Regulation of 2'-5'-oligoadenylate synthetase 1 (OAS1) by dsRNA

dsRNA 对 2-5-寡腺苷酸合成酶 1 (OAS1) 的调节

• 批准号: 9397149
• 负责人: Samantha Lynne Schwartz
• 金额: $ 4.4万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9397149
• 关键词: A549; Active Sites; Address; Affinity; Antiviral Agents; Binding; Binding Sites; Biochemical; Biological Assay; Biophysics; Biosensor; Catalytic Domain; Cells; Consensus Sequence; Coupled; Crystallization; Data; Detection; Deuterium; Development; Double-Stranded RNA; Enzyme Kinetics; Enzymes; Family; Foundations; Goals; Human; Hydrogen; Immobilization; Immune; In Vitro; Infection; Innate Immune System; Interferometry; Kinetics; Lead; Life Cycle Stages; Ligase; Link; Masks; Mass Spectrum Analysis; Measures; Mediating; Messenger RNA; Modeling; Molecular; Molecular Conformation; Molecular Profiling; Mutate; Mutation; Pathway interactions; Play; Point Mutation; Predisposition; Process; Production; Proteins; RNA; RNA Binding; RNA Degradation; RNA-Protein Interaction; Regulation; Ribonucleases; Ribosomal RNA; Roentgen Rays; Role; Second Messenger Systems; Signal Transduction; Site; Streptavidin; Structure; Therapeutic; Transcript; Variant; Viral; Virus; Virus Diseases; Virus Replication; Work; X-Ray Crystallography; analog; base; design; effective therapy; experimental study; extracellular; human disease; in vitro activity; insight; novel; oligoadenylate; pathogen; polymerization; sensor; tool

PROJECT SUMMARY The innate immune system is a broad set of critical intracellular and extracellular processes that limit viral infectivity. In order to provide its essential first line of defenses against pathogens, the innate immune system must be able to accurately distinguish “self” from foreign molecules. Misregulation of the innate immune system can cause increased persistence and susceptibility to viral infection and human diseases, such as interferonopathies. The 2’-5’-oligoadenylate synthetase (OAS) family of enzymes are important innate immune sensors of cytosolic double-stranded RNA (dsRNA). Attesting to the importance of the OAS/RNase L pathway, viruses have developed ways to evade OAS. Previous structural studies have revealed that dsRNA binding allosterically induces structural changes in OAS1 that reorganize the catalytic site to drive synthesis of 2’-5’- oligoadenylates from ATP. These 2’-5’-oligoadenylate secondary messengers activate a single known target, the latent ribonuclease (RNase L). Active RNase L in turn degrades viral and cellular RNA to halt viral replication. Although X-ray crystal structures have given some insight into how OAS1 is activated by dsRNA, we still understand very little about how specific features of the dsRNA contribute to the level of OAS1 activation. To address which specific features of dsRNA are required for potent OAS1 activation, we designed dsRNA hairpin variants, based on the RNA duplex used in the structural studies. Remarkably, while a single point mutation on one strand resulted in complete loss of OAS1 activity, the equivalent mutation on the opposite strand led to increased OAS1 activity. Despite these stark differences in ability to activate OAS1, both variants appear to bind OAS1 with similar affinity. Given these preliminary findings, I hypothesize that dsRNAs may contain competing OAS1 binding sites with remarkably different capacities to activate the protein in a context dependent manner. However, the molecular signatures defining these sites as activating and non-activating are unknown. The goal of this project is to determine how specific sequences in dsRNA, and their context, control regulation of OAS1 in the following two Specific Aims. Aim 1. To use complementary assays of OAS1 activity in vitro and in human cells to determine the features of dsRNA that lead to potent activation of OAS1. Aim 2. To use biochemical, biophysical, and structural approaches to define the molecular mechanism(s) by which the dsRNA hairpin variants differ in their effects upon OAS1 activation. These experiments will reveal new insights into the regulation of OAS1 by dsRNA. In doing so, I will enhance our understanding of host-pathogen interactions, such as how viruses might circumvent the OAS1/RNase L pathway by masking activating motifs to evade detection. My results will furthermore provide new insights into cellular translational control in the context of infection and potentially strengthen the foundations necessary to design effective treatments for viral infection.

项目摘要 先天性免疫系统是一组广泛的关键细胞内和细胞外过程，其限制病毒感染。 传染性为了提供其抵御病原体的基本第一道防线，先天免疫系统 必须能够准确区分"自我"和外来分子。先天免疫失调 系统可导致对病毒感染和人类疾病的持续性和易感性增加， 干扰素病2'-5'-寡腺苷酸合成酶（OAS）家族是重要的先天性免疫酶， 细胞溶质双链RNA（dsRNA）的传感器。证明了OAS/RNase L途径的重要性， 病毒已经开发出了逃避OAS的方法。以前的结构研究表明，dsRNA结合 变构诱导OAS 1的结构变化，其重组催化位点以驱动2'-5'- ATP的寡腺苷酸。这些2'-5'-寡腺苷酸第二信使激活单个已知靶标， 潜在核糖核酸酶（RNase L）。活性RNA酶L反过来降解病毒和细胞RNA， 复制的尽管X射线晶体结构已经对OAS1如何被dsRNA激活给出了一些见解， 我们仍然对dsRNA的特定特征如何影响OAS1水平知之甚少， activation.为了解决有效的OAS1激活所需的dsRNA的特定特征，我们设计了 dsRNA发夹变体，基于结构研究中使用的RNA双链体。值得注意的是， 一条链上的点突变导致OAS1活性完全丧失， 相反链导致OAS1活性增加。尽管激活OAS1的能力存在明显差异，但两者都 变体似乎以相似的亲和力结合OAS 1。根据这些初步发现，我假设 dsRNA可能含有竞争性OAS 1结合位点，这些位点具有显著不同的激活OAS 1的能力。 蛋白质以依赖于上下文的方式。然而，将这些位点定义为激活位点的分子特征 和非活化的是未知的。该项目的目标是确定dsRNA中的特定序列， 及其背景下，OAS1的控制监管有以下两个具体目标。目标1.使用互补 体外和人细胞中的OAS 1活性测定，以确定导致有效的 激活OAS1。目标2.使用生物化学、生物物理学和结构方法来定义分子 dsRNA发夹变体在其对OAS 1活化的作用上不同的机制。这些 这些实验将揭示dsRNA对OAS1调控的新见解。为此，我将加强我们的 了解宿主-病原体相互作用，例如病毒如何绕过OAS1/RNase L 通过掩盖激活基序来逃避检测。我的研究结果将进一步提供新的见解， 感染背景下的细胞翻译控制，并可能加强必要的基础， 为病毒感染设计有效的治疗方法。