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A protease digestion strategy was incorporated into single-point SUPREX (stability of unpurified proteins from rates of H/D exchange), which is an H/D exchange- and mass spectrometry-based assay for the detection of protein-ligand binding. Single-point SUPREX is an abbreviated form of SUPREX in which protein-ligand binding interactions are detected by measuring the increase in a protein’s thermodynamic stability upon ligand binding. The new protease digestion protocol provides a noteworthy increase in the efficiency of single-point SUPREX because peptide masses can be determined with greater precision than intact protein masses in the MALDI readout of single-point SUPREX. The protocol was evaluated in test screens on two model protein systems, including cyclophilin A (CypA) and the minor allele variant of human alanine:glyoxylate aminotransferase (AGTmi). The test screening results obtained on both proteins revealed that the peptide readout of the single-point SUPREX-protease digestion protocol was more efficient than the intact protein readout of the original single-point SUPREX protocol at discriminating hits and non-hits. In addition to this improvement in screening efficiency, the protease digestion strategy described here is expected to significantly increase the generality of the single-point SUPREX assay.

An H/D exchange- and MALDI mass spectrometry-based screening assay was applied to search for novel ligands that bind to cyclophilin A, a potential therapeutic and diagnostic target in lung cancer. The assay is based on SUPREX (stability of unpurified proteins from rates of H/D exchange), which exploits the H/D exchange properties of amide protons to measure the increase in a protein's thermodynamic stability upon ligand binding in solution. The current study evaluates the throughput and efficiency with which 880 potential ligands from the Prestwick Chemical Library could be screened for binding to cyclophilin A. Screening was performed at a rate of 3 min/ligand using a conventional MALDI mass spectrometer. False positive and false negative rates, based on a set of control data, were as low as 0% and 9%, respectively. Based on the 880-member library screening, a false positive rate of 0% was observed when a 2-tier selection strategy was implemented. Although novel ligands for cyclophilin A were not discovered, cyclosporin A, a known ligand to CypA and a blind control in the library, was identified as a hit. We also describe a new strategy to eliminate some of the complications related to back exchange that can arise in screening applications of SUPREX.

In this report, the model proteins staphylococcal nuclease and ubiquitin were used to test the applicability of two new hydrogen/deuterium exchange (HX) electrospray ionization mass spectrometry (ESI-MS) methods for estimating protein folding energies. Both methods use the H/D exchange of globally protected amide protons (amide protons which are buried in the hydrophobic core) to elucidate protein folding energies. One method is a kinetic-based method and the other is equilibrium-based. The first method, the HX ESI-MS kinetic-based approach is conceptually identical to SUPREX (stability of unpurified proteins from rates of H/D exchange) method but is based on ESI-MS rather than MALDI-MS (matrix assisted laser desorption mass spectrometry). This method employs the time-dependence of H/D exchange using various denaturant concentrations to extract folding energies. Like SUPREX, this approach requires the assumption of EX2 exchange kinetics. The second method, which we call a protein equilibrium population snapshot (PEPS) by HX ESI-MS uses data collected only for a single time point (usually the shortest possible) to obtain a snapshot of the open and closed populations of the protein. The PEPS approach requires few assumptions in the derivation of the equations used for calculation of the folding energies. The extraction of folding energies from mass spectral data is simple and straightforward. The PEPS method is applicable for proteins that follow either EX1 or EX2 HX mechanisms. In our experiments the kinetic-based method produced less accurate ΔGH2O and mGdHCl values for wild-type staphylococcal nuclease and mutants undergoing H/D exchange by EX1, as would be expected. Better results were obtained for ubiquitin which undergoes HX by an EX2 mechanism. Using the PEPS method we obtained ΔGH2O and mGdHCl values that were in good agreement with literature values for both staphylococcal nuclease (EX1) and ubiquitin (EX2). We also show that the observation of straight lines in linear extrapolation method (LEM) plots is not a reliable indicator of the validity of the data obtained using the LEM approach.

