The adaptor protein Crk in immune responses

免疫反应中的接头蛋白 Crk

基本信息

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
项目类别：
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

项目摘要

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. !

项目概要自然杀伤 (NK) 细胞在人体对感染和恶性肿瘤的免疫反应中发挥着重要作用。这些细胞如何有效区分患病组织和健康组织是未解决的关键问题之一当今免疫学的问题。拟议的工作旨在确定小型企业的机制接头蛋白 CT10 激酶 (Crk) 调节因子，及其磷酸化，控制 NK 细胞的激活和抑制通过使用人类 NK 细胞和新型 NK 细胞特异性条件敲除小鼠。长期目标是利用这些知识和本文开发的新型成像技术来揭示 NK 的分子基础细胞激活和抑制，并开发人类原发性免疫缺陷疾病的新疗法以及癌症和病毒感染等慢性疾病。 NK细胞通过极化释放杀死靶细胞通过称为免疫突触 (IS) 的细胞与细胞接触的特殊区域来释放裂解颗粒。通过之前对细胞毒性（Liu, D. et al., Immunity, 2009, Cover Article）和抑制性（Liu, D. et al., 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。这种双重作用背后的分子机制然而，其作用仍不清楚。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，这反过来决定了 Crk 的能力与关键的下游信号分子相互作用，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检验这些假设：1) 定义 Crk 样 (CrkL) 蛋白控制 NK 细胞激活的精确分子机制和抑制。拟议的工作将把尖端的单分子成像技术引入到领域 NK细胞研究。实验将确定 Crk 在 IS 上何时何地被磷酸化，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的作用来自部分迪乔治综合征（pDGS）患者的 NK 细胞。通过研究最常见的一种（1 在 3,000 名新生儿中）免疫缺陷疾病、pDGS（主要由 CrkL 单倍体不足引起），我们将确定 CrkL 功能丧失如何影响 NK 细胞介导的细胞毒性； 3）判断是Crk还是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 敲除小鼠已经生成，我们将确定 Crk 在 NK 细胞介导的病毒免疫反应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。拟议的工作涉及关键信号参与者和监管机制，并生成一个新颖的模型系统，其中确定 Crk 作为主调控分子的作用。它与直接临床意义广泛相关原发性免疫缺陷疾病、病毒感染和癌症的治疗。！