Cyclophilin A (CypA/Ppia) is an 18kD, 8-stranded beta-barrel, with a solvent-exposed hydrophobic pocket.The immunosuppressive drug cyclosporine grips the pocket and with CypA creates a composite surface that binds and inhibits calcineurin. The pocket cradles proline-containing peptides, including an exposed proline on HIV-1 Capsid, and catalyzes cis-trans interconversion of the peptidyl-prolyl bond. To identify the native function of CypA we generated mice lacking Ppia. Ppia1' mice develop allergic pathology with tissue infiltration by eosinophils and mast cells, and increased CD4+ T-helper type II (Th2) cytokine production. We hypothesized that the pathology resulted from increased activity of Itk, a tyrosine kinase upstream of PLC gamma (and therefore of calcineurin) that specifically promotes Th2 function. CypA bound Itk via the PPIase active site. Mutation of a conformationally heterogeneous proline in Itk (P287G) disrupted CypA binding and conferred Th2 hyperactivity on wild-type CD4+ T cells. Thus, CypA regulates CD4+ T cells signal transduction in the absence of cyclosporine via a regulatory proline residue in Itk.
Aim 1 : Ppia4' mice exhibit strain-dependent lethality. A dominant suppressor of lethality in 129/SvEv mice will be mapped and cloned.
Aim 2 : The functional significance of CypA peptidyl-prolyl isomerase activity for inhibition of Itk and Th2 cytokine expression will be tested using a panel of rationally-designed CypA mutants, as well as mutants screened in a yeast strain that is dependent on CypA PPIase activity.
Aim 3 : Ppia*'* memory CD4+ Th2 cells secrete enormous quantities of IL2. We will determine if this phenotype also results from effects of CypA on Itk and we will elucidate the post-transcriptional mechanism underlying it. These studies will provide the first detailed assessment of the biological function of CypA, shed light on a novel mechanism of protein kinase regulation characterized by a conformational switch in Itk, and advance basic knowledge of protein-folding, T cell signaling, cytokine expression, and CD4+ T cell differentiation. New information concerning these basic biological processes will help understand the etiology of such diseases as asthma and AIDS, and, therefore, is critical for the development of much-needed medical therapies. ? ? ?
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