Viruses are obligate intracellular parasites and therefore their replication completely depends on host cell factors. In case of the hepatitis C virus (HCV), a positive-strand RNA virus that in the majority of infections establishes persistence, cyclophilins are considered to play an important role in RNA replication. Subsequent to the observation that cyclosporines, known to sequester cyclophilins by direct binding, profoundly block HCV replication in cultured human hepatoma cells, conflicting results were obtained as to the particular cyclophilin (Cyp) required for viral RNA replication and the underlying possible mode of action. By using a set of cell lines with stable knock-down of CypA or CypB, we demonstrate in the present work that replication of subgenomic HCV replicons of different genotypes is reduced by CypA depletion up to 1,000-fold whereas knock-down of CypB had no effect. Inhibition of replication was rescued by over-expression of wild type CypA, but not by a mutant lacking isomerase activity. Replication of JFH1-derived full length genomes was even more sensitive to CypA depletion as compared to subgenomic replicons and virus production was completely blocked. These results argue that CypA may target an additional viral factor outside of the minimal replicase contributing to RNA amplification and assembly, presumably nonstructural protein 2. By selecting for resistance against the cyclosporine analogue DEBIO-025 that targets CypA in a dose-dependent manner, we identified two mutations (V2440A and V2440L) close to the cleavage site between nonstructural protein 5A and the RNA-dependent RNA polymerase in nonstructural protein 5B that slow down cleavage kinetics at this site and reduce CypA dependence of viral replication. Further amino acid substitutions at the same cleavage site accelerating processing increase CypA dependence. Our results thus identify an unexpected correlation between HCV polyprotein processing and CypA dependence of HCV replication.

Author Summary

Owing to limited genetic information, viruses have to exploit host cells to achieve efficient production of virus progeny. Host cell factors and pathways therefore play an important role for virus replication and thus represent a possible target for antiviral therapy. In case of the hepatitis C virus (HCV), an RNA virus infecting liver cells and causing chronic liver disease, host cell cyclophilins were shown to play an important role in replication. Pharmacological inhibition of cyclophilins, which are catalysts of protein folding, causes profound inhibition of HCV replication, but neither the underlying mechanism by which cyclophilins contribute to viral replication, nor the exact nature of the cyclophilin are known. In this study we demonstrate that HCV replication and presumably also virus particle assembly requires cyclophilin A (CypA), which can be blocked by the cyclosporine analogue DEBIO-025. We identify mutations affecting proteolytic cleavage of the viral polyprotein that render HCV replication less dependent on CypA and thus cause DEBIO-025 resistance. Studies with additional mutants reveal a correlation between polyprotein cleavage kinetics and CypA dependence. Our results support a model by which CypA activates the viral replicase in a manner that depends on the kinetics with which the viral polyprotein is cleaved.

Cyclophilins are peptidyl-prolyl cis-trans isomerases involved in catalyzing conformational changes and accelerating the rate of protein folding and refolding in several cellular systems. In the present study, we analyzed the expression pattern and intracellular distribution of the cellular isomerase cyclophilin A (CypA) during vaccinia virus (VV) infection. An impressive increase in CypA stability was observed, leading to a practically unchanged accumulation of CypA during infection, although its synthesis was completely inhibited at late times. By confocal microscopy, we observed that CypA went through an intense reorganization in the cell cytoplasm and colocalized with the virosomes late in infection. CypA relocation to viral factories required the synthesis of viral postreplicative proteins, and treatment of infected cells with cyclosporine (CsA) prevented CypA relocation, clearly excluding the virosomes from CypA staining. Immunoelectron microscopy of VV-infected cells showed that CypA was incorporated into VV particles during morphogenesis. Biochemical and electron microscopic assays with purified virions confirmed that CypA was encapsidated within the virus particle and localized specifically in the core. This work suggests that CypA may develop an important role in VV replication.

Background

Cyclophilin A (CypA) represents a potential target for antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication, although the mechanism through which CypA modulates HIV-1 infectivity still remains unclear. The interaction of HIV-1 viral protein R (Vpr) with the human peptidyl prolyl isomerase CypA is known to occur in vitro and in vivo. However, the nature of the interaction of CypA with Pro-35 of N-terminal Vpr has remained undefined.

