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%的相同性，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，许多免疫系统的细胞类型在TOX缺陷小鼠中无法发育，包括CD 4 T淋巴细胞，自然杀伤（NK）细胞和淋巴结器官形成所需的专门细胞类型。这种惊人的生物学特性为这项研究增添了特别的意义。在这里，我们专注于TOX DNA结合HMG盒结构域及其在NK细胞发育过程中调节基因表达的作用。这是蛋白质的高度进化保守区域，并且是定义蛋白质的四成员TOX亚家族的区域。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发挥重要作用。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）基于我们确定的TOX HMG-盒结构域的晶体结构，验证该结构域在与DNA结合时发生构象变化的新假设;（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）确定此类突变如何改变蛋白质的功能。我们有一个双管齐下的方法来评估蛋白质作为基因表达调节因子的功能。首先，我们已经开发了一种创新的测定法，该测定法依赖于TOX在容易转染的细胞系中上调细胞表面标志物表达的能力，使得能够进行相对简单的定量分析。 功能测定。其次，我们将使用TOX缺陷的骨髓祖细胞来测量在表达突变形式的TOX蛋白后NK细胞发育和基因表达的恢复。总之，这些研究将提供对这种蛋白质的结构-功能关系的关键见解，因为它涉及NK细胞发育，并形成未来开发作为探针和治疗剂的TOX家族蛋白质的构象依赖性小分子抑制剂或激活剂的基础。

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.