Biochemistry of SAMHD1-mediated innate immunity responses

摘要 SAMHD 1是HD结构域水解酶家族的哺乳动物成员，催化 脱氧核苷酸三磷酸（dNTPs）三磷酸和非磷酸化核苷，这被认为是 是细胞dNTPs受控耗竭的主要途径。发现SAMHD 1是一种免疫 一种限制非循环免疫细胞中逆转录病毒复制并调节干扰素信号传导的因子 dNTP缺失可能是先天性抗病毒免疫的防御机制。存在这种 机制意味着SAMHD 1的酶活性必须受到先天免疫途径的控制 SAMHD 1的细胞调节是理解细胞功能的关键。 抗病毒免疫与dNTP代谢的关系。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，以阐明SAMHD 1的生化调节 决定其免疫功能。本项目将探索两种新的监管机制， 从我们的初步工作，并建立他们的贡献，SAMHD 1介导的抗逆转录病毒状态的非- 循环免疫细胞这些研究将阐明不同的分子线索和细胞的 信号通路改变了髓样和静息T细胞对HIV感染的易感性，从而阐明了 SAMHD 1在dNTP代谢和抗病毒防御界面功能的生物学意义。中 继续与Diaz-Griffero博士的实验室合作，我们将追求两个主要的具体目标。目标1 我们将探索核酸结合在SAMHD 1免疫功能中的作用，阐明SAMHD 1的结构和功能， SAMHD 1与寡核苷酸的高亲和力相互作用的生物化学决定因素，并确定 核酸种类调节SAMHD 1活性及其原因。我们的初步数据表明硫代磷酸酯 核酸中的连接可以作为一种与抗病毒相关的分子模式或第二信使， 免疫力在目标2中，我们将阐明SAMHD 1的氧化还原转化与酶促转化之间的联系机制。 活性和免疫功能。我们的初步研究表明， SAMHD 1可能提供了对活性氧（ROS）在调节先天性巨噬细胞中的新作用的认识。 抗病毒免疫我们将确定SAMHD 1的氧化还原活性半胱氨酸采样的氧化还原状态， 这些转化如何改变蛋白质的生化特性，并探索SAMHD 1 活性由细胞中特定的ROS来源和信号传导途径控制。

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.