Alport syndrome is a hereditary glomerular disease caused by mutation in type IV collagen α3–α5 chains (α3–α5(IV)), which disrupts trimerization, leading to glomerular basement membrane degeneration. Correcting the trimerization of α3/α4/α5 chain is a feasible therapeutic approach, but is hindered by lack of information on the regulation of intracellular α(IV) chain and the absence of high-throughput screening (HTS) platforms to assess α345(IV) trimer formation. Here, we developed sets of split NanoLuc-fusion α345(IV) proteins to monitor α345(IV) trimerization of wild-type and clinically associated mutant α5(IV). The α345(IV) trimer assay, which satisfied the acceptance criteria for HTS, enabled the characterization of intracellular- and secretion-dependent defects of mutant α5(IV). Small interfering RNA-based and chemical screening targeting the ER identified several chemical chaperones that have potential to promote α345(IV) trimer formation. This split luciferase-based trimer formation assay is a functional HTS platform that realizes the feasibility of targeting α345(IV) trimers to treat Alport syndrome.

中文翻译：

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基于分裂荧光素酶的三聚体形成分析作为高通量筛选平台治疗阿尔波特综合征的平台。

Alport综合征是一种遗传性肾小球疾病，由IV型胶原α3–α5链（α3–α5（IV））的突变引起，该突变破坏三聚化，导致肾小球基底膜变性。纠正α3/α4/α5链的三聚是一种可行的治疗方法，但由于缺乏有关细胞内α（IV）链调控的信息以及缺乏评估α345（IV）的高通量筛选（HTS）平台而受到阻碍）三聚体的形成。在这里，我们开发了多套拆分的NanoLuc-fusionα345（IV）蛋白，以监测野生型和临床相关突变体α5（IV）的α345（IV）三聚化。满足HTS接受标准的α345（IV）三聚体测定能够表征突变体α5（IV）的细胞内和分泌依赖性缺陷。针对ER的基于RNA的小型干扰物和化学筛选确定了几种化学分子伴侣，它们可能促进α345（IV）三聚体的形成。这种基于萤光素酶的三聚体分裂测定方法是功能性的HTS平台，可实现靶向α345（IV）三聚体治疗Alport综合征的可行性。