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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.
Liddle syndrome is an autosomal dominant, salt-sensitive hypertensive syndrome, the molecular pathogenesis that epithelial sodium channel (epithelial Na + channel, ENaC) β subunit or γ subunit of the cytoplasm Low-frequency point mutations or deletions in the carboxy-terminal region of the kidney lead to an increase in sodium reabsorption in the distal convoluted tubules.This study proposed a concept of treating Liddle syndrome at the molecular level by constructing a cell line that recognizes patients with Liddle’s syndrome The ENYC protein mutant PY motif of the artificial ubiquitin ligase E3, to bind and degrade the mutant ENaC protein, so that the renal distal tubule epithelial membrane ENaC expression quantity and activity recovery.While identifying PY mutant E3 , Random peptide library can be obtained by screening random peptide library with PY mutant in patients, and then replacing it with the WW domain of Nedd4, a natural ligand protein of PY motif, to obtain a new artificial E3. A novel Lyddle syndrome mutant Y620H was used as bait protein to screen a novel random peptide library. A random peptide with at least two PY mutants (Y620H and P618L) was obtained. ENaC degradation of mutant artificial E3 accumulated significant experimental data.