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Activating mutations in MEK: from molecules to morphologies

激活 MEK 突变：从分子到形态

基本信息

批准号：
8884927
负责人：
REBECCA D. BURDINE
金额：
$ 44.97万
依托单位：
PRINCETON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-15 至 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8884927
关键词：
Adult Affect Antineoplastic Agents Binding Biochemical Biological Biological Assay Birth Cardiac Myoblasts Cell Count Congenital Heart Defects Cultured Cells DNA Sequence Alteration Defect Development Drosophila genus Embryo Enzymes Event Experimental Models Face Germ-Line Mutation Goals Heart Human Image Joints Kinetics Knock-in Mouse Life Light Literature MAP Kinase Gene MAPK Signaling Pathway Pathway MEKs Malignant Neoplasms Maps Mass Spectrum Analysis Modeling Monitor Morphogenesis Morphology Mus Mutate Mutation Neurocognitive Organ Pathway interactions Patients Pattern Phenotype Phosphorylation Predisposition Proteins Publishing Reaction Regulation Research Research Personnel Signal Transduction Testing Tissues Tube Variant Work Zebrafish base cardiogenesis design extracellular genome sequencing in vivo insight migration postnatal programs public health relevance quantitative imaging reconstitution research study three dimensional structure

项目摘要

DESCRIPTION (provided by applicant): Our work is designed to provide fundamental understanding of RASopathies, a large class of developmental abnormalities caused by the germline mutations in components of the RAS/MAPK pathway. Patients with these conditions display a broad spectrum of phenotypes, including heart defects, short stature, facial dysmorphisms, and neurocognitive delays. Whole genome sequencing of RASopathies provides new sequence variants in the well characterized components of the RAS pathway, but the functional consequences of these sequence variations is poorly understood. We will quantitatively characterize the functional and phenotypic consequences of the activating mutations in MEK, a core component of the RAS pathway. Focusing on the same group of mutations, we will first determine their effects on the regulation and enzymatic activity of MEK (Aim 1). This will be done using mass spectrometry-based kinetic assays with purified proteins. Next, we will quantify the consequences of activating mutations for the kinetics of RAS signaling in vivo (Aim 2). This will be done using a quantitative imaging approach, with which we will monitor RAS signaling in Drosophila embryos. Finally, we will determine how the same mutations influence RAS-dependent tissue morphogenesis (Aim 3). This will be done using live imaging experiments in zebrafish, focusing on heart development as a model of a morphogenetic event that is commonly affected in RASopathies. Our studies should provide mechanistic insights into the biochemical and morphogenetic effects of mutations identified in a large group of human developmental abnormalities and highlight the importance of using multiple models and quantitative approaches for answering a specific biological question.

描述（由申请人提供）：我们的工作旨在提供对RAS病的基本理解，RAS病是由RAS/MAPK通路组分中的种系突变引起的一大类发育异常。患有这些疾病的患者表现出广泛的表型，包括心脏缺陷、身材矮小、面部畸形和神经认知延迟。RASopathies的全基因组测序提供了RAS途径的充分表征的组分中的新序列变体，但是这些序列变体的功能后果知之甚少。我们将定量表征MEK激活突变的功能和表型后果，MEK是RAS途径的核心组成部分。聚焦于同一组突变，我们将首先确定它们对MEK的调节和酶活性的影响（目的1）。这将使用基于质谱的纯化蛋白质动力学测定来完成。接下来，我们将量化激活突变对体内RAS信号传导动力学的影响（目的2）。这将使用定量成像方法来完成，我们将监测果蝇胚胎中的RAS信号。最后，我们将确定相同的突变如何影响RAS依赖的组织形态发生（目的3）。这将使用斑马鱼的活体成像实验来完成，重点是心脏发育作为RASopathies中常见的形态发生事件的模型。我们的研究应该提供机制的见解，在一大群人类发育异常的突变的生化和形态发生的影响，并强调使用多种模型和定量方法来回答一个特定的生物学问题的重要性。