A calcineurin-independent mechanism of angiogenesis inhibition by a nonimmunosuppressive cyclosporin A analog.

Cyclosporin A (CsA) is a widely used immunosuppressant drug. Its immunosuppressive activity occurs through the inhibition of the protein phosphatase calcineurin via formation of a ternary complex with cyclophilin A (CypA). CsA also inhibits endothelial cell proliferation and angiogenesis. This has been thought to occur through calcineurin inhibition as well. However, CsA is also a potent inhibitor of cyclophilins, a class of prolyl isomerases. Because calcineurin inhibition requires binding, and therefore inhibition of CypA, the relative contributions of calcineurin and cyclophilin inhibition in antiangiogenesis have not been addressed. We have taken a chemical biology approach to explore this question by dissociating the two activities of CsA at the molecular level. We have identified a nonimmunosuppressive analog of CsA that does not inhibit calcineurin but maintains inhibition of endothelial cell proliferation and in vivo angiogenesis. The same analog also maintains inhibition of all cyclophilin isoforms tested. We also show that a second, structurally distinct, cyclophilin inhibitor is sufficient to block endothelial cell proliferation. These results suggest that the inhibition of cyclophilins may play a larger role in the antiangiogenic activity of CsA than previously believed, and that cyclophilins may be potential antiangiogenic drug targets.

The protein-free IANUS peptide array uncovers interaction sites between Escherichia coli parvulin 10 and alkyl hydroperoxide reductase.

The reliable identification of interacting structural elements without prior isolation of interacting proteins can be achieved by using the novel fluorescence resonance energy transfer-coupled IANUS (Induced orgANization of strUcture by matrix-assisted togethernesS) peptide array. Here we report that parvulin 10 (Par10), an abundant Escherichia coli peptidyl prolyl cis/trans isomerase (PPIase), physically interacts with the alkyl hydroperoxide reductase subunit C (AhpC) in bacterial cell extracts, as determined by affinity chromatography and chemical cross-linking experiments. A Par10-negative E. coli strain showed increased sensitivity toward hydrogen peroxide compared to the wild-type strain. The IANUS experiment revealed three segments of the peroxiredoxin AhpC chain as potential Par10 binding partners. Inhibition of the Par10 PPIase activity by the corresponding AhpC-derived peptides as well as NMR data of (15)N-labeled Par10 in the presence of the AhpC(115-132) peptide or full-length AhpC confirmed that the putative Par10 active site is involved in the Par10-AhpC interaction. Moreover, NMR-based docking calculations as well as NOESY exchange peaks between the proline cis and trans isomers revealed the Asp125-Pro126 moiety of the AhpC segment G115-A132 as a substrate for Par10 enzymatic action. On the basis of these data, we conclude that Par10 catalytic activity is involved in the cellular protection against oxidative stress.