN and other MFS-as...

Arrhythmogenic cardiomyopathy (ACM) is a genetic disorder characterized by the progressive substitution of functional myocardium with noncontractile fibro-fatty tissue contributing to ventricular arrhythmias and sudden cardiac death. Cyclophilin A (CyPA) is a ubiquitous protein involved in several pathological mechanisms, which also characterize ACM (i.e., fibrosis, inflammation, and adipogenesis). Nevertheless, the involvement of CyPA in ACM cardiac remodeling has not been investigated yet. Thus, we first evaluated CyPA expression levels in the right ventricle (RV) tissue specimens obtained from ACM patients and healthy controls (HC) by immunohistochemistry. Then, we took advantage of ACM- and HC-derived cardiac mesenchymal stromal cells (C-MSC) to assess CyPA modulation during adipogenic differentiation. Interestingly, CyPA was more expressed in the RV sections obtained from ACM vs. HC subjects and positively correlated with the adipose replacement extent. Moreover, CyPA was upreg...

Prolyl-peptidyl isomerases (PPIases) are enzymes that are found in all living organisms. They form an essential part of the cellular protein folding homeostasis machinery. PPIases are associated with many important human diseases, e.g. cardiovascular disease, cancer and Alzheimer's. The development of novel PPIase inhibitors has been limited by the lack of a rapid, laboratory-based assay for these enzymes, as their substrates and products are challenging to distinguish. A well

In studies of gene expression in acute ischemic heart tissue, internal reference genes need to show stable expression per-unit-living tissue to hinder dead cells from biasing real-time RT-PCR data. Until now, this important issue has not been appropriately investigated. We hypothesized that the expression of seven internal reference genes would show stable per-unit-living tissue expression in Langendorff-perfused rat hearts subjected to ischemia-reperfusion. This was found for cyclophilin A, GAPDH, RPL-32, and PolR2A mRNA, with GAPDH showing the highest degree of stability ( R = 0.11), suggesting unchanged rates of mRNA transcription in live cells and complete degradation of mRNA from dead cells. The infarct size-dependent degradation of GAPDH was further supported by a close correlation between changes in GAPDH mRNA and changes in RNA quality measured as RNA integrity number (R = 0.90, P < 0.05). In contrast, β-actin and 18S rRNA showed stable expression per-unit-weight tissue a...

The title molecule N-(2,4-Dihydroxy-3,3-dimethylbutanoyl)-β-alanine or Vitamin B5 and its 23 derivatives were selected for theoretical investigations viz geometry optimization, ADME profiling, binding affinity using quantum-mechanical calculations and modeling simulation tools. Geometry optimization by Gaussian 09 program revealed the stability and electrophilic nature of the investigated molecules. In order to depict the charge density distributions, the contour maps of HOMO-LUMO and the associated chemical descriptors such as chemical potential (µ), electronegativity (χ), electrophilicity (ω), hardness (η) and softness (σ) were explored. The docked molecules showed strong propensity for binding to 2HQ6 cancer protein active sites. Vit B5-CH=CF2 and Vit B5-CCl3 showed low binding energies of -5.861 and -5.478 kcal/mol respectively with low inhibition constant value (1.43 M). The molecular docking and the druglikeness assessments were concomitant with the antiviral, antibacterial, and anticancer activities of the investigated molecules. Studies on the natural bond orbital (NBO), Mulliken population, and Fukui functions were analyzed. Further, the interactions between the derivatives and other molecules were studied using Hirshfeld surface analysis.

