Generation of an ITK Biosensor Tool Box

ITK 生物传感器工具箱的生成

• 批准号: 7842629
• 负责人: CONSTANTINE D TSOUKAS
• 金额: $ 18.44万
• 依托单位: SAN DIEGO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-15 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7842629
• 关键词: Actins; Activated Lymphocyte; Antigen Receptors; Antigen-Presenting Cells; Autoimmunity; Binding; Biochemical Process; Biosensor; Boxing; Cell Communication; Cells; Complex; Cytoplasm; Cytoskeleton; Data; Dimerization; Disease; Environment; Event; Family; Fluorescence; Fluorescence Resonance Energy Transfer; Generations; Head; Image Analysis; Immune response; In Vitro; Interphase Cell; Lymphocyte Activation; Lymphocyte Function; Modification; Molecular Conformation; Nuclear Magnetic Resonance; Pathogenesis; Phosphorylation; Phosphotransferases; Play; Production; Proline-Rich Domain; Protein Tyrosine Kinase; Proteins; Regulation; Rest; Role; SH3 Domains; Signal Transduction; Site; Structure; System; T-Cell Activation; T-Cell Development; T-Lymphocyte; T-bet protein; Tail; Testing; Thymus Gland; Time; allergic response; base; cytokine; design; dimer; emt protein-tyrosine kinase; improved; in vivo; insight; pathogen; public health relevance; research study; tool

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.

描述（由申请人提供）：胸腺来源（T）淋巴细胞的激活对于病原体的免疫反应、自身免疫和过敏反应至关重要。 T 细胞通过抗原受体 (TCR) 的激活涉及一个复杂的生化事件过程，该过程严重依赖于其他信号传导效应器中的蛋白酪氨酸激酶。一种称为诱导性 T 细胞激酶 (ITK) 的酪氨酸激酶已被证明在 T 细胞发育和激活中至关重要。 ITK的完整结构尚未解析。然而，通过对其孤立域的 NMR 分析，获得了对 ITK 结构的有价值的见解。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 结构域的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有利，而后者在相对高的浓度下稳定。这些预测的结构尚未在相关的体内系统中得到证实。因此，在本申请中，我们建议通过生成一组生物传感器构建体来测试基于 NMR 的 ITK 结构预测，这些生物传感器构建体在 ITK 的氨基末端或羧基末端或两者上表达具有青色荧光蛋白（CFP）或黄色荧光蛋白（YFP）的嵌合 ITK 分子。这些构建体将在 T 细胞中表达，并通过使用荧光共振能量转移 (FRET) 分析，我们将评估 ITK 是否发生在分子内折叠或分子间二聚化或两者兼而有之。鉴于我们之前的发现，在静息状态下，ITK存在于细胞质中，并且在TCR诱导的激活后易位至T细胞-APC接触位点，我们假设ITK在静息状态下将主要处于细胞质中的分子内构象（相对较低的浓度），但在TCR接合后，当ITK易位至接触位点时，分子间二聚化（头至 头或头到尾）将以时间依赖的方式取代折叠构象。公共卫生相关性：声明 其发病机制具有免疫学基础的疾病通常涉及功能异常的活化淋巴细胞。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。我们建议在这里研究的诱导型 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，并将其与体外实验获得的信息进行比较。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，并提高我们设计控制 ITK 和其他类似分子在疾病条件下作用的方法的能力。