Cyclophilin A Function in the Immune System

描述(申请人提供)：亲环素A(CypA/Ppia)是一种18kD的8链β-桶，有一个暴露在溶剂中的疏水口袋。免疫抑制药物环孢素抓住口袋，与CypA形成结合和抑制钙调神经磷酸酶的复合表面。口袋托架容纳含有Pro的多肽，包括HIV-1衣壳上暴露的一个Pro，并催化多肽-Pro键的顺反相互转化。为了确定CypA的天然功能，我们建立了缺乏PpIA的小鼠。Ppia1‘小鼠出现变态反应性病理，嗜酸性粒细胞和肥大细胞渗入组织，并增加了CD4T辅助II型(Th2)细胞因子的产生。我们假设病理是由于ITK的活性增加所致，ITK是PLC伽马上游的一种酪氨酸激酶(因此也是钙调神经磷酸酶)，它特异性地促进Th2功能。CypA通过PPIase活性位点与ITK结合。ITK(P287G)中一种构象异质性的脯氨酸突变破坏了CypA结合，并赋予野生型CD4T细胞Th2过度活性。因此，在没有环孢素的情况下，CypA通过ITK中的一个调节的Pro残基来调节CD4T细胞的信号转导。目的1：Ppia4‘小鼠表现出应变依赖致死性。129/SvEv小鼠致死率的显性抑制基因将被定位和克隆。目的：利用一组合理设计的CypA突变体，以及在依赖CypA PPIase活性的酵母菌株中筛选的突变体，验证CypA肽基-Pro异构酶活性对抑制ITK和Th2细胞因子表达的功能意义。目的3：Ppia*‘*Memory CD4Th2细胞分泌大量IL2。我们将确定这种表型是否也是CypA对ITK影响的结果，并将阐明其背后的转录后机制。这些研究将首次详细评估CypA的生物学功能，阐明以ITK构象开关为特征的蛋白激酶调节的新机制，并增进对蛋白质折叠、T细胞信号、细胞因子表达和CD4T细胞分化的基础知识。有关这些基本生物过程的新信息将有助于理解哮喘和艾滋病等疾病的病因，因此对开发急需的医疗疗法至关重要。

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.