Results

Characterization of the interactions of human CypA with N-terminal peptides of HIV-1 Vpr has been achieved using a combination of nuclear magnetic resonace (NMR) exchange spectroscopy and surface plasmon resonance spectroscopy (SPR). NMR data at atomic resolution indicate prolyl cis/trans isomerisation of the highly conserved proline residues Pro-5, -10, -14 and -35 of Vpr are catalyzed by human CypA and require only very low concentrations of the isomerase relative to that of the peptide substrates. Of the N-terminal peptides of Vpr only those containing Pro-35 bind to CypA in a biosensor assay. SPR studies of specific N-terminal peptides with decreasing numbers of residues revealed that a seven-residue motif centred at Pro-35 consisting of RHFPRIW, which under membrane-like solution conditions comprises the loop region connecting helix 1 and 2 of Vpr and the two terminal residues of helix 1, is sufficient to maintain strong specific binding.

Conclusions

Only N-terminal peptides of Vpr containing Pro-35, which appears to be vital for manifold functions of Vpr, bind to CypA in a biosensor assay. This indicates that Pro-35 is essential for a specific CypA-Vpr binding interaction, in contrast to the general prolyl cis/trans isomerisation observed for all proline residues of Vpr, which only involve transient enzyme-substrate interactions. Previously suggested models depicting CypA as a chaperone that plays a role in HIV-1 virulence are now supported by our data. In detail the SPR data of this interaction were compatible with a two-state binding interaction model that involves a conformational change during binding. This is in accord with the structural changes observed by NMR suggesting CypA catalyzes the prolyl cis/trans interconversion during binding to the RHFP35RIW motif of N-terminal Vpr.

Molecular chaperones are a highly diverse group of proteins that recognize and bind unfolded proteins in order to facilitate protein folding and prevent non-specific protein aggregation. The mechanisms by which chaperones bind their protein substrates have been studied for decades. However, there are few reports on the affinity of molecular chaperones for their unfolded protein substrates. Thus, little is known about the relative binding affinities of different chaperones and about the relative binding affinities of chaperones for different unfolded protein substrates. Here we describe the application of SUPREX (stability of unpurified proteins from rates of H/D exchange), an H/D exchange and MALDI-based technique, to study the binding interaction between the molecular chaperone Hsp33 and four different unfolded protein substrates including citrate synthase, lactate dehydrogenase, malate dehydrogenase, and aldolase. The results of our studies suggest that the cooperativity of the Hsp33 folding/unfolding reaction increases upon binding with denatured protein substrates. This is consistent with the burial of significant hydrophobic surface area in Hsp33 when it interacts with its substrate proteins. The SUPREX derived Kd-values for Hsp33 complexes with four different substrates were found to be all within a range of 3-300 nM.

Background

The capsid (CA) protein of HIV-1 binds with high affinity to the host protein cyclophilin A (CypA). This binding positively affects some early stage of the viral life-cycle because prevention of binding either by drugs that occupy that active site of cyclophilin A, by mutation in HIV-1 CA, or RNAi that knocks down intracellular CypA level diminishes viral infectivity. The closely related lentivirus, SIVcpz also binds CypA, but it was thought that this interaction was limited to the HIV-1/SIVcpz lineage because other retroviruses failed to interact with CypA in a yeast two-hybrid assay.

Results

We find that diverse lentiviruses, FIV and SIVagmTAN also bind to CypA. Mutagenesis of FIV CA showed that an amino acid that is in a homologous position to the proline at amino acid 90 of HIV-1 CA is essential for FIV interactions with CypA.

Conclusion

These results demonstrate that CypA binding to lentiviruses is more widespread than previously thought and suggest that this interaction is evolutionarily important for lentiviral infection.

