Cyclophilin A Function in the Immune System

描述（由申请人提供）：亲环素A（CypA/Ppia）是一个18 kD，8链β-桶，具有暴露于溶剂的疏水口袋。免疫抑制药物环孢菌素抓住口袋，并与CypA产生结合和抑制钙调磷酸酶的复合表面。口袋摇篮含脯氨酸的肽，包括暴露在HIV-1衣壳上的脯氨酸，并催化肽基-脯氨酰键的顺式-反式相互转化。为了鉴定CypA的天然功能，我们产生了缺乏Ppia的小鼠。Ppia 1+小鼠发生变应性病理，伴有嗜酸性粒细胞和肥大细胞的组织浸润，以及增加的CD 4 + T辅助II型（Th 2）细胞因子产生。我们假设病理学是由Itk活性增加引起的，Itk是PLC γ上游的酪氨酸激酶（因此也是钙调神经磷酸酶），特异性促进Th 2功能。CypA通过PPIase活性位点结合Itk。Itk（P287 G）中构象异质性脯氨酸的突变破坏了CypA结合，并赋予野生型CD 4 + T细胞Th 2超活性。因此，CypA通过Itk中的调节性脯氨酸残基在不存在环孢菌素的情况下调节CD 4 + T细胞信号转导。目的1：PPia 4+小鼠表现出品系依赖性致死性。将对129/SvEv小鼠中致死性的显性抑制基因进行定位和克隆。目标二：将使用一组合理设计的CypA突变体以及在依赖于CypA PPI酶活性的酵母菌株中筛选的突变体来测试CypA肽基-脯氨酰异构酶活性对Itk和Th 2细胞因子表达的抑制的功能意义。目的3：血小板记忆性CD 4 + Th 2细胞分泌大量的IL 2。我们将确定这种表型是否也是CypA对Itk的影响的结果，并阐明其转录后机制。这些研究将首次详细评估CypA的生物学功能，阐明以Itk构象开关为特征的蛋白激酶调节的新机制，并推进蛋白折叠，T细胞信号传导，细胞因子表达，和CD 4 + T细胞分化。有关这些基本生物过程的新信息将有助于了解哮喘和艾滋病等疾病的病因，因此，对于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.