Cyclophilin B (CyPB), encoded by PPIB, is an ER-resident peptidyl-prolyl cis-trans isomerase (PPIase) that functions independently and as a component of the collagen prolyl 3-hydroxylation complex. CyPB is proposed to be the major PPIase catalyzing the rate-limiting step in collagen folding. Mutations in PPIB cause recessively inherited osteogenesis imperfecta type IX, a moderately severe to lethal bone dysplasia. To investigate the role of CyPB in collagen folding and post-translational modifications, we generated Ppib 2/2 mice that recapitulate the OI phenotype. Knock-out (KO) mice are small, with reduced femoral areal bone mineral density (aBMD), bone volume per total volume (BV/TV) and mechanical properties, as well as increased femoral brittleness. Ppib transcripts are absent in skin, fibroblasts, femora and calvarial osteoblasts, and CyPB is absent from KO osteoblasts and fibroblasts on western blots. Only residual (2-11%) collagen prolyl 3-hydroxylation is detectable in KO cells and tissues. Collagen folds more slowly in the absence of CyPB, supporting its ratelimiting role in folding. However, treatment of KO cells with cyclosporine A causes further delay in folding, indicating the potential existence of another collagen PPIase. We confirmed and extended the reported role of CyPB in supporting collagen lysyl hydroxylase (LH1) activity. Ppib 2/2 fibroblast and osteoblast collagen has normal total lysyl hydroxylation, while increased collagen diglycosylation is observed. Liquid chromatography/mass spectrometry (LC/MS) analysis of bone and osteoblast type I collagen revealed site-specific alterations of helical lysine hydroxylation, in particular, significantly reduced hydroxylation of helical crosslinking residue K87. Consequently, underhydroxylated forms of di-and trivalent crosslinks are strikingly increased in KO bone, leading to increased total crosslinks and decreased helical hydroxylysine-to lysine-derived crosslink ratios. The altered crosslink pattern was associated with decreased collagen deposition into matrix in culture, altered fibril structure in tissue, and reduced bone strength. These studies demonstrate novel consequences of the indirect regulatory effect of CyPB on collagen hydroxylation, impacting collagen glycosylation, crosslinking and fibrillogenesis, which contribute to maintaining bone mechanical properties.

The Crk adaptor protein, a critical modifier of multiple signaling pathways, is overexpressed in many cancers where it contributes to tumor progression and metastasis. Recently, we have shown that Crk interacts with the peptidyl prolyl cis-trans isomerase, Cyclophilin A (CypA; PP1A) via a G 219 P 220 Y 221 (GPY) motif in the carboxyl-terminal linker region of Crk, thereby delaying pY221 phosphorylation and preventing downregulation of Crk signaling. Here, we investigate the physiologic significance of the CypA/Crk interaction and query whether CypA inhibition affects Crk signaling in vitro and in vivo. We show that CypA, when induced under conditions of hypoxia, regulates Crk pY221 phosphorylation and signaling in cancer cell lines. Using nuclear magnetic resonance spectroscopy, we show that CypA binds to the Crk GPY motif via the catalytic PPII domain of CypA, and small-molecule nonimmunosuppressive inhibitors of CypA (Debio-025) disrupt the CypA-CrkII interaction and restores phosphorylation of Crk Y221. In cultured cell lines, Debio-025 suppresses cell migration, and when administered in vivo in an orthotopic model of triplenegative breast cancer, Debio-025 showed antitumor efficacy either alone or in combination with anti-PD-1 mAb, reducing both tumor volume and metastatic lung dispersion. Furthermore, when analyzed by NanoString immune profiling, treatment of Debio-025 with anti-PD-1 mAb increased both T-cell signaling and innate immune signaling in tumor microenvironment. Implications: These data suggest that pharmacologic inhibition of CypA may provide a promising and unanticipated consequence in cancer biology, in part by targeting the CypA/CrkII axis that regulates cell migration, tumor metastasis, and host antitumor immune evasion.

The Crk adaptor protein, a critical modifier of multiple signaling pathways, is overexpressed in many cancers where it contributes to tumor progression and metastasis. Recently, we have shown that Crk interacts with the peptidyl prolyl cis-trans isomerase, Cyclophilin A (CypA; PP1A) via a G219P220Y221 (GPY) motif in the carboxyl-terminal linker region of Crk, thereby delaying pY221 phosphorylation and preventing downregulation of Crk signaling. Here, we investigate the physiologic significance of the CypA/Crk interaction and query whether CypA inhibition affects Crk signaling in vitro and in vivo. We show that CypA, when induced under conditions of hypoxia, regulates Crk pY221 phosphorylation and signaling in cancer cell lines. Using nuclear magnetic resonance spectroscopy, we show that CypA binds to the Crk GPY motif via the catalytic PPII domain of CypA, and small-molecule nonimmunosuppressive inhibitors of CypA (Debio-025) disrupt the CypA–CrkII interaction and restores phosphory...