Although cyclophilin A (CypA) has been reported to be over-expressed in cancer cells and solid tumors, its expression and role in glioblastomas have not been studied. Herein, we show that expression of CypA in human glioblastoma cell lines and tissues is significantly higher than in normal human astrocytes and normal counterparts of brain tissue. To determine the role of over-expressed CypA in glioblastoma, stable RNA interference (RNAi)-mediated knockdown of CypA (CypA KD) was performed in gliobastoma cell line U87vIII (U87MG · ΔEGFR). CypA KD stable single clones decrease proliferation, infiltration, migration, and anchorage-independent growth in vitro and with slower growth in vivo as xenografts in immunodeficient nude mice. We have also observed that knockdown of CypA inhibits expression of interleukin-8 (IL-8), a tumorigenic and proangiogenic cytokine. Conversely, enforced expression of CypA in the CypA KD cell line, Ud-12, markedly enhanced IL-8 transcripts and restored Ud-12 proliferation, suggesting that CypA-mediated IL-8 production provides a growth advantage to glioblastoma cells. CypA knockdown-mediated inhibition of IL-8 is due to reduced activity of NF-κB, which is one of the major transcription factors regulating IL-8 expression. These results not only establish the relevance of CypA to glioblastoma growth in vitro and in vivo, but also suggest that small interfering RNA-based CypA knockdown could be an effective therapeutic approach against glioblastomas.

The nonimmunosuppressive cyclophilin (Cyp) inhibitor SCY-635 blocks hepatitis C virus (HCV) replication both in vitro and in vivo and represents a novel potent anti-HCV agent. However, its mechanism of action remains to be fully elucidated. A growing body of evidence suggests that cyclophilin A (CypA) is absolutely necessary for HCV replication and that the HCV nonstructural 5A (NS5A) protein serves as a main viral ligand for CypA. In this study, we examined the effect of SCY-635 on HCV replication. Specifically, we asked whether SCY-635 blocks HCV replication by targeting CypA-NS5A interactions. We also investigated the possibility that HCV can escape SCY-635 selection pressure and whether this resistance influences either CypA-NS5A interactions or the dependence of HCV on CypA. We found not only that SCY-635 efficiently inhibits HCV replication, but it is sufficient alone to clear HCV replicon-containing cells. We found that SCY-635 prevents CypA-NS5A interactions in a dose-dependent manner. SCY-635 prevents the contact between CypA and NS5A derived from genotypes 1 to 3. Together, these data suggest that NS5A-CypA interactions control HCV replication and that SCY-635 blocks viral replication by preventing the formation of these complexes. We also found that NS5A mutant proteins found in SCY-635-resistant HCV replicons behave similarly to wild-type NS5A in terms of both CypA binding and SCY-635-mediated dissociation and inhibition of CypA binding. However, the NS5A mutations found in SCY-635-resistant HCV replicons rescued viral replication in CypA-knockdown cells, suggesting that the NS5A mutations, which arose in vitro under SCY-635 selection, do not alter the binding affinity of CypA for NS5A. These specific mutations in NS5A eliminate the dependence of HCV RNA replication on the expression of host CypA

Cyclophilin A (CypA) is a typical member of the cyclophilin family of peptidyl-prolyl isomerases and is involved in the replication of several viruses. Previous studies indicate that CypA interacts with influenza virus M1 protein and impairs the early stage of the viral replication. To further understand the molecular mechanism by which CypA impairs influenza virus replication, a 293T cell line depleted for endogenous CypA was established. The results indicated that CypA inhibited the initiation of virus replication. In addition, the infectivity of influenza virus increased in the absence of CypA. Further studies indicated that CypA had no effect on the stages of virus genome replication or transcription and also did not impair the nuclear export of the viral mRNA. However, CypA decreased the viral protein level. Additional studies indicated that CypA enhanced the degradation of M1 through the ubiquitin/proteasome-dependent pathway. Our results suggest that CypA restricts influenza virus replication through accelerating degradation of the M1 protein.

