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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.

描述（申请人提供）：囊性纤维化（CF）中最普遍的疾病相关突变是囊性纤维化跨膜传导调节因子（CFTR）中苯丙氨酸508（F508 del）的缺失，这导致呼吸道、胃肠道、胃肠道和胃肠道中蛋白质的错误加工和顶端定位减少。胰腺和其它上皮我们的初步证据表明，核糖体蛋白L12（Rp 112）的抑制纠正了F508 delCFTR的折叠缺陷和功能性细胞表面表达。许多最近的和备受瞩目的出版物表明，核糖体不仅调节肽合成和一级氨基酸结构，而且在蛋白质折叠过程中起着关键的“伴侣样”作用[14-18]。因此，重要的是理解Rpl 12在共翻译折叠期间介导与新生F508 del多肽相互作用的机制，并测试Rpl 12作为患有CF的个体的新治疗靶标的可行性。特异性Ai 1：表征Rpl 12敲低对F508 del CFTR加工的影响。在siRNA介导的Rpl 12抑制后，我们将测量F508 del表达、成熟和功能的稳态水平。此外，我们将确定是否废除其他核糖体蛋白位于60 S亚基P柄（在Rpl 12附近）也介导救援F508删除处理。具体目标2：确定Rpl 12抑制拯救F508 del CFTR功能的机制。我们将使用前沿技术（多聚核糖体分析，核糖体分析，RNA-Seq）来定量解决核糖体组装，翻译效率，延伸和足迹响应Rpl 12敲低。这些研究将鉴定CFTR内通过缺失F508而改变的特定亚结构域，并测试Rpl 12抑制作为克服这种性质的缺陷的手段。具体目标3：通过开发RPL 12敲除、条件性敲除或单倍不足小鼠来确定体内相关性。RPL 12小鼠模型将与CFTRF 508 del小鼠杂交以测定胃肠道和呼吸道内CF表型表现的改善。这将包括 CFTR表达的研究（生化分析）、组织病理学、免疫组织化学和功能（短路电流测量）以及来自上气道的体内生物电测量。成功完成所提出的实验将：（1）建立一种新的，特异性的核糖体蛋白作为F508 del CFTR折叠和蛋白质构象的基本贡献者，（2）证明在共翻译过程中核糖体质量控制的体内作用。蛋白质折叠的背景下，一个重要的人类疾病，和（3）提供新的证据，核糖体伴侣蛋白质的功能在蛋白质生物合成。在小鼠体内的实验也将提供一个重要的基础，为未来的研究，旨在调节核糖体动力学作为一个实验性的治疗策略，这种疾病的患者。