Cyclophilin A (CypA) is overexpressed in a number of human cancer types, but the mechanisms by which the protein promotes oncogenic properties of cells are not understood. Here we demonstrate that CypA binds the CrkII adaptor protein and prevents it from switching to the inhibited state. CrkII influences cell motility and invasion by mediating signaling through its SH2 and SH3 domains. CrkII Tyr221 phosphorylation by the Abl or EGFR kinases induces an inhibited state of CrkII by means of an intramolecular SH2-pTyr221 interaction, causing signaling interruption. We show that the CrkII phosphorylation site constitutes a binding site for CypA. Recruitment of CypA sterically restricts the accessibility of Tyr221 to kinases, thereby suppressing CrkII phosphorylation and promoting the active state. Structural, biophysical and in vivo data show that CypA augments CrkII-mediated signaling. A strong stimulation of cell migration is observed in cancer cells wherein both CypA and CrkII are gre...

Proline switches, controlled by cis-trans isomerization, have emerged as a particularly effective regulatory mechanism in a wide range of biological processes. Here we report the structures of both the cis and trans conformers of a proline switch in Crk signaling protein. Proline isomerization toggles Crk between two conformations: an autoinhibitory, stabilized by the intramolecular association of two tandem SH3 domains in the cis form, and an uninhibited, activated conformation promoted by the trans form. In addition to acting as a structural switch the heterogeneous proline recruits cyclophilin A, which accelerates the interconversion rate between the isomers thereby regulating the kinetics of Crk activation. The data provide atomic insight into the mechanisms that underpin the functionality of this binary switch and elucidate its remarkable efficiency. The results also reveal novel SH3 binding surfaces highlighting the binding versatility and expanding the non-canonical ligand repertoire of this important signaling domain.

Lentiviruses are widespread in a variety of vertebrates, often associated with chronic disease states. However, until the recent discovery of the prehistoric endogenous lentiviruses in rabbits (RELIK) and lemurs (PSIV), it was thought that lentiviruses had no capacity for germline integration and were only spread horizontally in an exogenous fashion. The existence of RELIK and PSIV refuted these ideas, revealing lentiviruses to be present in a range of mammals, capable of germline integration, and far more ancient than previously thought. Using Gag sequences reconstructed from the remnants of these prehistoric lentiviruses, we have produced chimeric lentiviruses capable of infecting nondividing cells and determined structures of capsid domains from PSIV and RELIK. We show that the structures from these diverse viruses are highly similar, containing features found in modern-day lentiviruses, including a functional cyclophilin-binding loop. Together, these data provide evidence for an ancient capsidcyclophilin interaction preserved throughout lentiviral evolution.

The highly conserved family of cyclophilins comprises multifunctional chaperones that interact with proteins and RNAs, facilitating the dynamic assembly of multimolecular complexes involved in various cellular processes. Cyclophilin A (CypA), the predominant member of this family, exhibits peptidyl-prolyl cis-trans isomerase activity. This enzymatic function aids with the folding and activation of protein structures and often serves as a molecular regulatory switch for large multimolecular complexes, ensuring appropriate inter-and intra-molecular interactions. Here, we investigated the involvement of CypA in the nucleus, where it plays a crucial role in supporting the assembly and trafficking of heterogeneous ribonucleoproteins (RNPs). We reveal that CypA is enriched in the nucleolus, where it colocalizes with the pseudouridine synthase dyskerin, the catalytic component of the multifunctional H/ACA RNPs involved in the modification of cellular RNAs and telomere stability. We show that dyskerin, whose mutations cause the X-linked dyskeratosis (X-DC) and the Hoyeraal-Hreidarsson congenital ribosomopathies, can directly interact with CypA. These findings, together with the remark that substitution of four dyskerin prolines are known to cause X-DC pathogenic mutations, lead us to indicate this protein as a CypA client. The data presented here suggest that this chaperone can modulate dyskerin activity influencing all its partecipated RNPs.

