Modulation of Ribosome Dynamics Rescues F508del CFTR Maturational Arrest

核糖体动力学调节挽救 F508del CFTR 成熟停滞

基本信息

批准号：
9051505
负责人：
Kathryn E Oliver
金额：
$ 2.54万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-04-13 至 2016-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9051505
关键词：
Address Amino Acids Apical Bicarbonates Biochemical Biogenesis Biological Assay Biological Models Birth Breeding Caucasians Cell Culture Techniques Cell model Cell surface Cells Chimera organism Chloride Ion Chlorides Critical Pathways Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Data Defect Development Diagnosis Disease Elongation Factor Endoplasmic Reticulum Epithelial Epithelium Foundations Future Gastrointestinal tract structure Harvest Histopathology Human Immunohistochemistry Individual Investigational Therapies Ion Transport Kinetics Knock-out Knockout Mice Lead Libraries Life Lung Transplantation Measurement Measures Mediating Messenger RNA Methods Modeling Molecular Chaperones Molecular Conformation Mus Mutation Nature North America Organ Pancreas Pathogenesis Pathway interactions Patients Peptide Synthesis Phenotype Phenylalanine Physiologic Monitoring Physiologic pulse Play Polyribosomes Population Positioning Attribute Printing Process Protein Conformation Proteins Publications Quality Control Reproductive system Respiratory System Respiratory tract structure Ribosomal Proteins Ribosomes Role Saccharomyces cerevisiae Safety Small Interfering RNA Structure Technology Testing Therapeutic Therapeutic Intervention Tissues Translations Transplant Recipients VX-770 VX-809 Western Blotting Work Yeasts airway epithelium clinically significant combinatorial cystic fibrosis patients flexibility foot gastrointestinal gastrointestinal system gene product genome-wide improved in vivo insight knock-down mouse model new therapeutic target novel novel strategies phenomics polypeptide protein degradation protein folding protein misfolding public health relevance research study respiratory response ribosome profiling small molecule stable cell line therapeutic target transcriptome sequencing

项目摘要

DESCRIPTION (provided by applicant): The most prevalent disease-associated mutation in cystic fibrosis (CF) is deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR), which results in misprocessing and reduced apical localization of the protein in respiratory, gastrointestinal, pancreatic and other epithelia Our preliminary evidence demonstrates that suppression of ribosomal protein L12 (Rpl12) corrects the folding defect and functional cell surface expression of F508del CFTR. A number of recent and high-profile publications indicate that the ribosome not only regulates peptide synthesis and primary amino acid structure, but also plays a crucial "chaperone-like" role during protein folding [14-18]. Therefore, it is important to understand the mechanism by which Rpl12 mediates interaction with nascent F508del polypeptide during co- translational folding and test the feasibility of Rpl12 as a novel therapeutic target for individuals living with CF. Specific Ai 1: Characterize the effects of Rpl12 knockdown on F508del CFTR processing. Following siRNA-mediated Rpl12 inhibition, we will measure steady state levels of F508del expression, maturation, and function. In addition, we will determine whether abrogation of other ribosomal proteins located on the 60S subunit P stalk (in close proximity to Rpl12) also mediate rescue of F508del processing. Specific Aim 2: Ascertain the mechanism by which Rpl12 suppression rescues F508del CFTR function. We will use leading-edge technology (polysome analyses, ribosome profiling, RNA-Seq) to quantitatively address ribosome assembly, translation efficiency, elongation, and foot printing in response to Rpl12 knockdown. These studies will identify specific sub-domains within CFTR that are altered by deletion of F508, and test Rpl12 suppression as a means to overcome defects of this nature. Specific Aim 3: Determine in vivo relevance by development of RPL12 knockout, conditional knockout, or haploinsufficient mice. RPL12 mouse models will be cross-bred to CFTRF508del mice to assay for improvements in CF phenotyptic manifestations within the gastrointestinal and respiratory tracts. This will include studies of CFTR expression (biochemical analyses), histopathology, immunohistochemistry, and function (short circuit current measurements), and in vivo bioelectric measurements from the upper airways. Successful completion of the proposed experiments will: (1) establish a novel, specific ribosomal protein as a fundamental contributor to F508del CFTR folding and protein conformation, (2) demonstrate the in vivo role of ribosomal quality control during co-translational protein folding in the context of an important human disorder, and (3) provide new evidence for ribosomal-chaperone function during protein biogenesis. The in vivo experiments in mice will also furnish an important foundation for future studies aimed at modulating ribosome kinetics as an experimental therapeutic strategy for patients with this disease.

DESCRIPTION (provided by applicable): The most prevalent disease-associated mutation in cystic fibrosis (CF) is deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR), which results in missprocessing and reduced apical localization of the protein in respiratory, gastrointestinal, pancreatic and other epithelia Our初步证据表明，核糖体蛋白L12（RPL12）的抑制纠正了F508DEL CFTR的折叠缺陷和功能细胞表面表达。许多近期和备受瞩目的出版物表明，核糖体不仅调节肽合成和原代氨基酸结构，而且在蛋白质折叠过程中也起着至关重要的“伴侣样”作用[14-18]。因此，重要的是要了解RPL12在共同翻译过程中介导与新生F508DEL多肽相互作用的机制，并测试RPL12作为CF患者的新型热目标的可行性。特定AI 1：表征RPL12敲低对F508DEL CFTR处理的影响。 siRNA介导的RPL12抑制后，我们将测量F508DEL表达，成熟和功能的稳态水平。此外，我们将确定是否废除了位于60年代亚基P茎上的其他核糖体蛋白（靠近RPL12）是否也媒体营救了F508DEL加工。特定目标2：确定RPL12抑制响应F508DEL CFTR函数的机制。我们将使用领先技术（多层分析，核糖体分析，RNA-Seq）来定量解决核糖体组装，翻译效率，伸长率，伸长率和脚印，以响应RPL12敲低。这些研究将确定CFTR中特定的子域因删除F508而改变，并测试RPL12抑制作用是克服这种性质缺陷的手段。特定目标3：通过开发RPL12敲除，有条件敲除或单倍弹性小鼠来确定体内相关性。 RPL12小鼠模型将跨繁殖到CFTRF508DEL小鼠，以断言胃肠道和呼吸道内CF表现症状表现的改善。这将包括 CFTR表达（生物化学分析），组织病理学，免疫组织化学和功能（短路电流测量值）以及从上呼吸道的体内生物电测量的研究。成功完成所提出的实验将：（1）建立一种新型的特异性核糖体蛋白，作为F508DEL CFTR折叠和蛋白质构象的基本贡献，（2）证明了共同体转换过程中核糖体质量控制的体内作用在重要的人类疾病的背景下，蛋白质折叠，（3）在蛋白质生物发生过程中为核糖体 - 链蛋白功能提供了新的证据。小鼠的体内实验还将为未来研究的重要基础，旨在调节核糖体动力学作为该疾病患者的实验疗法策略。