一种新的遗传性皮肤病致病基因－KLHL24泛素连接酶的作用机制及相关药物筛选

This project started from human clinical data and identified the gene KLHL24 as a novel causative gene for a hereditary skin disease - Epidermolysis bullosa (EB). We found the disease-causing mutations are all in the start codon of KLHL24, resulting in the translation from the second AUG and an N-terminal 28 amino acids truncated version of the protein (KLHL24-ΔN28). We observed that KLHL24-ΔN28 is significantly more stable than the wild type protein in cells. KLHL24 encodes a substrate receptor for a Cul3-Rbx1 type ubiquitin ligase, for which I identified Keratin 14 as a potential substrate. It is known that loss-of-function of Keratin 14 can cause EB, therefore we hypothesize that the stabilized mutant KLHL24-ΔN28 leads to over degradation of Keratin 14, which causes EB. In this project, we will: (1) study the molecular mechanism of the stabilization of KLHL24-ΔN28; (2) prove Keratin 14 is a real substrate of KLHL24; (3) establish cell and animal disease models with KLHL24 knock-inmutations; (4) screening for small molecule inhibitors of KLHL24. Our research can lead to a molecular mechanism of specific degradation of keratins, improve our understanding of the regulation of ubiquitin ligases and provide drug candidates targeting the ubiquitin proteasome pathway.

本课题从临床病例发现了遗传性皮肤病－大疱性表皮松解症（EB) 的新型致病基因 KLHL24。该基因的致病突变都发生在起始密码子，导致翻译生成一种N端缺失28个氨基酸的蛋白。我们发现这个蛋白的稳定性比野生型蛋白显著增强。KLHL24编码一种Cul3-Rbx1型泛素连接酶的底物结合蛋白。我们找到了它的一种可能底物：角蛋白 Keratin 14。Keratin 14的功能缺失已知能导致EB，因此我们认为KLHL24突变体的稳定性增强导致了Keratin 14的过度降解而致病。我们将：（1）研究KLHL24突变体稳定性增强的分子机制； （2）确定Keratin 14是KLHL24的一个泛素化底物；（3）建立KLHL24突变Knock-in的细胞和动物疾病模型；（4）筛选KLHL24的小分子抑制剂。我们的研究为角蛋白的降解找到了可能的机制，对了解泛素连接酶的稳定性调节及针对性的药物开发有重要价值。

我们从一种遗传性皮肤病-大疱性表皮松解症的新型致病基因KLHL24的致病机制研究出发，发现了皮肤角蛋白Keratin 14是KLHL24的一个泛素化底物。KLHL24的功能增强点突变导致Keratin 14的过度降解，从而引起皮肤脆弱的症状。我们进一步确定了Keratin 14的泛素化位点和KLHL24结合区域。我们通过构建KLHL24突变Knock-in小鼠，进一步用体内实验验证了Keratin 14被KLHL24功能增强型突变导致过度降解的机制。为了进一步发现KLHL24小分子抑制剂作为治疗的药物，我们进行了小分子筛选并发现一个小分子抑制剂候选物能在细胞水平抑制KLHL24的活性并稳定Keratin 14。我们后续还发现了KLHL24突变导致病人成年后得心肌炎的机制，并发现KLHL24在心脏中的一个泛素化底物。

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