Objective: This study is aimed at investigating the changes in serum CypC levels and their relationship with cardiovascular events at 12 months of follow-up in coronary artery disease (CAD) patients. Methods: The study included a total of 125 subjects (40 patients with acute CAD, 40 patients with chronic CAD, and 45 control volunteers) and we analyzed plasma CypC levels from baseline to 6 and 12 months for a better understanding of its behavior in atherosclerosis. Results: Serum CypC levels were shown to be gradually increased in CAD patients (30.63 pg/mL ± 3.77 at baseline, 38.70 pg/mL ± 6.41 at 6 months [p = 0.25], and 47.27 pg/mL ± 5.65 at 12 months [p = 0.007]). In addition, serum CypC levels during the follow-up were a significant predictor of CAD (c-statistic 0.76 at 6 months and 0.89 at 12 months; p < 0.001). Despite it, there was no significant association between CypC and cardiovascular events, but serum CypC levels tended to be higher in patients suffering cardiovascular events during the follow-up (29.02 pg/mL ± 6.39 vs. 79.96 pg/mL ± 22.18; p = 0.029). In this regard, plasma levels of high-sensitivity C-reactive protein (hsCRP) > 2.3 mg/L plus NT-proBNP > 300 pg/mL together were significant predictors of cardiovascular events during the follow-up in CAD patients with CypC levels >17.5 pg/mL (p = 0.048). Conclusions: Taken together, our results suggest that serum CypC levels increase during the follow-up in CAD patients and could be a novel biomarker with a possible prognostic value in combination with hsCRP and NT-proBNP.

Background: Diabetes is a group of metabolic diseases marked by hyperglycemia caused by insulin secretion, action, or both. Diabetes chronic hyperglycemia is associated with long-term damage, dysfunction, & failure of numerous organs, most notably eyes, kidneys, nerves, heart, & blood vessels. Aim and objectives: This research aimed to evaluate the association between Urinary Cyclophilin A & diabetic nephropathy in children& adolescents with Type 1 Diabetes Mellitus (T1DM). Subjects and Methods: Sixty children& adolescents were recruited consecutively from those attending diabetes outstudied case clinic in Suez Canal University Children's Hospital. Thirty years old & gender-matched children & adolescents were recruited as the control group. Results: Concerning urinary CyPA, we found that those with microalbuminuria had the highest urinary CyPA levels when compared to others. This was statistically significant (p<0.001). On performing post hoc analysis, we found that variation could be explained by the difference among group B included normoalbuminuric diabetic children and adolescents and group C included healthy children and adolescents (p<0.001), among group A included microalbuminuric diabetic children and adolescents &group C (p<0.001). Conclusion: Urinary CyPA can be used as an early indicator for Diabetic nephropathy because important great levels of urinary CyPA were detected in T1DM studied cases with DN before the presence of albuminuria.

Objective: New device therapies have expanded the strategies for treating heart failure (HF) patients. Unloading of the heart with a left ventricular assist device (LVAD) can lead to the reversal of many remodeling changes whose underlying mechanisms are not yet completely known. Molecular analysis might play a role in obtaining further insight into the regulatory mechanisms of this process. A critical step in an RT-PCR study is the selection of reference genes for data normalization. This study aimed to determine an optimal combination of stably expressed reference genes in different regions of the human heart in order to study the effects of LVAD implants on cardiac remodeling, and in particular to check their reliability on the evaluation of pro-inflammatory cytokine expression. Design and methods: We validated nine of the most commonly used reference genes in human myocardium samples obtained at heart transplantation from patients with LVAD implant (n = 30 from a total of six patients) and from heart transplant (HT from a total of seven patients) recipients as controls (n = 35). Samples from both left (LV) and right (RV) ventricles were analyzed. The normalization strategy was tested by analyzing mRNA expression of IL-6, IL-8 and TNF-a, whose protein levels were measured by immunometric assay. Results: The most stable gene combinations changed according to the experimental groups (the LVAD and HT groups and the different myocardial regions). Considering all the cardiac samples as a whole, the three most stably expressed genes were PPIA, RPL13A, and YWHAZ (M = 0.70). Using the best normalization strategy, a significant increase in IL-6, IL-8 mRNA expression was observed in LVAD samples compared to HT (p < 0.0001). Similar results were obtained by protein analysis. Conclusions: Our results underline the importance of always selecting reference genes for the specific system studied. The most appropriate normalization strategy is of pivotal importance for understanding the molecular mechanisms associated with the pathophysiology of HF, such as inflammation.

