Generation of an ITK Biosensor Tool Box

描述(由申请人提供)：胸腺来源(T)淋巴细胞的激活在对病原体的免疫反应、自身免疫和过敏反应中至关重要。T细胞通过其抗原受体(TCR)激活涉及一个复杂的生化过程，该过程主要依赖于蛋白酪氨酸激酶和其他信号效应器。其中一种被称为诱导型T细胞激酶(ITK)的酪氨酸激酶在T细胞的发育和激活过程中起着至关重要的作用。ITK的完整结构尚未解决。然而，通过对ITK的孤立结构域的核磁共振分析，已经获得了对ITK结构的有价值的见解。这一分析预测了ITK的两种稳定构象。一种是SH3结构域与上游富含脯氨酸区域结合形成的分子内折叠，另一种是SH2和SH3结构域相互作用导致的相互二聚化。前一种结构在相对较低的浓度下有利，而后者在相对较高的浓度下是稳定的。这些预测的结构还没有在相关的体内系统中得到证实。因此，在这个应用中，我们建议通过生成一组生物传感器构建来测试基于核磁共振的ITK结构预测，这些构建物在ITK的氨基或羧基末端或两者都表达嵌合的ITK分子与青色荧光蛋白(CFP)或黄色荧光蛋白(YFP)。这些结构将在T细胞中表达，并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，还是两者兼有。鉴于我们以前的发现，在静息状态下，ITK存在于细胞质中，当TCR诱导的激活移位到T细胞-APC接触部位时，我们假设静息状态ITK主要以分子内构象(相对较低的浓度)存在于细胞质中，但当TCR参与时，当ITK移位到接触部位时，分子间二聚化(头到头或头到尾)将以一种时间依赖的方式取代折叠的构象。公共卫生相关性：疾病的发病机制具有免疫学基础，通常涉及功能异常的激活淋巴细胞。被称为酪氨酸激酶的细胞内分子是淋巴细胞激活的关键调节因素。我们建议在这里研究的可诱导T细胞激酶(ITK)代表了其中一种酪氨酸激酶。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。我们的数据将使我们更好地理解ITK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.