SPLUNC1-Derived Peptides as ENaC Antagonists

作为 ENaC 拮抗剂的 SPLUNC1 衍生肽

基本信息

批准号：
8295783
负责人：
ROBERT TARRAN
金额：
$ 36.33万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-05-10 至 2016-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8295783
关键词：
Acids Active Sites Affect Alanine Amino Acids Bicarbonates Binding Binding Sites Biological Assay Breathing Cell membrane Cessation of life Cleaved cell Crystallization Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Data Dissociation Dose Epithelial Equilibrium Exposure to Extracellular Domain Failure Fluorescence Resonance Energy Transfer Homeostasis Human Hydration status Incidence Infection Lead Learning Left lung Length Leukocyte Elastase Measures Modeling Molecular Models Mucous body substance Mutate Mutation N-terminal Oranges Patients Peptide Hydrolases Peptides Proteomics Pulmonary Cystic Fibrosis Recurrence Resistance Saline Serine Protease Side Site Sodium Chloride Structure Surface Surface Plasmon Resonance Testing Ubiquitination Water absorption airway surface liquid base cystic fibrosis airway cystic fibrosis airway epithelia data modeling density epithelial Na+ channel extracellular improved inhibitor/antagonist knock-down molecular modeling mutant novel prevent small hairpin RNA

项目摘要

DESCRIPTION (provided by applicant): The extracellular loops of ¿ and ? ENaC can be roteolytically cleaved at multiple sites by furin-type convertases, serine proteases and neutrophil elastase, leading to activation of the channel and increased Na+ absorption. In cystic fibrosis (CF) airways, ENaC is abnormally hyperactive due in part to excessive proteolytic cleavage, which contributes to a depletion of airway surface liquid (ASL) volume. This in turn may lead to mucus stasis and increased incidence of airway infections that frequently lead to the death of the patient. We have recently identified SPLUNC1 as a potent inhibitor of ENaC that binds to the extracellular side of ENaC and diminishes plasma membrane ENaC levels. We have also shown that when SPLUNC1 is knocked down by shRNA, normal human bronchial epithelial cultures can no longer regulate ENaC activity. We hypothesize that SPLUNC1 limits Na+ absorption by reducing ENaC surface densities, preventing exposure to extracellular proteases. We have identified the active site of SPLUNC1, and have synthesized an 18 amino acid peptide based on this site, called S18, which specifically binds to ¿ENaC. Although S18 robustly inhibits ENaC in NL and CF airway epithelia, SPLUNC1 fails to regulate ENaC in CF airway epithelia. CF ASL is acidic due to the lack of CFTR-associated HCO3- secretion. Molecular modeling indicates that SPLUNC1 may undergo a pH- induced conformational change that "buries" its active site, preventing binding to ENaC in the acidic CF ASL. Thus, we propose to (i) solve the crystal structure of SPLUNC1 to further refine this model, (ii) determine the mechanism whereby extracellular SPLUNC1 can reduce ENaC surface density and (iii) determine why SPLUNC1 fails to function in CF airways. S18-like peptides may be therapeutically beneficial in the treatment of CF lung disease. Furthermore, our data suggest that inhaled hypertonic HCO3- may offer significant advantages over hypertonic saline for restoring CF mucus clearance. PUBLIC HEALTH RELEVANCE: CF mucus is dehydrated due to a genetically induced imbalance in salt and water transport in the lungs, leaving the airways prone to mucus plugging and recurrent infections. In this application, we propose to understand how CF airways can regulate salt and water transport to maintain mucus clearance. We also propose to develop novel peptides that can inhibit excessive salt absorption in CF airways to improve CF mucus hydration.

描述（由应用程序提供）：»和？ ENAC可以通过弗林型转化酶，丝氨酸蛋白酶和中性粒细胞在多个位点上裂解。弹性酶，导致通道激活并增加Na+损失。在囊性纤维化（CF）气道中，ENAC基本上是由于过量的蛋白水解裂解而过度活跃，这有助于部署气道表面液体（ASL）体积。反过来，这可能导致粘液停滞和气道感染事件增加，这些事件经常导致患者死亡。我们最近将Splunc1鉴定为ENAC的潜在抑制剂，该抑制剂与ENAC的细胞外侧结合并减少质膜ENAC水平。我们还表明，当shRNA击倒Splunc1时，正常的人支气管上皮培养物无法再调节ENAC活性。我们假设Splunc1通过降低ENAC表面密度来限制Na+受苦，从而阻止暴露于细胞外蛋白酶。我们已经确定了Splunc1的活性位点，并基于该位点（称为S18）合成了18个氨基酸肽，尽管S18在NL和CF Airway上皮elia中稳健抑制ENAC，但Spluncc1在CF Airway Epithelia中无法调节ENAC。 CF ASL由于缺乏CFTR相关的HCO3分泌而具有酸性。分子建模表明，Splunc1可能会发生pH诱导的会议变化，以“掩埋”其活性位点，以防止在酸性CF ASL中与ENAC结合。这是我们建议（i）求解Splunc1的晶体结构以进一步完善该模型，（ii）确定细胞外splunc1可以降低ENAC表面密度的机制，并且（iii）确定为什么Spluncc1在CF Airways中无法正常运行。 S18样的胡椒体可能对CF肺部疾病的治疗产生热有益。此外，我们的数据表明，遗传的高渗HCO3-可能比高渗盐水具有显着优势来恢复CF粘液清除率。公共卫生相关性：CF粘液由于肺部盐和水运输的遗传诱导不平衡而脱水，使气道容易受到粘液堵塞和复发感染。在此应用中，我们建议了解CF气道如何调节盐和水运输以保持粘液清除。我们还建议开发新的肽，这些肽可以抑制CF气道中过量的盐分滥用，以改善CF粘液水合。