Cyclophilin A (CypA) binds to the HIV-1 capsid to facilitate reverse transcription and nuclear entry and counter the antiviral activity of TRIM5α. Interestingly, recent
studies suggest that the capsid enters the nucleus of an infected cell and uncoats prior
to integration. We have previously reported that the capsid protein regulates HIV-1
integration. Therefore, we probed whether CypA-capsid interaction also regulates this
post-nuclear entry step. First, we challenged CypA-expressing (CypA+/+) and CypA-depleted (CypA-/-) cells with HIV-1 and quantified the levels of provirus. CypA-depletion
significantly reduced integration, an effect that was independent of CypA’s effect on
reverse transcription, nuclear entry, and the presence or absence of TRIM5α. In addition, cyclosporin A, an inhibitor that disrupts CypA-capsid binding, inhibited proviral
integration in CypA+/+ cells but not in CypA-/- cells. HIV-1 capsid mutants (G89V and P90A)
deficient in CypA binding were also blocked at the integration step in CypA+/+ cells but
not in CypA-/- cells. Then, to understand the mechanism, we assessed the integration
activity of the HIV-1 preintegration complexes (PICs) extracted from acutely infected
cells. PICs from CypA-/- cells retained lower integration activity in vitro compared to those
from CypA+/+ cells. PICs from cells depleted of both CypA and TRIM5α also had lower
activity, suggesting that CypA’s effect on PIC was independent of TRIM5α. Finally, CypA
protein specifically stimulated PIC activity, as this effect was significantly blocked by CsA.
Collectively, these results provide strong evidence that CypA directly promotes HIV-1
integration, a previously unknown role of this host factor in the nucleus of an infected
cell.

SUMMARY By immunological screening of a cDNA library derived from protoscoleces of Echinococcus granulosus with IgE from patients with cystic echinococcosis (CE) and allergic manifestations, we isolated a protein identical to E. granulosus cyclophilin. The protein, named EA21, has close homology with Malassezia furfur cyclophilin allergen (Mal f 6) and with human cyclophilin. Using immunoblotting (IB) with a polyclonal antibody specific to EA21, we identified E. granulosus cyclophilin both in protoscoleces and in sheep hydatid fluid. Of the 58 sera from patients with CE, 29 (50%) were IgE positive to EA21, whereas, despite the high sequence homology, none were IgE positive to Mal f 6 or human cyclophilin. Only 26 of the 58 patients (45%) had IgG specific to EA21, whereas all patients (100%) had IgG specific to Mal f 6 and human cyclophilin. IB analysis showed that serum IgE-binding reactivity to EA21 differed significantly in patients with and without allergic reactions (20 of 25, 8...

Cyclophilin A is a conserved peptidyl-prolyl cis-trans isomerase (PPIase) best known as the cellular receptor of the immunosuppressant cyclosporine A. Despite significant effort, evidence of developmental functions of cyclophilin A in non-plant systems has remained obscure. Mutations in a tomato (Solanum lycopersicum) cyclophilin A ortholog, DIAGEOTROPICA (DGT), have been shown to abolish the organogenesis of lateral roots; however, a mechanistic explanation of the phenotype is lacking. Here, we show that the dgt mutant lacks auxin maxima relevant to priming and specification of lateral root founder cells. DGT is expressed in shoot and root, and localizes to both the nucleus and cytoplasm during lateral root organogenesis. Mutation of ENTIRE/IAA9, a member of the auxin-responsive Aux/IAA protein family of transcriptional repressors, partially restores the inability of dgt to initiate lateral root primordia but not the primordia outgrowth. By comparison, grafting of a wild-type scion...

