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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

基本信息

批准号：
10414028
负责人：
Alexei Korennykh
金额：
$ 31.51万
依托单位：
PRINCETON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-05-01 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10414028
关键词：
Address Ankyrin Repeat Area Bacillus anthracis Bacteria Binding Biochemical Biochemistry Cancer Biology Cells Cellular Stress Cellular biology 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

项目摘要

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.

项目概要该提案研究了两种相关受体的结构和功能机制蛋白激酶家族、RNase L（目标 1、2）和 Gcn2（目标 3）。这些受体普遍表达在人体组织中调节蛋白质合成（翻译）以减轻细胞压力。 RNase L 感觉检测由双链 RNA (dsRNA) 引起的应激，而 Gcn2 检测由营养限制引起的应激。哺乳动物细胞对 dsRNA 的存在高度敏感。双链RNA分子通常很少见，但由于富含 dsRNA 的内源重复序列激增，在病毒感染的细胞和癌细胞中广泛存在元素，这使得研究 dsRNA 反应变得至关重要。然而，我们对此了解甚少 dsRNA-RNase L 途径。本提案的目标 1 和 2 的目标是了解分子总体调节 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 除了具有抗癌和抗病毒作用外，它还是一种抗脂肪生成受体，对于治疗可能很重要预防传染病、肿瘤和代谢性疾病。在目标 1 中，我们将阐明其机制通过获得潜在状态的 RNase L 结构来调节 RNase L。我们的假设是 RNase L 形成明确的潜在结构，限制无法控制的 RNase L 信号传导以保护健康组织。我们将使用冷冻电子显微镜（cryo-EM）进行结构分析来检验这一假设。在目标 2 中，我们将使用我们小组开发了细胞生物学、生物化学和创新的 RNA-seq 方法，以建立 RNase L 的翻译调控机制。2019 年，我们发表了 RNase L 的发现主动翻译 mRNA 的核酸内切裂解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提供了对这种 mRNA 衰减的进一步见解，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 的作用是与翻译相结合。我们将在调查中彻底检验这一假设。在目标 3 中，我们将工作扩展到调节全局翻译的相关受体 Gcn2。 GCN2 是一个人们对应激激酶的机制知之甚少，它位于人类激酶组中的 RNase L 附近。 Gcn2 是对于蛋白质稳态、记忆功能和癌症代谢很重要。 Gcn2 作为饥饿传感器结合不带电荷的 tRNA 后，会抑制整体蛋白质合成，从而缓解营养缺乏。这由于缺乏有关 Gcn2 的结构信息，人们对 Gcn2 激活的机制知之甚少。调节 tRNA 感应域。我们将采用冷冻电镜来确定传感器域的结构。我们的研究将提供新知识并有助于建立综合机制了解应激激酶调节翻译。我们的工作还将贡献一种新方法，LRtcB RNA-seq，作为研究哺乳动物 mRNA 衰减的工具。

项目成果

期刊论文数量（13）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

A mutation in the viral sensor 2'-5'-oligoadenylate synthetase 2 causes failure of lactation.

DOI：
10.1371/journal.pgen.1007072
发表时间：
2017-11
期刊：
PLoS genetics
影响因子：
4.5
作者：
Oakes SR;Gallego-Ortega D;Stanford PM;Junankar S;Au WWY;Kikhtyak Z;von Korff A;Sergio CM;Law AMK;Castillo LE;Allerdice SL;Young AIJ;Piggin C;Whittle B;Bertram E;Naylor MJ;Roden DL;Donovan J;Korennykh A;Goodnow CC;O'Bryan MK;Ormandy CJ
通讯作者：
Ormandy CJ

Crystal Structure of Human Nocturnin Catalytic Domain.