Generation of an ITK Biosensor Tool Box

描述（由申请人提供）：胸腺源性(T)淋巴细胞的激活在对病原体、自身免疫和过敏反应的免疫反应中至关重要。T细胞通过其抗原受体（TCR）激活涉及一个复杂的生化事件过程，该生化事件严重依赖于蛋白酪氨酸激酶和其他信号效应器。其中一种酪氨酸激酶被称为诱导性T细胞激酶（ITK），已被证明在T细胞的发育和激活中都是至关重要的。ITK的完整结构还没有解决。然而，对ITK的结构有价值的见解已经通过对其孤立区域的核磁共振分析获得。该分析预测了ITK的两种稳定构象。一种是由于SH3结构域与上游富含脯氨酸的区域结合而产生的分子内折叠，而另一种是由于SH2和SH3结构域相互作用而产生的相互二聚化。前一种结构在相对低的浓度下是有利的，而后一种结构在相对高浓度下是稳定的。这些预测的结构尚未在相关的体内系统中得到证实。因此，在本应用中，我们建议通过生成一组生物传感器构建体来测试基于核磁共振的ITK结构预测，这些生物传感器构建体在ITK的氨基端或羧基端表达与青色荧光蛋白（CFP）或黄色荧光蛋白（YFP）嵌合的ITK分子。这些结构将在T细胞中表达，通过荧光共振能量转移（FRET）分析，我们将评估ITK是发生在分子内折叠还是分子间二聚化，还是两者兼而有。鉴于我们之前的研究结果，在静息状态下ITK存在于细胞质中，在TCR诱导的激活下易位到T细胞- apc接触位点，我们假设ITK在静息状态下主要在细胞质中（相对低浓度）处于分子内构象，但在TCR作用下，当ITK易位到接触位点时，分子间二聚化（头对头或头对尾）将以时间依赖的方式取代折叠构象。公共卫生相关：声明发病机制有免疫学基础的疾病通常涉及功能异常的活化淋巴细胞。被称为酪氨酸激酶的细胞内分子是淋巴细胞活化的关键调节因子。诱导性T细胞激酶（ITK），我们建议在这里研究，代表这些酪氨酸激酶之一。我们建议研究ITK在细胞内的行为方式，并将其与来自体外实验的信息进行比较。我们的数据将有助于更好地理解ITK的结构和功能之间的关系，并提高我们设计方法来控制ITK和其他类似分子在疾病条件下的作用的能力。

DESCRIPTION (provided by applicant): Activation of thymus-derived (T) lymphocytes is critical in the immune response to pathogens, autoimmunity, and in allergic responses. Activation of T cells through their antigen receptors (TCR) involves a complex process of biochemical events critically dependent on protein tyrosine kinases among other signaling effectors. One such tyrosine kinase known as the Inducible T cell kinase (ITK) has been shown to be critical in both T cell development and activation. The complete structure of ITK has not been resolved. However, valuable insights into the structure of ITK have been obtained by NMR analysis of its isolated domains. This analysis predicts two stable conformations of ITK. One is an intramolecular fold resulting from the binding of the SH3 domain to an upstream proline-rich region, whereas the other is a reciprocal dimerization due to the interaction of the SH2 and SH3 domains. The former structure is favored at relatively low concentrations whereas the latter is stable at relatively high concentrations. These predicted structures have not been confirmed in a relevant in vivo system. Therefore, in this application we propose to test the NMR-based predictions of the structure of ITK by generating a panel of biosensor constructs that express chimeric ITK molecules with Cyan Fluorescence Protein (CFP) or Yellow Fluorescence Protein (YFP) at either the amino- or carboxyl-terminus of ITK, or both. These constructs will be expressed in T cells and by using Fluorescence Resonance Energy Transfer (FRET) analysis we will assess whether ITK occurs in an intramolecular fold or an intermolecular dimerization or both. In view of our previous findings that at the resting state ITK is found in the cytoplasm, and upon TCR-induced activation translocates to the T cell-APC contact site, we hypothesize that ITK will be primarily in an intramolecular conformation in the cytoplasm (relatively low concentration) at the resting state, but upon TCR engagement, when ITK translocates to the contact site, intermolecular dimerization (head to head or head to tail) will replace the folded conformation in a time-dependent fashion. Public Health Relevance: Statement Diseases whose pathogenesis has an immunological basis often involve activated lymphocytes that function abnormally. Intracellular molecules known as Tyrosine Kinases are key regulators of lymphocyte activation. The Inducible T cell Kinase (ITK), we propose to study here, represents one of these tyrosine kinases. We propose to study the way ITK behaves inside cells and compare it to information that has been derived from in vitro experiments. Our data will result in better understanding of the relationship between the structure and function of ITK and improve our ability to design ways to control the action of ITK, and other similar molecules, in disease conditions.