Cyclosporin A Associated Helicase-Like Protein Facilitates the Association of Hepatitis C Virus RNA Polymerase with Its Cellular Cyclophilin B

BACKGROUND: Cyclosporin A (CsA) is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV) genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. PRINCIPAL FINDINGS: Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL), possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB), known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. CONCLUSIONS: We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology

Functional cyclophilin D moderates platelet adhesion, but enhances the lytic resistance of fibrin

In the course of thrombosis, platelets are exposed to a variety of activating stimuli classified as ‘strong’ (e.g. thrombin and collagen) or ‘mild’ (e.g. ADP). In response, activated platelets adhere to injured vasculature, aggregate, and stabilise the three-dimensional fibrin scaffold of the expanding thrombus. Since ‘strong’ stimuli also induce opening of the mitochondrial permeability transition pore (MPTP) in platelets, the MPTP-enhancer Cyclophilin D (CypD) has been suggested as a critical pharmacological target to influence thrombosis. However, it is poorly understood what role CypD plays in the platelet response to ‘mild’ stimuli which act independently of MPTP. Furthermore, it is unknown how CypD influences platelet-driven clot stabilisation against enzymatic breakdown (fibrinolysis). Here we show that treatment of human platelets with Cyclosporine A (a cyclophilin-inhibitor) boosts ADP-induced adhesion and aggregation, while genetic ablation of CypD in murine platelets enhances adhesion but not aggregation. We also report that platelets lacking CypD preserve their integrity in a fibrin environment, and lose their ability to render clots resistant against fibrinolysis. Our results indicate that CypD has opposing haemostatic roles depending on the stimulus and stage of platelet activation, warranting a careful design of any antithrombotic strategy targeting CypD

Transgenic Overexpression of Active Calcineurin in β-Cells Results in Decreased β-Cell Mass and Hyperglycemia

BACKGROUND:Glucose modulates beta-cell mass and function through an initial depolarization and Ca(2+) influx, which then triggers a number of growth regulating signaling pathways. One of the most important downstream effectors in Ca(2+) signaling is the calcium/Calmodulin activated serine threonine phosphatase, calcineurin. Recent evidence suggests that calcineurin/NFAT is essential for beta-cell proliferation, and that in its absence loss of beta-cells results in diabetes. We hypothesized that in contrast, activation of calcineurin might result in expansion of beta-cell mass and resistance to diabetes. METHODOLOGY/PRINCIPAL FINDINGS:To determine the role of activation of calcineurin signaling in the regulation of pancreatic beta-cell mass and proliferation, we created mice that expressed a constitutively active form of calcineurin under the insulin gene promoter (caCn(RIP)). To our surprise, these mice exhibited glucose intolerance. In vitro studies demonstrated that while the second phase of Insulin secretion is enhanced, the overall insulin secretory response was conserved. Islet morphometric studies demonstrated decreased beta-cell mass suggesting that this was a major component responsible for altered Insulin secretion and glucose intolerance in caCn(RIP) mice. The reduced beta-cell mass was accompanied by decreased proliferation and enhanced apoptosis. CONCLUSIONS:Our studies identify calcineurin as an important factor in controlling glucose homeostasis and indicate that chronic depolarization leading to increased calcineurin activity may contribute, along with other genetic and environmental factors, to beta-cell dysfunction and diabetes