Regulation of Atopic Immune Responses by the Prolyl lsomerase Cyclophillin A

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， 过敏需要定义。过敏反应是由过多的辅助性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。这项建议的长远目标是 定义辅助性T细胞内控制过敏产生的特定细胞内信号事件- 促进细胞因子如IL-4。我们已经确定了环孢菌素受体的重要作用， 亲环素A（CypA）在调节辅助性T细胞应答中的作用。CypA改变了肽键的结构 与脯氨酸残基相邻，因此可以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，这两种细胞类型具有 在过敏反应中的关键作用。这些小鼠的辅助T细胞过度产生IL-4和其他过敏反应- 并显示关键信号分子磷脂酶C-γ 1的活化增加 （PLCgl）。已知的PLCgl调节剂是Itk，其是控制IL-4表达的酪氨酸激酶。CypA相互作用 与Itk中的脯氨酸残基;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的其它因子之间的接触 activation.基于这些发现，我们的中心假设是CypA作为Itk活性的阻遏物发挥作用， 从而限制辅助T细胞表达IL-4。为了探索这一假设，我们将探讨以下问题 具体目的：1）鉴定Itk和CypA之间相互作用以及Itk自身所需的氨基酸， 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突变对功能的影响; CypA和Itk 具有改变的功能的突变蛋白将在辅助性T细胞中表达，以评估它们对IL-4的影响。 3）确定CypA和Itk在过敏性疾病中的作用; 分析CypA或Itk活性调节哮喘小鼠模型中哮喘发展的作用; 4） 确定CypA是否调节表达过敏促进因子的辅助性T细胞的发育 细胞因子;细胞培养系统将用于定义CypA的调节靶标。

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 onIL-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.