【摘要】 Liddle综合征是一种常染色体显性、盐敏感型的高血压综合征,其分子发病机制研究认为是上皮钠离子通道(epithelial Na+channel,ENaC)的β亚基或γ亚基的胞质侧羧基端区域低频率的点突变或缺失突变导致肾远曲小管钠离子重吸收增加.本研究提出了一种从分子水平上治疗Liddle综合征的设想,即构建一种可识别Liddle综合征患者中ENaC蛋白突变的PY模体的人工泛素连接酶E3,使其结合并降解突变的ENaC蛋白,从而使肾远曲小管上皮细胞膜上ENaC的表达数量和活性恢复.而识别PY突变体的E3,可通过用患者中的PY突变体筛选随机多肽文库获得与之结合的随机肽段,用其替换PY模体天然配体蛋白Nedd4的WW结构域,从而得到一个新的人工E3.本研究中以一种Liddle综合征突变型Y620H为诱饵蛋白,筛选新型随机多肽文库,获得了1个至少能与2种PY突变体(Y620H和P618L)特异性结合的随机肽段,为进一步构建可降解ENaC突变体的人工E3积累了重要的实验数据.更多还原

【Abstract】 Liddle syndrome is an autosomal dominant trait links to salt-sensitive hypertension. The pathogenesis associated with to the deletion or point mutations of a PY motif in the cytoplasmic C-terminus at either the β or γ subunit of the epithelial Na+channel( ENaC),therefore lead to constitutively increase of channel activity and Na+reabsorption in the distal nephron. We proposed a strategy for the molecular therapy of Liddle syndrome by creating an artificial ubiquitin ligase( E3) to recognize Liddle Syndrome-associated mutations and degrade ENaC by ubiquitination,theus to recover channel activity and Na+reabsorption from abnormal states. The artificial E3 was constructed by replacing the WW domain of Nedd4( the native binding partner of PY motif in ENaC) with a binding peptide of PY motif mutations obtained from the screenings of a random peptide libraries. A specific peptide from yeast two-hybrid screening of random peptide library was obtained using a Liddle mutation,Y620 H,as bait. This novel peptide recognized two Liddle syndrome mutations,Y620 H and P618 L. These data provide the important basis for the construction of the functional artificial E3.更多还原