SUMMARY By immunological screening of a cDNA library derived from protoscoleces of Echinococcus granulosus with IgE from patients with cystic echinococcosis (CE) and allergic manifestations, we isolated a protein identical to E. granulosus cyclophilin. The protein, named EA21, has close homology with Malassezia furfur cyclophilin allergen (Mal f 6) and with human cyclophilin. Using immunoblotting (IB) with a polyclonal antibody specific to EA21, we identified E. granulosus cyclophilin both in protoscoleces and in sheep hydatid fluid. Of the 58 sera from patients with CE, 29 (50%) were IgE positive to EA21, whereas, despite the high sequence homology, none were IgE positive to Mal f 6 or human cyclophilin. Only 26 of the 58 patients (45%) had IgG specific to EA21, whereas all patients (100%) had IgG specific to Mal f 6 and human cyclophilin. IB analysis showed that serum IgE-binding reactivity to EA21 differed significantly in patients with and without allergic reactions (20 of 25, 8...

Cyclophilins are prolyl isomerases with multitude of functions in different cellular processes and pathological conditions. Cyclophilin A (PpiA) of Mycobacterium tuberculosis is secreted during infection in intraphagosomal niche. However, our understanding about the evolutionary origin, secretory mechanism or the interactome of M. tuberculosis PpiA is limited. This study demonstrates through phylogenetic and structural analyses that PpiA has more proximity to human cyclophilins than the prokaryotic counterparts. We report a unique N-terminal sequence (MADCDSVTNSP) present in pathogenic mycobacterial PpiA and absent in non-pathogenic strains. This sequence stretch was shown to be essential for PpiA secretion. The overexpression of full-length PpiA from M. tuberculosis in non-pathogenic Mycobacterium smegmatis resulted in PpiA secretion while truncation of the N-terminal stretch obstructed the secretion. In addition, presence of an ESX pathway substrate motif in M. tuberculosis PpiA suggested possible involvement of Type VII secretion system. Site-directed mutagenesis of key residues in this motif in full-length PpiA also hindered the secretion in M. smegmatis. Bacterial twohybrid screens with human lung cDNA library as target were utilized to identify interaction partners of PpiA from host repertoire, and a number of substrates with functional representation in iron storage, signal transduction and immune responses were detected. The extensive host interactome coupled with the sequence and structural similarity to human cyclophilins is strongly suggestive of PpiA being deployed by M. tuberculosis as an effector mimic against the host cyclophilins.

Background: Hyperelastosis cutis is an inherited autosomal recessive connective tissue disorder. Affected horses are characterized by hyperextensible skin, scarring, and severe lesions along the back. The disorder is caused by a mutation in cyclophilin B. Results: The crystal structures of both wild-type and mutated (Gly6->Arg) horse cyclophilin B are presented. The mutation neither affects the overall fold of the enzyme nor impairs the catalytic site structure. Instead, it locally rearranges the flexible N-terminal end of the polypeptide chain and also makes it more rigid. Conclusions: Interactions of the mutated cyclophilin B with a set of endoplasmic reticulum-resident proteins must be affected.

