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。为了开发减轻这些疾病严重程度的治疗剂，需要定义驱动过敏的生物过程。过敏反应是由过量的辅助性T细胞对抗原的反应引发的;因此这些细胞是新疗法的有吸引力的靶点。该提议的长期目标是确定辅助性T细胞内控制促变态反应细胞因子如IL-4产生的特异性细胞内信号传导事件。我们已经确定了环孢菌素受体亲环素A（CypA）在调节辅助性T细胞反应中的重要作用。CypA改变了脯氨酸残基附近的肽键结构，因此可能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，这两种细胞在过敏反应中起关键作用。来自这些小鼠的辅助T细胞过度产生IL-4和其他过敏相关细胞因子，并显示关键信号分子磷脂酶C-γ 1（PLCg 1）的活化增加。已知的PLCgl调节剂是Itk，其是控制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自缔合，这被假定为下调Itk活性，并且还抑制Itk与促进PLCgl活化的其他因子之间的接触。基于这些发现，我们的中心假设是CypA作为Itk活性的阻遏物起作用，从而限制辅助性T细胞表达IL-4。为了探索这一假说，我们将追求以下具体目标：1）鉴定Itk和CypA之间相互作用以及Itk自缔合所需的氨基酸;生物化学测定将用于评估Itk和CypA中的氨基酸变化如何影响蛋白质-蛋白质相互作用; 2）确定CypA和Itk中的突变对功能的影响;具有改变的功能的CypA和Itk突变蛋白将在辅助T细胞中表达以评估它们对IL-4表达和PLC gl活化的影响; 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该疾病的小鼠模型中的哮喘发展; 4）确定CypA是否调节表达促变应性细胞因子的辅助性T细胞的发展;将使用细胞培养系统来确定CypA的调节靶。

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.