Generation of an ITK Biosensor Tool Box

ITK 生物传感器工具箱的生成

• 批准号: 7661189
• 负责人: CONSTANTINE D TSOUKAS
• 金额: $ 22.18万
• 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 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7661189
• 关键词: Actins; Activated Lymphocyte; Allergic; 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; base; cytokine; design; dimer; emt protein-tyrosine kinase; improved; in vivo; insight; pathogen; public health relevance; research study; response; 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（由申请方提供）：胸腺衍生（T）淋巴细胞的活化在对病原体的免疫应答、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）的活化涉及生物化学事件的复杂过程，其在其它信号传导效应物中关键地依赖于蛋白酪氨酸激酶。一种被称为诱导性T细胞激酶（ITK）的酪氨酸激酶已被证明在T细胞发育和活化中至关重要。ITK的完整结构尚未解决。然而，有价值的见解ITK的结构已获得通过NMR分析其孤立的域。该分析预测了ITK的两种稳定构象。一个是由SH 3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，而另一个是由于SH 2和SH 3结构域的相互作用而导致的相互二聚化。前一种结构在相对低的浓度下是有利的，而后者在相对高的浓度下是稳定的。这些预测的结构尚未在相关的体内系统中得到证实。因此，在本申请中，我们提出通过产生一组生物传感器构建体来测试ITK结构的基于NMR的预测，所述生物传感器构建体表达在ITK的氨基末端或羧基末端或两者处具有青色荧光蛋白（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，并且通过使用荧光共振能量转移（FRET）分析，我们将评估ITK是否以分子内折叠或分子间二聚化或两者发生。鉴于我们先前的发现，即在静息状态下，ITK存在于细胞质中，并且在TCR诱导的活化后易位至T细胞-APC接触位点，我们假设ITK将主要在细胞质中处于分子内构象（相对低的浓度），但在TCR接合时，当ITK易位至接触位点时，分子间二聚化（头对头或头对尾）将以时间依赖性方式取代折叠构象。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剂。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。我们的数据将使我们更好地理解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。