The Na þ-Ca 2þ exchanger (NCX) is a major Ca 2þ regulating protein. It is almost ubiquitously expressed. Cyclophilins (Cyps) make up a class of proteins that are involved in protein folding via their peptidyl prolyl cis-trans isomerase (PPIase) and chaperone domains. They are also the cellular receptors of cyclosporin A (CsA). Binding of CsA to cyclophilins inhibits both PPIase and chaperone activities. We have shown that treatment of transfected HEK 293 cells expressing the Na þ-Ca 2þ exchanger NCX1 with CsA results in downregulation of surface expression and transport activity, without any reduction in the total level of cell NCX1 protein [Kimchi-Sarfaty, C., et al. (2002) J. Biol. Chem. 277 (4), 2505-2510]. In this work, we show that knockdown of cell CypA using targeting siRNA (without any CsA treatment) results in a reduction in the level of NCX1 surface expression, a decrease in the level of Na þ-dependent Ca 2þ uptake, and no change in the total amount of cell NCX1 protein in NCX1.5-transfected HEK 293 cells and nontransfected H9c2 cells that express NCX1.1 naturally. It also reduced Na þ-dependent Ca 2þ fluxes measured by changes in Fluo-4 AM fluorescence in single NCX1.5-transfected HEK 293 and single H9c2 cells. Knockdown of CypB had no significant effect on either transport activity, surface expression, NCX1 cell protein expression, or Ca 2þ fluxes. Overexpression of CypA or its R55A mutant, which exhibits a substantially reduced PPIase activity, alleviated the reduction of NCX1 surface expression caused by CsA treatment, suggesting that the PPIase domain was probably not mandatory for NCX1 functional expression. We suggest that CypA plays a role in the functional expression of NCX1 protein.

The Na þ-Ca 2þ exchanger (NCX) is a major Ca 2þ regulating protein. It is almost ubiquitously expressed. Cyclophilins (Cyps) make up a class of proteins that are involved in protein folding via their peptidyl prolyl cis-trans isomerase (PPIase) and chaperone domains. They are also the cellular receptors of cyclosporin A (CsA). Binding of CsA to cyclophilins inhibits both PPIase and chaperone activities. We have shown that treatment of transfected HEK 293 cells expressing the Na þ-Ca 2þ exchanger NCX1 with CsA results in downregulation of surface expression and transport activity, without any reduction in the total level of cell NCX1 protein [Kimchi-Sarfaty, C., et al. (2002) J. Biol. Chem. 277 (4), 2505-2510]. In this work, we show that knockdown of cell CypA using targeting siRNA (without any CsA treatment) results in a reduction in the level of NCX1 surface expression, a decrease in the level of Na þ-dependent Ca 2þ uptake, and no change in the total amount of cell NCX1 protein in NCX1.5-transfected HEK 293 cells and nontransfected H9c2 cells that express NCX1.1 naturally. It also reduced Na þ-dependent Ca 2þ fluxes measured by changes in Fluo-4 AM fluorescence in single NCX1.5-transfected HEK 293 and single H9c2 cells. Knockdown of CypB had no significant effect on either transport activity, surface expression, NCX1 cell protein expression, or Ca 2þ fluxes. Overexpression of CypA or its R55A mutant, which exhibits a substantially reduced PPIase activity, alleviated the reduction of NCX1 surface expression caused by CsA treatment, suggesting that the PPIase domain was probably not mandatory for NCX1 functional expression. We suggest that CypA plays a role in the functional expression of NCX1 protein.

Mycobacterium tuberculosis (M. tb) has two peptidyl-prolyl isomerases (Ppiases) PpiA and PpiB, popularly known as cyclophilin A and cyclophilin B. The role of cyclophilins in processes such as signaling, cell surface recognition, chaperoning, and heat shock response has been well-documented. We present evidence that M. tb Ppiases modulate the host immune response. ELISA results revealed significant presence of antibodies to M. tb Ppiases in patient sera as compared to sera from healthy individuals. Treatment of THP-1 cells with increasing concentrations of rPpiA, induced secretion of pro-inflammatory cytokines TNF-α and IL-6. Alternatively, treatment with rPpiB inhibited secretion of TNF-α and induced secretion of IL-10. Furthermore, heterologous expression of M. tb PpiA and PpiB in Mycobacterium smegmatis increased bacterial survival in THP-1 cells as compared to those transformed with the vector control. Our results demonstrate that M. tb Ppiases are immunogenic proteins that can possibly modulate host immune response and enhance persistence of the pathogen within the host by subverting host cell generated stresses.