Structure/Function Analysis of TOX, a Key Regulator of NK Cell Development

NK 细胞发育的关键调节因子 TOX 的结构/功能分析

基本信息

批准号：
8702947
负责人：
JONATHAN G KAYE
金额：
$ 25.05万
依托单位：
CEDARS-SINAI MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-02-01 至 2016-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8702947
关键词：
Acute Address Affect Affinity Amino Acid Sequence Binding Biological Assay Biology Bone Marrow Bone Marrow Cell Transplantation Bone Marrow Transplantation Boxing CD4 Positive T Lymphocytes Cell Differentiation process Cell Line Cell Lineage Cell surface Cells Cellular biology Chromatin DNA DNA Binding DNA Binding Domain DNA Minor Groove Binding Data Development Employee Strikes Failure Family Future Gene Expression Genomics Goals HMG-Box HMGB Proteins Helper-Inducer T-Lymphocyte Human Immune Immune system In Vitro Laboratories Lymphoid Tissue Measures Molecular Conformation Molecular Mechanisms of Action Mus Mutagenesis Mutation Natural Killer Cells Nuclear Protein Organogenesis Pattern Physiological Play Population Property Protein Family Protein Region Proteins Regulation Regulator Genes Reporting Research Role Series Shapes Site Solutions Stem cells Structure Structure-Activity Relationship Study models T-Lymphocyte T-Lymphocyte and Natural Killer Cell Tertiary Protein Structure Testing Therapeutic Thymocyte Selection Time Viral Work base cancer immunotherapy cell type complex biological systems data modeling follow-up immune function in vivo inhibitor/antagonist innovation insight lymph nodes member mutant novel novel strategies progenitor programs protein function public health relevance reconstitution research study residence restoration small molecule structural biology tool tumor

项目摘要

DESCRIPTION (provided by applicant): The long-term goal of this work is to understand how nuclear protein TOX regulates development and function of the immune system. This protein's amino acid sequence is 94% identical between mice and humans, suggesting a highly conserved function. Dr. Kaye's laboratory has previously demonstrated that many cell types of the immune system fail to develop in TOX-deficient mice, including CD4 T lymphocytes, natural killer (NK) cells, and a specialized cell type that is required for the lymph node organogenesis. This striking biology adds particular significance to this study. We focus here on the TOX DNA binding HMG-box domain and its role in regulating gene expression during NK cell development. This is a highly evolutionarily conserved region of the protein, and one that defines the four-member TOX subfamily of proteins. NK cells, innate immune cells with cytolytic activity, play important roles in anti-viral and tumor surveillance immune functions. Understanding the regulation of NK cell development has significance for potential enhancement of immune reconstitution following bone marrow transplantation and for development of cell-based immunotherapies for cancer. The major aims of this work are (1) to test a novel hypothesis that the TOX HMG-box domain undergoes conformational change upon binding to DNA, based on the crystal structure of this domain as determined by us, (2) to determine how targeted mutations of this protein domain influence DNA binding, and (3) to determine how such mutations alter the function of the protein. We have a two-pronged approach to assess function of the protein as a regulator of gene expression. First, we have developed an innovative assay that relies on the ability of TOX to upregulate expression of a cell surface marker in an easily transfectable cell line, enabling a relatively simple quantitative assay for function. Second, we will use bone marrow progenitor cells deficient in TOX to measure restoration of NK cell development, and gene expression, upon expression of mutant forms of the TOX protein. Together, these studies will provide key insights into structure-function relationships of this protein as it relates NK cell development and form the basis for future development of conformation- dependent small molecule inhibitors or activators of the TOX-family of proteins as probes and therapeutics.

描述（由申请人提供）：这项工作的长期目标是了解核蛋白毒素如何调节免疫系统的发展和功能。该蛋白质的氨基酸序列在小鼠和人类之间相同94％，表明具有高度保守的功能。 Kaye博士的实验室先前已经证明，许多免疫系统的细胞类型无法在缺陷型小鼠中发展，包括CD4 T淋巴细胞，天然杀伤（NK）细胞以及淋巴结器官所需的专用细胞类型。这种引人注目的生物学为这项研究增添了重要意义。我们在这里专注于TOX DNA结合HMG-box结构域及其在调节NK细胞发育过程中基因表达中的作用。这是蛋白质的高度进化保守区域，也是蛋白质的四成员托克斯亚科。 NK细胞，具有胞溶活性的先天免疫细胞，在抗病毒和肿瘤监测免疫功能中起重要作用。了解NK细胞发育的调节对于骨髓移植后免疫重建的潜在增强和开发基于细胞的癌症的免疫疗法具有重要意义。这项工作的主要目的是（1）测试一个新的假设，即托克斯HMG-box域在与DNA结合时经历构象变化，基于我们确定的该结构域的晶体结构，（2）确定该蛋白质结构域的靶向突变如何影响DNA的靶向突变，并如何影响DNA结合，并（3）确定这种突变的蛋白质功能如何改变该蛋白质的功能。我们有一种两种普通的方法来评估蛋白质作为基因表达的调节剂的功能。首先，我们开发了一种创新的测定法，该测定依赖于托克斯在易于转染的细胞系中上调细胞表面标记的能力，从而实现了相对简单的定量功能分析。其次，我们将使用缺乏毒To的骨髓祖细胞来测量NK细胞发育的恢复和基因表达，以表达托克斯蛋白的突变形式。这些研究将共同提供对该蛋白质的结构功能关系的关键见解，因为它与NK细胞的发育相关联并构成了依赖构象依赖性的小分子抑制剂或蛋白质作为蛋白质作为探针和治疗疗法的构型小分子抑制剂或活化剂的基础。