The adaptor protein Crk in immune responses

免疫反应中的接头蛋白 Crk

• 批准号: 10084254
• 负责人: Dongfang Liu
• 金额: $ 50.69万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-02-13 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10084254
• 关键词: Actins; Adaptor Signaling Protein; Affect; Algorithms; Apoptosis; Automobile Driving; Biochemical; Biological Models; Birth; Cell Line; Cell physiology; Cell-Mediated Cytolysis; Cells; Chronic; Chronic Disease; Clinical; Complex; Custom; Cytoplasmic Granules; Cytoskeleton; Data; DiGeorge Syndrome; Disease; Energy Transfer; Event; Fluorescence; Generations; Goals; Host Defense; Human; Image; Imaging Techniques; Imaging technology; Immune; Immune response; Immune system; Immunity; Immunologic Deficiency Syndromes; Immunologics; Immunology; Immunoprecipitation; Infection; Integrins; Killer Cells; Knockout Mice; Knowledge; Lipid Bilayers; Lytic; Malignant Neoplasms; Memory; Microscopy; Modeling; Molecular; Molecular Conformation; Murid herpesvirus 1; NK Cell Activation; Natural Killer Cells; Outcome; Pathway interactions; Patients; Phosphorylation; Phosphotransferases; Play; Protein Family; Proteins; Receptor Signaling; Regulation; Research; Resolution; Role; Shapes; Signal Transduction; Signaling Molecule; Small Interfering RNA; Structure; Synapses; System; Techniques; Testing; Tissues; Transfection; Viral Cancer; Virus; Virus Diseases; Work; antibody-dependent cell cytotoxicity; cell killing; cell mediated immune response; conditional knockout; congenital immunodeficiency; cytotoxic; experimental study; fighting; immune function; immunological synapse; immunoregulation; in vivo; kinase inhibitor; live cell microscopy; molecular imaging; natural antibodies; novel; novel imaging technique; population based; proto-oncogene protein c-crk; receptor; reconstitution; recruit; single molecule; spatiotemporal

Project Summary Natural killer (NK) cells play an important role in the human immune response to infection and malignancy. How these cells effectively distinguish between diseased and healthy tissue is one of the key unsolved problems in immunology today. The proposed work seeks to identify the mechanism(s) by which the small adaptor protein CT10 regulator of kinase (Crk), and its phosphorylation, control NK cell activation and inhibition by using both human NK cells and novel NK cell-specific conditional knockout mice. The long-term goal is to use this knowledge and the novel imaging techniques developed herein to uncover the molecular basis of NK cell activation and inhibition, and to develop new treatments for human primary immunodeficiency diseases and chronic diseases such as cancer and viral infection. NK cells kill target cells through the polarized release of lytic granules through a specialized region of cell-cell contact known as the immunological synapse (IS). Through previous studies of the cytotoxic (Liu, D. et al., Immunity, 2009, Cover Article) and inhibitory (Liu, D. et al., Immunity, 2012) IS, we discovered that Crk plays an essential upstream role at the IS, influencing signaling events required for both activation and inhibition. The molecular mechanisms underlying this dual role, however, remain unclear. We hypothesize that receptor-driven, integrin-influenced phosphorylation of Crk acts as a molecular switch, driving a conformational change, which in turn determines Crk's ability to interact with critical downstream signaling molecules and ultimately shapes the actin cytoskeleton into a functional IS. Guided by strong preliminary data, we will test these hypotheses via three Specific Aims: 1) Define the precise molecular mechanisms by which Crk-like (CrkL) protein controls NK cell activation and inhibition. The proposed work will bring cutting-edge single molecule imaging technology to the field of NK cell research. Experiments will determine where and when Crk is phosphorylated at the IS, as well as how it interacts with key receptors, signaling molecules, and the actin cytoskeleton; 2) Determine the role of CrkL in NK cells from patients with partial DiGeorge syndrome (pDGS). By studying one of the most common (1 in 3,000 births) immunodeficiency diseases, pDGS (mainly caused by CrkL haploinsufficiency), we will determine how loss of CrkL function affects NK cell-mediated cytotoxicity; 3) Determine whether Crk or CrkL is required for NK function in vivo. Leveraging novel NK cell-specific Crk knockout mice that we have already generated, we will determine Crk's in vivo roles in NK cell-mediated immune responses to viral infection and cancer, including a newly identified role in memory NK cell generation. The proposed work involves key signaling players and regulatory mechanisms and generates a novel model system in which to determine Crk's role as a master regulatory molecule. It is broadly relevant with direct clinical implications for the treatment of primary immunodeficiency diseases, viral infections, and cancer. !

项目摘要 自然杀伤（NK）细胞在人类对感染和恶性肿瘤的免疫应答中起着重要作用。 这些细胞如何有效地区分病变和健康组织是尚未解决的关键问题之一 免疫学的问题。拟议的工作旨在确定小企业 衔接蛋白CT 10调节激酶（Crk）及其磷酸化，控制NK细胞的活化和抑制 通过使用人类NK细胞和新型NK细胞特异性条件性敲除小鼠。长期目标是 利用这些知识和本文开发的新成像技术来揭示NK的分子基础 细胞激活和抑制，并开发用于人类原发性免疫缺陷疾病的新疗法 以及慢性疾病如癌症和病毒感染。NK细胞通过极化释放杀伤靶细胞 通过一个称为免疫突触（IS）的细胞-细胞接触的专门区域。 通过先前的细胞毒性研究（Liu，D.例如，Immunity，2009，封面文章）和抑制性（Liu，D. 例如，Immunity，2012）IS，我们发现Crk在IS中起着重要的上游作用，影响 激活和抑制所需的信号事件。这种双重的分子机制 然而，作用仍然不明确。我们假设受体驱动的整合素影响的磷酸化， Crk充当分子开关，驱动构象变化，这反过来又决定了Crk的能力， 与关键的下游信号分子相互作用，并最终将肌动蛋白细胞骨架塑造成 功能性IS在强有力的初步数据的指导下，我们将通过三个具体目标来测试这些假设：1） 定义Crk样（CrkL）蛋白控制NK细胞活化的精确分子机制 和抑制。这项拟议中的工作将把尖端的单分子成像技术带到生物医学领域。 NK细胞研究。实验将确定Crk在IS磷酸化的位置和时间，以及如何磷酸化。 它与关键受体、信号分子和肌动蛋白细胞骨架相互作用; 2）确定CrkL的作用 部分DiGeorge综合征（pDGS）患者的NK细胞。通过研究一个最常见的（1 在3,000例新生儿中）免疫缺陷疾病，pDGS（主要由CrkL单倍不足引起），我们将 确定CrkL功能的丧失如何影响NK细胞介导的细胞毒性; 3）确定Crk或CrkL 是体内NK功能所必需的。利用我们现有的新型NK细胞特异性Crk敲除小鼠， 我们将确定Crk在NK细胞介导的对病毒的免疫应答中的体内作用。 感染和癌症，包括新发现的记忆NK细胞生成中的作用。拟议工作 涉及关键的信号传导参与者和调节机制，并产生了一个新的模型系统， 确定Crk作为主调节分子的作用。它与以下直接临床意义广泛相关： 治疗原发性免疫缺陷疾病、病毒感染和癌症。 !