SPLUNC1-Derived Peptides as ENaC Antagonists

描述(申请人提供)：？和？的胞外环ENAC可被呋喃型转化酶、丝氨酸蛋白酶和中性粒细胞在多个位置进行旋转切割 弹性酶，导致通道激活，增加钠的吸收。在囊性纤维化(CF)的呼吸道中，ENaC异常过度活跃，部分原因是过度的蛋白分解，这导致了呼吸道表面液体(ASL)的耗竭。这反过来可能导致粘液淤积和增加呼吸道感染的发生率，从而经常导致患者死亡。我们最近发现SPLunc1是一种有效的ENaC抑制剂，它与ENaC的胞外侧结合，并降低质膜ENaC水平。我们还表明，当SPLunc1被shRNA击倒时，正常的人支气管上皮细胞培养物不再能调节ENaC的活性。我们假设SPLuc1通过减少ENaC表面密度，防止接触细胞外蛋白酶来限制Na的吸收。我们已经确定了SPLunc1的活性部位，并在此基础上合成了一个与ENaC特异结合的18个氨基酸的多肽，称为S18。虽然S18对NL和CF上皮细胞的ENaC有强烈的抑制作用，但SPLuc1不能调节CF上皮细胞的ENaC。Cf ASL是酸性的，因为缺乏CFTR相关的HCO3-分泌。分子模拟表明，SPLunc1可能经历了pH诱导的构象变化，“埋葬”了它的活性部位，阻止了与酸性CFASL中的ENaC的结合。因此，我们建议(I)解决SPLunc1的晶体结构以进一步完善这一模型，(Ii)确定细胞外SPLunc1降低ENaC表面密度的机制，以及(Iii)确定SPLunc1在CF呼吸道中无法发挥作用的原因。S18样多肽可能在治疗慢性肺病方面有一定的应用价值。此外，我们的数据表明，吸入高渗HCO3-可能比高渗盐水在恢复CF粘液清除方面具有显著优势。 与公共卫生相关：由于遗传原因导致肺部盐分和水的运输不平衡，导致CF粘液脱水，使呼吸道容易粘液堵塞和反复感染。在这一应用中，我们建议了解CF气道如何调节盐和水的运输以维持粘液清除。我们还建议开发新的多肽来抑制CF呼吸道的过度盐分吸收，以改善CF粘液的水化。

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.