Upon cerebral hypoxia-ischemia (HI), apoptosis-inducing factor (AIF) can move from mitochondria to nuclei, participate in chromatinolysis, and contribute to the execution of cell death. Previous work (Cande, C., N. Vahsen, I. Kouranti, E. Schmitt, E. Daugas, C. Spahr, J. Luban, R.T. Kroemer, F. Giordanetto, C. Garrido, et al. 2004. Oncogene. 23:1514–1521) performed in vitro suggests that AIF must interact with cyclophilin A (CypA) to form a proapoptotic DNA degradation complex. We addressed the question as to whether elimination of CypA may afford neuroprotection in vivo. 9-d-old wild-type (WT), CypA+/−, or CypA−/− mice were subjected to unilateral cerebral HI. The infarct volume after HI was reduced by 47% (P = 0.0089) in CypA−/− mice compared with their WT littermates. Importantly, CypA−/− neurons failed to manifest the HI-induced nuclear translocation of AIF that was observed in WT neurons. Conversely, CypA accumulated within the nuclei of damaged neurons after HI, and this nuclear translocation of CypA was suppressed in AIF-deficient harlequin mice. Immunoprecipitation of AIF revealed coprecipitation of CypA, but only in injured, ischemic tissue. Surface plasmon resonance revealed direct molecular interactions between recombinant AIF and CypA. These data indicate that the lethal translocation of AIF to the nucleus requires interaction with CypA, suggesting a model in which two proteins that normally reside in separate cytoplasmic compartments acquire novel properties when moving together to the nucleus.

Background

Cyclophilin A (CypA) expression is associated with malignant phenotypes in many cancers. However, the role and mechanisms of CypA in liver fluke-associated cholangiocarcinoma (CCA) are not presently known. In this study, we investigated the expression of CypA in CCA tumor tissues and CCA cell lines as well as regulation mechanisms of CypA in tumor growth using CCA cell lines.

Methods

CypA expression was determined by real time RT-PCR, Western blot or immunohistochemistry. CypA silence or overexpression in CCA cells was achieved using gene delivery techniques. Cell proliferation was assessed using MTS assay or Ki-67 staining. The effect of silencing CypA on CCA tumor growth was determined in nude mice. The effect of CypA knockdown on ERK1/2 activation was assessed by Western blot.

Results

CypA was upregulated in 68% of CCA tumor tissues. Silencing CypA significantly suppressed cell proliferation in several CCA cell lines. Likewise, inhibition of CypA peptidyl-prolyl cis-trans isomerase (PPIase) activity using cyclosporin A (CsA) decreased cell proliferation. In contrast, overexpression of CypA resulted in 30% to 35% increases in proliferation of CCA cell lines. Interestingly, neither silence nor overexpression of CypA affected cell proliferation of a non-tumor human cholangiocyte cell line, MMNK1. Suppression of CypA expression attenuated ERK1/2 activity in CCA M139 cells by using both transient and stable knockdown methods. In the in vivo study, there was a 43% reduction in weight of tumors derived from CypA-silenced CCA cell lines compared with control vector CCA tumors in mice; these tumors with stable CypA silencing showed a reduced cell proliferation.

Conclusions

CypA is upregulated in majority of CCA patients' tissues and confers a significant growth advantage in CCA cells. Suppression of CypA expression decreases proliferation of CCA cell lines in vitro and reduces tumor growth in the nude mouse model. Inhibition of CypA activity also reduces CCA cell proliferation. The ERK1/2 pathway may be involved in the CypA-mediated CCA cell proliferation. Thus, CypA may represent an important new therapeutic target for liver fluke-associated CCA.

NS5A plays a critical, yet poorly defined, role in hepatitis C virus genome replication. The protein consists of three domains, each of which is able to bind independently to the 3′ untranslated region (UTR) of the viral positive strand genomic RNA. The peptidyl-prolyl isomerase cyclophilin A (CypA) binds to domain II, catalyzing cis-trans isomerization. CypA inhibitors such as cyclosporine (CsA) have been shown to inhibit hepatitis C virus (HCV) replication. We show here that CypA stimulated domain II RNA binding activity, and this stimulation was abrogated by CsA. An isomerase mutant of CypA (H126Q) failed to bind to domain II and did not stimulate RNA binding. Finally, we demonstrate that the RNA binding of two domain II mutants, the D316E and D316E/Y317N mutants, previously shown to exhibit CypA independence for RNA replication, was unaffected by CypA. This study provides an insight into the molecular mechanism of CypA activity during HCV replication and further validates the use of CypA inhibitors in HCV therapy.

