Structure and Function of Kinase Family Receptors Regulating Translation

调节翻译的激酶家族受体的结构和功能

• 批准号: 10237208
• 负责人: Alexei Korennykh
• 金额: $ 31.51万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10237208
• 关键词: Address; Ankyrin Repeat; Antiviral Agents; Area; Bacillus anthracis; Bacteria; Binding; Biochemical; Biochemistry; Cancer Biology; Cells; Cellular Stress; Cellular biology; Cleaved cell; Code; Complex; Coupled; Cryoelectron Microscopy; Crystallization; Dangerousness; Data; Dependence; Disease; Double-Stranded RNA; Elements; Enzymes; Eukaryotic Cell; Exhibits; FRAP1 gene; Family; Foundations; Geographic state; Goals; Heat shock proteins; Human; Inflammatory; Influenza; Initiator Codon; Interferons; Investigation; Knowledge; L Forms; Laboratories; Malignant Neoplasms; Mammalian Cell; Mediating; Memory; Messenger RNA; Metabolic; Metabolic syndrome; Metastatic Prostate Cancer; Methods; Molecular; Molecular Biology; N-terminal; Nutrient; Obesity; Pathologic Processes; Pathway interactions; Phosphotransferases; Play; Protein Biosynthesis; Protein Family; Protein Kinase; Proteins; Publishing; Regulation; Research; Ribonucleases; Ribosomes; Role; Signal Transduction; Site; Starvation; Stress; Structure; Surface; Terminator Codon; Testing; Tissues; Transfer RNA; Translating; Translational Regulation; Translations; Virus; West Nile virus; Work; anti-cancer; base; biological adaptation to stress; cancer cell; carcinogenesis; cohesion; dimer; human tissue; innovation; insight; lipid biosynthesis; mRNA Decay; migration; neoplastic; novel; paralogous gene; pathogenic virus; preference; prevent; programs; prostate cancer cell; prostate cancer metastasis; proteostasis; public health relevance; receptor; receptor function; response; sensor; stress kinase; therapeutic target; tool; transcriptome sequencing; tumor metabolism

Project Summary This proposal investigates structural and functional mechanisms of two related receptors from the family of protein kinases, RNase L (Aims 1, 2) and Gcn2 (Aim 3). These receptors are ubiquitously expressed in human tissues and regulate protein synthesis (translation) to mitigate cellular stresses. RNase L senses stress caused by double-stranded RNA (dsRNA), whereas Gcn2 detects stress caused by nutrient limitation. Mammalian cells are highly sensitive to the presence of dsRNA. DsRNA molecules are normally rare, but are widespread in virus-infected cells and in cancer cells due to upsurge of dsRNA-rich endogenous repeat elements, which makes studying dsRNA responses critically important. Yet, we have a poor understanding of the dsRNA-RNase L pathway. Our goal in Aims 1 and 2 of this proposal is to understand the molecular mechanism regulating RNase L generally and to extensively characterize the novel mechanism of translational regulation by RNase L, discovered by our laboratory. This work is biomedically significant because RNase L, in addition to having anticancer and antiviral roles, is an antilipogenic receptor that could be important for treating and preventing infectious, neoplastic, and metabolic illnesses. In Aim 1 we will elucidate the mechanism of RNase L regulation by obtaining the structure of RNase L in its latent state. Our hypothesis is that RNase L forms a defined latent structure that restricts uncontrollable RNase L signaling to protect healthy tissues. We will test this hypothesis using structural analysis by cryo electron microscopy (cryo-EM). In Aim 2, we will use cell biology, biochemistry and an innovative RNA-seq approach developed in our group to establish the mechanism of translation regulation by RNase L. In 2019, we published our discovery that RNase L performs endonucleolytic cleavage of actively translating mRNAs as a strategy to control cell-wide protein synthesis. Preliminary data in this proposal provide further insights into this mRNA decay and show that RNase L exhibits a distinctive preference for mRNA coding regions, leading us to the hypothesis that the action of RNase L is coupled to translation. We will test this hypothesis thoroughly in our investigation. In Aim 3, we extend our work to a related receptor regulating global translation, Gcn2. Gcn2 is a mechanistically poorly understood stress kinase located proximally to RNase L in the human kinome. Gcn2 is important for proteostasis, memory function, and cancer metabolism. Gcn2 serves as a sensor of starvation that, upon binding uncharged tRNAs, inhibits global protein synthesis to mitigate nutrient deficits. The mechanism of Gcn2 activation is poorly understood due to the absence of structural information about its regulatory tRNA-sensing domain. We will employ cryo-EM to determine the structure of the sensor domain. Our research will provide new knowledge and contribute to building a comprehensive mechanistic understanding of stress kinases regulating translation. Our work will also contribute a new method, LRtcB RNA-seq, as a tool for studying mammalian mRNA decay.

项目摘要 本研究旨在探讨两种相关受体的结构和功能机制， 蛋白激酶家族，RNase L（目的1，2）和Gcn 2（目的3）。这些受体普遍表达于 并调节蛋白质合成（翻译）以减轻细胞压力。RNase L传感器 Gcn 2检测由双链RNA（dsRNA）引起的胁迫，而Gcn 2检测由营养限制引起的胁迫。 哺乳动物细胞对dsRNA的存在高度敏感。双链RNA分子通常是罕见的， 但由于富含dsRNA的内源性重复序列的激增， 这使得研究dsRNA反应至关重要。然而，我们对 dsRNA-RNase L途径。我们在本提案的目标1和2中的目标是了解分子 一般调节RNase L的机制，并广泛表征翻译的新机制， 由我们实验室发现的RNase L调节。这项工作具有生物医学意义，因为RNase L在 除了具有抗癌和抗病毒作用外，是一种抗脂肪生成受体，对于治疗 以及预防传染病、肿瘤和代谢性疾病。在目标1中，我们将阐明 RNase L调节通过获得RNase L在其潜伏状态下的结构。我们的假设是RNase L 形成限定的潜在结构，限制不可控的RNase L信号传导以保护健康组织。我们 将通过冷冻电子显微镜（cryo-EM）的结构分析来验证这一假设。在目标2中，我们将使用 细胞生物学、生物化学和我们小组开发的创新RNA-seq方法， RNase L的翻译调控机制。在2019年，我们发表了我们的发现，RNase L执行 在一些实施方案中，核酸内切酶切割活跃翻译的mRNA作为控制细胞范围蛋白质合成的策略。 该提案中的初步数据为这种mRNA衰变提供了进一步的见解，并表明RNase L表现出 对mRNA编码区的独特偏好，使我们假设RNase L的作用是 再加上翻译。我们将在调查中彻底检验这一假设。 在目标3中，我们将我们的工作扩展到一个相关的受体调节全局翻译，Gcn 2。GCN 2是一个 在人类激酶组中，对位于RNase L附近的应激激酶的机制知之甚少。GCN 2是 对蛋白质稳定、记忆功能和癌症代谢很重要。GCN 2作为饥饿的传感器 在结合不带电荷的tRNA后，抑制整体蛋白质合成以减轻营养缺乏。的 Gcn 2激活的机制由于缺乏有关其结构信息而知之甚少。 调节性tRNA传感结构域。我们将采用低温EM来确定传感器域的结构。 我们的研究将提供新的知识，并有助于建立一个全面的机制， 了解调节翻译的应激激酶。我们的工作也将贡献一个新的方法，LRtcB RNA-seq，作为研究哺乳动物mRNA衰变的工具。