Cyclophilin A

ABSTRACTTripartite motif-containing protein 5 (TRIM5) restricts human immunodeficiency virus type 1 (HIV-1) in a species-specific manner by uncoating viral particles while activating early innate responses. Although the contribution of TRIM5 proteins to cellular immunity has not yet been studied, their interactions with the incoming viral capsid and the cellular proteasome led us to hypothesize a role for them. Here, we investigate whether the expression of two nonhuman TRIM5 orthologs, rhesus TRIM5α (RhT5) and TRIM-cyclophilin A (TCyp), both of which are potent restrictors of HIV-1, could enhance immune recognition of infected cells by CD8+T cells. We illustrate how TRIM5 restriction improves CD8+T-cell-mediated HIV-1 inhibition. Moreover, when TRIM5 activity was blocked by the nonimmunosuppressive analog of cyclosporine (CsA), sarcosine-3(4-methylbenzoate)–CsA (SmBz-CsA), we found a significant reduction in CD107a/MIP-1β expression in HIV-1-specific CD8+T cells. This finding under...

Invasion of human erythrocytes by Plasmodium falciparum merozoites involves multiple interactions between host receptors and their merozoite ligands. Here we report human Cyclophilin B as a receptor for PfRhopH3 during merozoite invasion. Localization and binding studies show that Cyclophilin B is present on the erythrocytes and binds strongly to merozoites. We demonstrate that PfRhopH3 binds to the RBCs and their treatment with Cyclosporin A prevents merozoite invasion. We also show a multi-protein complex involving Cyclophilin B and Basigin, as well as PfRhopH3 and PfRh5 that aids the invasion. Furthermore, we report identification of a de novo peptide CDP3 that binds Cyclophilin B and blocks invasion by up to 80%. Collectively, our data provide evidence of compounded interactions between host receptors and merozoite surface proteins and paves the way for developing peptide and small-molecules that inhibit the protein-protein interactions, individually or in toto, leading to abrog...

Internal protein dynamics are intimately connected to enzymatic catalysis. However, enzyme motions linked to substrate turnover remain largely unknown. We have studied dynamics of an enzyme during catalysis at atomic resolution using nuclear magnetic resonance relaxation methods. During catalytic action of the enzyme cyclophilin A, we detect conformational fluctuations of the active site that occur on a time scale of hundreds of microseconds. The rates of conformational dynamics of the enzyme strongly correlate with the microscopic rates of substrate turnover. The present results, together with available structural data, allow a prediction of the reaction trajectory.

Proline isomerization is a ubiquitous process that plays a key role in the folding of proteins and in the regulation of their functions. Different families of enzymes, known as "peptidyl-prolyl isomerases" (PPIases), catalyze this reaction, which involves the interconversion between the cis and trans isomers of the N-terminal amide bond of the amino acid proline. However, complete descriptions of the mechanisms by which these enzymes function have remained elusive. We show here that cyclophilin A, one of the most common PPIases, provides a catalytic environment that acts on the substrate through an electrostatic handle mechanism. In this mechanism, the electrostatic field in the catalytic site turns the electric dipole associated with the carbonyl group of the amino acid preceding the proline in the substrate, thus causing the rotation of the peptide bond between the two residues. We identified this mechanism using a combination of NMR measurements, molecular dynamics simu...

The immune system is alerted to cell death by molecules known as damage-associated molecular patterns (DAMPs). These molecules partly mediate acetaminophen-induced liver injury, an archetypal experimental model of sterile cell death and the commonest cause of acute liver failure in the western world. Cyclophilin A (CypA) is an intracellular protein that is proinflammatory when released by cells. We hypothesized that CypA is released from necrotic liver cells and acts as a DAMP to mediate acetaminophen-induced liver injury. Our data demonstrated that mice lacking CypA (Ppia(-/-)) were resistant to acetaminophen toxicity. Antagonism of the extracellular receptor for CypA (CD147) also reduced acetaminophen-induced liver injury. When injected into a wild-type mouse, necrotic liver from Ppia(-/-) mice induced less of an inflammatory response than did wild-type liver. Conversely, the host inflammatory response was increased when CypA was injected with necrotic liver. Antagonism of CD147 a...