Cyclophilin A (CypA) is a peptidyl-prolyl isomerase that binds to the capsid protein (CA) of human immunodeficiency virus type 1 (HIV-1) and by doing so facilitates HIV-1 replication. Although CypA is incorporated into HIV-1 virions by virtue of CypA-Gag interactions that occur during virion assembly, in this study we show that the CypA-CA interaction that occurs following the entry of the viral capsid into target cells is the major determinant of CypA's effects on HIV-1 replication. Specifically, by using normal and CypA-deficient Jurkat cells, we demonstrate that the presence of CypA in the target and not the virus-producing cell enhances HIV-1 infectivity. Moreover, disruption of the CypA-CA interaction with cyclosporine A (CsA) inhibits HIV-1 infectivity only if the target cell expresses CypA. The effect of CsA on HIV-1 infection of human cells varies according to which particular cell line is used as a target, and CA mutations that confer CsA resistance and dependence exert their effects only if target cells, and not if virus-producing cells, are treated with CsA. The differential effects of CsA on HIV-1 infection in different human cells appear not to be caused by polymorphisms in the recently described retrovirus restriction factor TRIM5α. We speculate that CypA and/or CypA-related proteins affect the fate of incoming HIV-1 capsid either directly or by modulating interactions with unidentified host cell factors.

Cyclophilin A (CypA) is the main member of the immunophilin superfamily that has peptidyl-prolyl cis-trans isomerase activity. CypA participates in protein folding, cell signaling, inflammation and tumorigenesis. Further, CypA plays critical roles in the replication of several viruses. Upon influenza virus infection, CypA inhibits viral replication by interacting with the M1 protein. In addition, CypA is incorporated into the influenza virus virions. Finally, Cyclosporin A (CsA), the main inhibitor of CypA, inhibits influenza virus replication through CypA-dependent and -independent pathways. This review briefly summarizes recent advances in understanding the roles of CypA during influenza virus infection.

Cyclophilin inhibitors currently in clinical trials for hepatitis C virus (HCV) are all analogues of cyclosporine (CsA). Sanglifehrins are a group of naturally occurring cyclophilin binding polyketides that are structurally distinct from the cyclosporines and are produced by a microorganism amenable to biosynthetic engineering for lead optimization and large-scale production by fermentation. Preclinical characterization of the potential utility of this class of compounds for the treatment of HCV revealed that the natural sanglifehrins A to D are all more potent than CsA at disrupting formation of the NS5A-CypA, -CypB, and -CypD complexes and at inhibition of CypA, CypB, and CypD isomerase activity. In particular, sanglifehrin B (SfB) was 30- to 50-fold more potent at inhibiting the isomerase activity of all Cyps tested than CsA and was also shown to be a more potent inhibitor of the 1b subgenomic replicon (50% effective concentrations [EC50s] of 0.070 μM and 0.16 μM in Huh 5-2 and Huh 9-13 cells, respectively). Physicochemical and mouse pharmacokinetic analyses revealed low oral bioavailability (F < 4%) and low solubility (<25 μM), although the half-lives (t1/2) of SfA and SfB in mouse blood after intravenous (i.v.) dosing were long (t1/2 > 5 h). These data demonstrate that naturally occurring sanglifehrins are suitable lead compounds for the development of novel analogues that are less immunosuppressive and that have improved metabolism and pharmacokinetic properties.

Background

The importance of enhancing influenza resistance in domestic flocks is quite clear both scientifically and economically. Chicken is very susceptible to influenza virus. It has been reported that human cellular cyclophilin A (CypA) impaired influenza virus infection in 293T cells. Whether chicken CypA (chCypA) inhibits influenza virus replication is not known. The molecular mechanism of resistance in chicken to influenza virus remains to be studied.

