Rescue of CFTR-deltaF508 misfolding by pharmacological and genetic suppressors

通过药理学和遗传抑制剂拯救 CFTR-deltaF508 错误折叠

• 批准号: 7500686
• 负责人: RYAN E TYLER
• 金额: $ 4.96万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-26 至 2010-09-25
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7500686
• 关键词: Affinity; Alleles; Binding; Biochemical; Biological Assay; Biological Factors; Biology; Cell Line; Cell membrane; Cell surface; Cells; Chinese Hamster Ovary Cell; Chloride Channels; Condition; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Detection; Disease; Drug Delivery Systems; Electrolytes; Endoplasmic Reticulum; Epithelial Cells; Fluorescence; Fluorescence Resonance Energy Transfer; Foundations; Genes; Genetic; Goals; Half-Life; Hereditary Disease; Human; Human Cell Line; Integral Membrane Protein; Laboratories; Licorice; Life; Ligand Binding; Localized; Measures; Membrane Proteins; Molecular Biology; Molecular Chaperones; Mutation; North America; Pancreatic ribonuclease; Pathway interactions; Peptides; Personal Satisfaction; Phenylalanine; Plant Extracts; Plant Roots; Positioning Attribute; Process; Processed Genes; Proteins; Quality Control; Reporter; Screening procedure; Signal Transduction; System; Technology; Temperature; Transcript; Translating; Ubiquitin; base; cystic fibrosis patients; enzyme reconstitution; high throughput screening; isoliquiritigenin; multicatalytic endopeptidase complex; mutant; neuronal cell body; novel; protein aminoacid sequence; protein function; small molecule; tool; trafficking

DESCRIPTION (provided by applicant): Cystic fibrosis is a life-threatening genetic disease characterized by the altered secretion of electrolytes by the secretory epithelial cells of the body. It is caused by mutations in the CF gene, which encodes the cystic fibrosis transmembrane conductance regulator (CFTR), a cell surface localized integral membrane protein that functions as chloride ion channel. The CF disease allele found in nearly 90% of cystic fibrosis patients in North America contains a trinucleotide deletion that removes the phenylalanine at position 508 of the CFTR peptide sequence. This protein, CFTR-deltaF508, is a conformational mutant that is recognized by the cellular protein quality control system and is eliminated via the ubiquitin-proteasome system in a process known as endoplasmic reticulum-associated degradation. However, the ability of CFTR-deltaF508 to function as a chloride channel under conditions that permit CFTR-deltaF508 to properly fold and traffic to the plasma membrane, such as at reduced incubation temperatures (<30¿C), indicate that the rescue of CFTRdeltaF508 folding may serve as a feasible target for the treatment of cystic fibrosis. The identification of small molecules that bind to and stabilize the native fold of CFTR-deltaF508 (pharmacological chaperones) or of proteins involved in CFTR-deltaF508 processing by gene-knockdown studies have been hindered by the lack of technology to directly measure CFTR-delta508 present on the cell surface that is suited to high throughput screening (HTS) platforms. To overcome these limitations we have developed a rapid and robust cell-based system to directly detect the rescue of misfolded CFTR-deltaF508 to the cell surface that combines high affinity ligand binding, enzymatic complementation/signal amplification and a sensitive FRET based fluorescence detection system. The capability of this system is exemplified in a preliminary HTS study, in which we identified a novel small molecule derived from plant extracts that rescues CFTRdeltaF508 misfolding. Therefore, we propose to use this tool to 1) assess the ability of previously identified modulators of CFTR channel activity to rescue CFTR-deltaF508 misfolding , 2) perform screen of a set of natural products extracts for potential compounds that correct the folding of CFTR-deltaF508 and 3) set up a system in human cell line amenable to genetic studies to identify suppressors of CFTR-deltaF508 ER retention and degradation. The short-term goals of this proposal are to provide a greater understanding of the CFTR-deltaF508 biology, to identify CFTR-deltaF508 pharmacological chaperones and to discover proteins that could serve as drug targets for the rescue of CFTR-deltaF508 trafficking. The broad objective of our studies is to uncover leads that could translate into new treatments for cystic fibrosis.

描述（由申请人提供）：囊性纤维化是一种危及生命的遗传性疾病，其特征是身体分泌上皮细胞的电解质分泌改变。它是由CF基因突变引起的，CF基因编码囊性纤维化跨膜传导调节因子（CFTR），这是一种细胞表面定位的整合膜蛋白，起氯离子通道的作用。在北美近90%的囊性纤维化患者中发现的CF疾病等位基因含有三核苷酸缺失，其去除CFTR肽序列的位置508处的苯丙氨酸。这种蛋白CFTR-deltaF 508是一种构象突变体，它被细胞蛋白质质量控制系统识别，并在称为内质网相关降解的过程中通过泛素-蛋白酶体系统消除。然而，CFTR-deltaF 508在允许CFTR-deltaF 508正确折叠并运输至质膜的条件下（例如在降低的孵育温度（<30 ℃）下）作为氯离子通道发挥作用的能力表明，拯救CFTR-deltaF 508折叠可作为治疗囊性纤维化的可行靶点。通过基因敲低研究鉴定结合并稳定CFTR-δ F508（药理学伴侣）的天然折叠的小分子或参与CFTR-δ F508加工的蛋白质，由于缺乏直接测量存在于细胞表面上的适合于高通量筛选（HTS）平台的CFTR-δ F508的技术而受到阻碍。为了克服这些局限性，我们开发了一种快速和稳健的基于细胞的系统，以直接检测错误折叠的CFTR-δ F508对细胞表面的拯救，该系统结合了高亲和力配体结合、酶互补/信号放大和灵敏的基于FRET的荧光检测系统。该系统的能力在初步HTS研究中得到了例证，在该研究中，我们鉴定了一种源自植物提取物的新型小分子，其拯救CFTRdeltaF 508错误折叠。因此，我们建议使用该工具来1）评估先前鉴定的CFTR通道活性调节剂拯救CFTR-δ F508错误折叠的能力，2）对一组天然产物提取物进行筛选，以寻找校正CFTR-δ F508折叠的潜在化合物，和3）在人类细胞系中建立适合遗传研究的系统，以鉴定CFTR-δ F508的抑制子。deltaF 508 ER保留和降解。该提案的短期目标是提供对CFTR-deltaF 508生物学的更好理解，以鉴定CFTR-deltaF 508药理学伴侣，并发现可用作拯救CFTR-deltaF 508贩运的药物靶标的蛋白质。我们研究的广泛目标是发现可能转化为囊性纤维化新疗法的线索。