Regulation of Atopic Immune Responses by the Prolyl lsomerase Cyclophillin A

描述（由申请人提供）：哮喘和其他过敏性疾病是工业化国家普遍存在的健康问题。为了开发治疗药物，减轻这些疾病的严重程度，驱动过敏的生物过程需要被定义。过敏反应是由辅助性T细胞对抗原的过度反应引发的；因此，这些细胞是新疗法的有吸引力的靶点。这项建议的长期目标是确定辅助性T细胞内控制过敏促进细胞因子如IL-4产生的特定细胞内信号事件。我们已经确定了环孢素受体亲环蛋白A （CypA）在调节辅助性T细胞反应中的重要作用。CypA可以改变邻近脯氨酸残基的肽键结构，从而通过诱导构象变化来调节蛋白质功能。缺乏CypA的小鼠会产生含有嗜酸性粒细胞和肥大细胞的炎症，这两种细胞类型在过敏反应中起着关键作用。来自这些小鼠的辅助性T细胞过量产生IL-4和其他与过敏相关的细胞因子，并表现出关键信号分子磷脂酶c - γ - 1 （PLCgl）的激活增加。已知PLCgl的调节因子是Itk，一种控制IL-4表达的酪氨酸激酶。CypA与Itk中的脯氨酸残基相互作用；这种相互作用促进Itk的自我关联，这被认为是下调Itk活性，也抑制Itk与其他促进PLCgl激活的因子之间的接触。基于这些发现，我们的中心假设是CypA作为Itk活性的抑制因子，从而限制了辅助性T细胞对IL-4的表达。为了探索这一假设，我们将追求以下具体目标：1)确定Itk与CypA相互作用和Itk自结合所需的氨基酸；生化分析将用于评估Itk和CypA中氨基酸的变化如何影响蛋白质之间的相互作用；2)确定CypA和Itk突变对功能的影响；功能改变的CypA和Itk突变蛋白将在辅助性T细胞中表达，以评估它们对IL-4表达和PLCgl激活的影响；3)确定CypA和Itk在变应性疾病中的作用；将分析CypA或Itk活性的变化如何调节哮喘小鼠模型的发展；4)确定CypA是否调节表达促过敏细胞因子的辅助性T细胞的发育；细胞培养系统将用于确定CypA的调控靶点。

DESCRIPTION (provided by applicant): Asthma and other allergic diseases are widespread health problems for industrialized nations. In order to develop therapeutic agents that lessen the severity of these diseases, the biological processes that drive allergy need to be defined. Allergic reactions are triggered by excessive helper T cell responses to antigens; thus these cells are attractive targets for new therapeutics. The long-term objective of this proposal is to define specific intracellular signaling events within helper T cells that control the production of allergy- promoting cytokines such as IL-4. We have identified an important role for the cyclosporine receptor cyclophilin A (CypA) in regulating helper T cell responses. CypA alters the structure of peptide bonds adjacent to proline residues, and may thus regulate protein function by inducing conformational changes. Mice lacking CypA develop inflammation that contains eosinophils and mast cells, two cell types that have key roles in allergic responses. Helper T cells from these mice overproduce IL-4 and other allergy- associated cytokines and show increased activation of the key signaling molecule phospholipase C-gamma1 (PLCgl). A known regulator of PLCgl is Itk, a tyrosine kinase that controls IL-4 expression. CypA interacts with a proline residue in Itk ; this interaction promotes Itk self-association, which is postulated to downregulate Itk activity, and also inhibits contacts between Itk and other factors that promote PLCgl activation. Based on these findings, our central hypothesis is that CypA functions as represser of Itk activity, thus limiting IL-4 expression by helper T cells. To explore this hypothesis, we will pursue the following specific aims: 1) Identify amino acids that are required for interaction between Itk and CypA and for Itk self- association; biochemical assays will be used to assess how amino acid changes in Itk and CypA affect protein-protein interactions; 2) Determine the effects of mutations in CypA and Itk on function; CypA and Itk mutant proteins with altered function will be expressed in helper T cells to assess their impact on IL-4 expression and PLCgl activation; 3) Determine the role of CypA and Itk in allergic disease; how changes in CypA or Itk activity modulate asthma development in mouse model for this disease will be analyzed; 4) Determine whether CypA regulates the development of helper T cells that express allergy-promoting cytokines; a cell culture system will be used to define regulatory targets for CypA.