Results

The chCypA gene was isolated and characterized in the present study. It contained an ORF of 498 bp encoding a polypeptide of 165 amino acids with an estimated molecular mass of 17.8 kDa sharing high identity with mammalian CypA genes. The chCypA demonstrated an anti-influenza activity as expected. ChCypA protein was shown to be able to specifically interact with influenza virus M1 protein. Cell susceptibility to influenza virus was reduced by over-expression of chCypA in CEF cells. The production of recombinant influenza virus A/WSN/33 reduced to one third in chCypA expressing cells comparing to chCypA absent cells. ChCypA was widely distributed in a variety of chicken tissues. It localized in cytoplasm of chicken embryo fibroblast (CEF) cells. Avian influenza virus infection induced its translocation from cytoplasm into nucleus. ChCypA expression was not significantly up-regulated by avian influenza virus infection. The present study indicated that chCypA was an inhibitory protein to influenza virus replication, suggesting a role as an intrinsic immunity factor against influenza virus infection.

Conclusion

The present data demonstrates that chCypA possesses anti-influenza virus activity which allows the consideration of genetic improvement for resistance to influenza virus in chickens.

Background

CD147, as a cellular receptor for cyclophilin A (CypA), is a multifunctional protein involved in tumor invasion, inflammation, tissue remodeling, neural function, and reproduction. Recent observations showing the expression of CD147 in leukocytes indicate that this molecule may have roles in inflammation.

Methods

In order to investigate the role of CD147 and its ligand in the pathogenesis of atherosclerosis, human atherosclerotic plaques were analyzed for the expression pattern of CD147 and CypA. The cellular responses and signaling molecules activated by the stimulation of CD147 were then investigated in the human macrophage cell line, THP-1, which expresses high basal level of CD147 on the cell surface.

Results

Staining of both CD147 and CypA was detected in endothelial cell layers facing the lumen and macrophage-rich areas. Stimulation of CD147 with its specific monoclonal antibody induced the expression of matrix metalloproteinase (MMP)-9 in THP-1 cells and it was suppressed by inhibitors of both ERK and NF-κB. Accordingly, the stimulation of CD147 was observed to induce phosphorylation of ERK, phosphorylation-associated degradation of IκB, and nuclear translocation of NF-κB p65 and p50 subunits.

Conclusion

These results suggest that CD147 mediates the inflammatory activation of macrophages that leads to the induction of MMP-9 expression, which could play a role in the pathogenesis of inflammatory diseases such as atherosclerosis.

Human immunodeficiency virus type 1 (HIV-1) requires the incorporation of cyclophilin A (CypA) for replication. CypA is packaged by binding to the capsid (CA) region of Gag. This interaction is disrupted by cyclosporine (CsA). Preventing CypA incorporation, either by mutations in the binding region of CA or by the presence of CsA, abrogates virus infectivity. Given that CypA possesses an isomerase activity, it has been proposed that CypA acts as an uncoating factor by destabilizing the shell of CA that surrounds the viral genome. However, because the same domain of CypA is responsible for both its isomerase activity and its capacity to be packaged, it has been challenging to determine if isomerase activity is required for HIV-1 replication. To address this issue, we fused CypA to viral protein R (Vpr), creating a Vpr-CypA chimera. Because Vpr is packaged via the p6 region of Gag, this approach bypasses the interaction with CA and allows CypA incorporation even in the presence of CsA. Using this system, we found that Vpr-CypA rescues the infectivity of viruses lacking CypA, either produced in the presence of CsA or mutated in the CypA packaging signal of CA. Furthermore, a Vpr-CypA mutant which has no isomerase activity and no capacity to bind to CA also rescues HIV-1 replication. Thus, this study demonstrates that the isomerase activity of CypA is not required for HIV-1 replication and suggests that the interaction of the catalytic site of CypA with CA serves no other function than to incorporate CypA into viruses.

Cyclophilin A (CypA) is a member of a family of cellular proteins that share a peptidyl prolyl cis-trans isomerase (PPIase) activity. CypA was previously reported to be required for the biochemical stability and function (specifically, induction of G2 arrest) of the human immunodeficiency virus type 1 (HIV-1) protein R (Vpr). In the present study, we examine the role of the Vpr-CypA interaction on Vpr-induced G2 arrest. We find that Vpr coimmunoprecipitates with CypA and that this interaction is disrupted by substitution of proline-35 of Vpr as well as incubation with the CypA inhibitor cyclosporine A (CsA). Surprisingly, the presence of CypA or its binding to Vpr is dispensable for the ability of Vpr to induce G2 arrest. Vpr expression in CypA−/− cells leads to induction of G2 arrest in a manner that is indistinguishable from that in CypA+ cells. CsA abolished CypA-Vpr binding but had no effect on induction of G2 arrest or Vpr steady-state levels. In view of these results, we propose that the interaction with CypA is independent of the ability of Vpr to induce cell cycle arrest. The interaction between Vpr and CypA is intriguing, and further studies should examine its potential effects on other functions of Vpr.