Cyclophilin A (CypA) is an immunophilin that acts as a receptor for the immunosuppressant drug cyclosporine A (CsA). CypA has emerged as a potential drug target for several inflammatory diseases, although the details of its mechanism are unclear. We examined the protective effects of CypA on inflammation in Raw 264.7 cells and animal models. A human CypA gene was fused with a protein transduction domain, PEP-1 peptide, to construct a cell permeable PEP-1-CypA protein. The protein expression level of cyclooxygenase-2 (COX-2) and cytokines was detected by Western blot, ELISA and mRNA level of COX-2 and cytokines were measured by RT-PCR. The nuclear factor-kappa B (NF-kB) and mitogen-activated protein kinase (MAPK) activation were analyzed by Western blot and electrophoretic mobility shift assay. Skin inflammation was detected with immunohistochemistry. Transduced PEP-1-CypA protein markedly inhibited lipopolysaccharide- and 12-O-tetradecanoyl phorbol-13-acetate-induced expression leve...

The CD147 protein is a ubiquitous multifunctional membrane receptor. Expression of CD147, which is regulated by sterol carrier protein, reportedly modulates amyloid-β (Aβ), the neurotoxic peptide implicated in neuronal degeneration in Alzheimer's disease (AD). Given that high fat/cholesterol is linked to amyloid deposition in AD, we investigated if cholesterol and/or Aβ can alter CD147 expression in rat cortical neuronal cultures. Water-soluble cholesterol and Aβ42 dose-dependently increased CD147 protein expression, but reduced FL-AβPP protein expression. Cholesterol and Aβ42 treatment also increased lactate dehydrogenase release but to varying degrees. Upregulation of CD147 expression was probably mediated by oxidative stress, as H2O2 (3 μM) also induced CD147 protein expression in neuronal cultures. In light of these findings, we investigated if CD147 induction was cytoprotective, a compensatory response to injury, or alternatively, a cell death signal. To this end, we used recombinant adenovirus to overexpress human CD147 (in SH-SY5Y cells and primary cortical neurons), and pre-treated cultures with or without recombinant cyclophilin A (rCYPA) protein, prior to Aβ42 exposure. We showed that increased CD147 expression protected against Aβ42, only when rCYPA protein was added to neuronal cultures. Together, our findings reveal potentially important relationships between cholesterol loading, CD147 expression, Aβ toxicity, and the putative involvement of CYPA protein in neuroprotection in AD.

Cyclosporin A (CsA) suppresses immune reaction by inhibiting calcineurin activity after forming complex with cyclophilins and is currently widely used as an immunosuppressive drug. Cyclophilin A (CypA) is the most abundantly and ubiquitously expressed family member of ...

Cyclosporin A (CSA) suppresses immune function by blocking the cyclophilin A and calcineurin/NFAT signaling pathways. In addition to immunosuppression, CSA has also been shown to have a wide range of effects in the cardiovascular system including disruption of heart valve development, smooth muscle cell proliferation, and angiogenesis inhibition. Circumstantial evidence has suggested that CSA might control Notch signaling which is also a potent regulator of cardiovascular function. Therefore, the goal of this project was to determine if CSA controls Notch and to dissect the molecular mechanism(s) by which CSA impacts cardiovascular homeostasis. We found that CSA blocked JAG1, but not Dll4 mediated Notch1 NICD cleavage in transfected 293T cells and decreased Notch signaling in zebrafish embryos. CSA suppression of Notch was linked to cyclophilin A but not calcineurin/NFAT inhibition since N-MeVal-4-CsA but not FK506 decreased Notch1 NICD cleavage. To examine the effect of CSA on vasc...

The Cyclophilin A (CypA)/Apoptosis Inducing Factor (AIF) complex is implicated in the DNA degradation in response to various cellular stress conditions, such as oxidative stress, cerebral hypoxia-ischemia and traumatic brain injury. The pro-apoptotic form of AIF (AIF(Δ1-121)) mainly interacts with CypA through the amino acid region 370-394. The AIF(370-394) synthetic peptide inhibits complex formation in vitro by binding to CypA and exerts neuroprotection in a model of glutamate-mediated oxidative stress. Here, the binding site of AIF(Δ1-121) and AIF(370-394) on CypA has been mapped by NMR spectroscopy and biochemical studies, and a molecular model of the complex has been proposed. We show that AIF(370-394) interacts with CypA on the same surface recognized by AIF(Δ1-121) protein and that the region is very close to the CypA catalytic pocket. Such region partially overlaps with the binding site of cyclosporin A (CsA), the strongest catalytic inhibitor of CypA. Our data point toward ...

Achieving greater understanding of the genomic influence on milk synthesis in dairy cows represents a daunting challenge. Bovine-specific microarrays have allowed for high-throughput gene expression analysis of the mammary transcriptome. However, real-time PCR (qPCR) still represents the method of choice for accurate expression profiling of small numbers of genes and verification of key microarray relationships. This method is extremely sensitive but requires data normalization to account for analytical errors. Ideally, ...