Biochemistry of SAMHD1-mediated innate immunity responses

摘要 SAMHD1 是 HD 结构域水解酶家族的哺乳动物成员，催化水解 脱氧核苷酸三磷酸（dNTP）转化为三磷酸和非磷酸化核苷，这被认为 是控制细胞 dNTP 消耗的主要途径。发现 SAMHD1 具有免疫功能 限制逆转录病毒在非循环免疫细胞中复制并调节干扰素信号传导的因素揭示 dNTP 耗尽可能作为先天抗病毒免疫的防御机制。存在这样的 机制表明 SAMHD1 的酶活性必须受到先天免疫途径的控制 SAMHD1 的细胞调节是理解功能的关键 抗病毒免疫与 dNTP 代谢之间的关系。在这里描述的研究中，我们将使用 我的实验室开发了独特的实验工具来阐明 SAMHD1 的生化调节 决定了其免疫功能。该项目将探索已经出现的两种新颖的监管机制 从我们的初步工作中，确定它们对 SAMHD1 介导的非逆转录病毒状态的贡献 循环免疫细胞。这些研究将揭示不同的分子线索和细胞如何以及可能为何 信号通路改变骨髓和静息 T 细胞对 HIV 感染的易感性，从而阐明 SAMHD1 在 dNTP 代谢和抗病毒防御界面功能的生物学意义。在一个 继续与 Diaz-Griffero 博士实验室合作，我们将追求两个主要的具体目标。目标 1 我们将探讨核酸结合在 SAMHD1 免疫功能中的作用，阐明结构和 SAMHD1 与寡核苷酸高亲和力相互作用的生化决定因素，并确定什么 核酸种类调节 SAMHD1 活性及其原因。我们的初步数据表明硫代磷酸酯 核酸中的连接可能充当危险相关的分子模式或抗病毒药物中的第二信使 免疫力。在目标 2 中，我们将阐明将 SAMHD1 的氧化还原转化与酶促反应联系起来的机制。 蛋白质的活性和免疫功能。我们的初步研究表明氧化还原调节 SAMHD1 可能有助于深入了解活性氧 (ROS) 在调节先天性中的新兴作用 抗病毒免疫力。我们将确定 SAMHD1 的氧化还原活性半胱氨酸采样哪些氧化还原态， 这些转变如何改变蛋白质的生化特性并探索 SAMHD1 是否 活性由细胞中特定的 ROS 来源和信号通路控制。

ABSTRACT SAMHD1, a mammalian member of the HD-domain hydrolase family of enzymes, catalyzes hydrolysis of deoxynucleotides triphosphates (dNTPs) to triphosphate and unphosphorylated nucleosides, which is thought to be the main pathway for controlled depletion of cellular dNTPs. Discoveries that SAMHD1 is an immune factor that restricts retroviral replication in non-cycling immune cells and regulates interferon signaling revealed that dNTP depletion may act as a defense mechanism of innate antiviral immunity. Existence of such mechanism implies that the enzymatic activity of SAMHD1 must be controlled by pathways of innate immune sensing and response, and that cellular regulation of SAMHD1 is key to understanding the functional relationship between antiviral immunity and dNTP metabolism. In the studies described here we will use unique experimental tools developed by my laboratory to elucidate how biochemical regulation of SAMHD1 determines its immune function. This project will explore two novel regulatory mechanisms that have emerged from our preliminary work and establish their contribution to the SAMHD1-mediated anti-retroviral state in non- cycling immune cells. The studies will shed light on how and possibly why different molecular clues and cellular signaling pathways alter susceptibility of myeloid and resting T cells to HIV infection, and thus elucidate the biological significance of SAMHD1 function at the interface of dNTP metabolism and antiviral defense. In a continued collaboration with the laboratory of Dr. Diaz-Griffero we will pursue two major specific aims. In Aim 1 we will explore the role of nucleic acid binding in the immune function of SAMHD1, elucidate structural and biochemical determinants of high-affinity interaction of SAMHD1 with oligonucleotides and determine what nucleic acid species regulate SAMHD1 activity and why. Our preliminary data suggest that phosphorothioate linkages in nucleic acids may act as a danger-associated molecular pattern or a second messenger in antiviral immunity. In Aim 2 we will elucidate the mechanism linking redox transformations of SAMHD1 to the enzymatic activity and the immune function of the protein. Our preliminary studies suggest that redox regulation of SAMHD1 may offer insight into the emerging role of reactive oxygen species (ROS) in modulating innate antiviral immunity. We will determine what redox states are sampled by the redox-active cysteines of SAMHD1, how these transformations alter the biochemical properties of the protein and explore whether SAMHD1 activity is controlled by specific sources of ROS and signaling pathways in the cell.