Cyclophilin A (CypA) is an important human immunodeficiency virus type 1 (HIV-1) cofactor in human cells. HIV-1 A92E and G94D capsid escape mutants arise during CypA inhibition and in certain cell lines are dependent on CypA inhibition. Here we show that dependence on CypA inhibition is due to high CypA levels. Restricted HIV-1 is stable, and remarkably, restriction is augmented by arresting cell division. Nuclear entry is not inhibited. We propose that high CypA levels and capsid mutations combine to disturb uncoating, leading to poor infectivity, particularly in arrested cells. Our data suggest a role for CypA in uncoating the core of HIV-1 to facilitate integration.

The mechanisms by which cyclophilin A (CypA) governs hepatitis C virus (HCV) replication remain unknown. Since CypA binds two essential components of the HCV replication complex (RC) – the polymerase NS5B and the phosphoprotein NS5A – we asked in this study whether CypA regulates their RC association. We found that CypA, via its isomerase pocket, locates in a protease-resistant compartment similar to that where HCV replicates. CypA association with this compartment is not mediated by HCV. Moreover, CypA depletion of RC does not influence NS5A and NS5B RC association, arguing against a model where CypA governs HCV replication by recruiting NS5A or NS5B into RC.

Background

Cyclophilin A (CypA) is a cytosolic protein possessing peptidyl-prolyl isomerase activity that was recently reported to be overexpressed in several cancers. Here, we explored the biology and molecular mechanism of CypA in non-small cell lung cancer (NSCLC).

Methods

The expression of CypA in human NSCLC cell lines was detected by real-time reverse transcription PCR. The RNA interference-mediated knockdown of CypA was established in two NSCLC cell lines (95C and A549). 239836 CypA inhibitor was also used to suppress CypA activity. Tumorigenesis was assessed based on cellular proliferation, colony formation assays, and anchorage-independent growth assays; metastasis was assessed based on wound healing and transwell assays.

Results

Suppression of CypA expression inhibited the cell growth and colony formation of A549 and 95C cells. CypA knockdown resulted in the inhibition of cell motility and invasion. Significantly, we show for the first time that CypA increased NSCLC cell invasion by regulating the activity of secreted matrix metallopeptidase 9 (MMP9). Likewise, suppression of CypA with 239836 CypA inhibitor decreased cell proliferation and MMP9 activity.

Conclusions

The suppression of CypA expression was correlated with decreased NSCLC cell tumorigenesis and metastasis.

Background

Cyclophilin A (CypA), a member of the immunophilin family, is a ubiquitously distributed intracellular protein. Recent studies have shown that CypA is secreted by cells in response to inflammatory stimuli. Elevated levels of extracellular CypA and its receptor, CD147 have been detected in the synovium of patients with RA. However, the precise process of interaction between CypA and CD147 in the development of RA remains unclear. This study aimed to investigate CypA secretion from fibroblast-like synoviocytes (FLS) isolated from mice with collagen-induced arthritis (CIA) and CypA-induced CD147 expression in mouse macrophages.

Findings

CIA was induced by immunization with type II collagen in mice. The expression and localization of CypA and CD147 was investigated by immunoblotting and immunostaining. Both CypA and CD147 were highly expressed in the joints of CIA mice. CD147 was expressed in the infiltrated macrophages in the synovium of CIA mice. In vitro, spontaneous CypA secretion from FLS was detected and this secretion was increased by stimulation with lipopolysaccharide. CypA markedly increased CD147 levels in macrophages.

Conclusions

These findings suggest that an interaction in the synovial joints between extracellular CypA and CD147 expressed by macrophages may be involved in the mechanisms underlying the development of arthritis.