Cyclophilin A Function in the Immune System

描述（由申请人提供）：亲环蛋白A （CypA/Ppia）是一种18kD， 8链β -桶，具有溶剂暴露的疏水性口袋。免疫抑制药物环孢素抓住口袋并与CypA形成结合并抑制钙调磷酸酶的复合表面。该口袋可容纳含有脯氨酸的肽，包括HIV-1衣壳上暴露的脯氨酸，并催化肽基-脯氨酸键的顺式-反式相互转化。为了确定CypA的天然功能，我们制造了缺乏Ppia的小鼠。Ppia1'小鼠出现过敏病理，组织被嗜酸性粒细胞和肥大细胞浸润，CD4+ t辅助型II （Th2）细胞因子的产生增加。我们假设病理是由Itk活性增加引起的，Itk是PLC γ上游的一种酪氨酸激酶（因此也是钙调磷酸酶），它特异性地促进了Th2的功能。CypA通过PPIase活性位点与Itk结合。Itk （P287G）构象异质脯氨酸的突变破坏了CypA的结合，并使野生型CD4+ T细胞产生Th2高活性。因此，在环孢素缺失的情况下，CypA通过Itk中的脯氨酸残基调控CD4+ T细胞的信号转导。目的1:Ppia4'小鼠表现出毒株依赖性致死。129/SvEv小鼠致死率的显性抑制基因将被定位和克隆。目的2:CypA肽基脯氨酸异构酶活性对Itk和Th2细胞因子表达抑制的功能意义将通过一组合理设计的CypA突变体以及在依赖于CypA PPIase活性的酵母菌株中筛选的突变体进行测试。目的3:Ppia记忆CD4+ Th2细胞分泌大量il - 2。我们将确定这种表型是否也是由CypA对Itk的影响引起的，我们将阐明其转录后机制。这些研究将首次详细评估CypA的生物学功能，揭示以Itk构象开关为特征的蛋白激酶调控新机制，并推进蛋白质折叠、T细胞信号传导、细胞因子表达和CD4+ T细胞分化的基础知识。有关这些基本生物学过程的新信息将有助于了解诸如哮喘和艾滋病等疾病的病因，因此，对开发急需的医学疗法至关重要。

DESCRIPTION (provided by applicant): 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.