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

描述（由申请人提供）：这项工作的长期目标是了解核蛋白TOX如何调节免疫系统的发育和功能。这种蛋白质的氨基酸序列在小鼠和人类之间有94%相同，这表明它具有高度保守的功能。Kaye博士的实验室先前已经证明，免疫系统的许多细胞类型无法在缺乏tox的小鼠中发育，包括CD4 T淋巴细胞、自然杀伤（NK）细胞和淋巴结器官发生所需的一种特殊细胞类型。这一惊人的生物学特性为这项研究增添了特别的意义。我们在这里重点研究TOX DNA结合HMG-box结构域及其在NK细胞发育过程中调节基因表达的作用。这是一个高度进化保守的蛋白质区域，它定义了四成员TOX蛋白亚家族。NK细胞是具有细胞溶解活性的天然免疫细胞，在抗病毒和肿瘤监视免疫功能中起着重要作用。了解NK细胞发育的调控对骨髓移植后免疫重建的潜在增强和基于细胞的癌症免疫疗法的发展具有重要意义。这项工作的主要目的是：(1)测试一个新的假设，即TOX HMG-box结构域在与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结合，以及(3)确定这种突变如何改变蛋白质的功能。我们有一种双管齐下的方法来评估蛋白质作为基因表达调节器的功能。首先，我们开发了一种创新的检测方法，该方法依赖于TOX在易于转染的细胞系中上调细胞表面标记物表达的能力，从而实现相对简单的定量分析

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.