KLHL24通过调控皮肤结构蛋白keratin5/plectin促进皮肤脆性增加的分子机制研究

Skin fragility is an important clinical feature of many dermatoses. Abnormal function and expression of skin structural protein is the main reason of skin fragility, but its mechanisms remain largely unknown. Our preliminary study identified KLHL24 gene as a new disease-causing gene of Epidermolysis Bullosa. We found the gain-of-function mutations of KLHL24 can lead to excessive ubiquitination and degradation of K14, a substrate of KLHL24, causing skin fragility. And we found KLHL24 might have undiscovered ubiquitination substrates or indirect regulatory molecules in the skin. Our recent results showed the protein level of K5 and plectin were largely reduced in the patient skin sample. Based on the results of preliminary experiments, we suppose K5 and plectin can be regulated by KLHL24 via directly ubiquitination followed by degradation or indirectly mechanism. And the reduced K5 and plectin protein level may lead to skin fragility by affecting the cytoskeleton structure. In this study, we will use protein stability and ubiquitination level detection, immunoprecipitation, quantitative proteomics and other experimental methods to elucidate the regulatory role of KLHL24 in K5 and plectin turnover, and the molecular mechanisms of KLHL24 in skin fragility. Our research may reveal the molecular mechanisms of skin fragility, and lay a foundation for exploring new treatment of dermatoses related to skin structural protein abnormality.

脆性增加是许多皮肤病的重要临床特征，皮肤结构蛋白功能和表达量异常是其主要原因，但其具体机制不清楚。我们前期鉴定了遗传性大疱性表皮松解症的新致病基因KLHL24，发现其功能增强型突变使K14被过度泛素化降解，导致皮肤脆性增加，而且KLHL24在皮肤中可能存在其它泛素化底物或间接调控分子。我们近期的实验发现KLHL24突变患者皮肤结构蛋白K5及plectin的蛋白量明显降低。基于预实验结果，我们猜想K5和plectin可能是KLHL24的直接泛素化底物或受其间接调控，两者的蛋白水平降低可通过影响细胞骨架结构导致皮肤脆性增加。本课题拟通过蛋白稳定性及泛素化水平检测、免疫共沉淀、定量蛋白质组学等实验方法，阐释KLHL24调控K5及plectin促进皮肤脆性增加的具体分子机制。本课题不仅有望揭示皮肤脆性增加的分子机制，也为探索皮肤结构蛋白异常相关皮肤病的新疗法奠定基础。

皮肤脆性增加以大疱性皮肤病为主的多种重症疾病的重要临床表现，多与皮肤结构蛋白的表达量或结构及功能异常相关，但具体机制尚不清楚。课题组前期通过全外显子测序确定了遗传性大疱性表皮松解症的一个新致病基因KLHL24，发现其功能增强型突变导致皮肤角质形成细胞内角蛋白K14被过度泛素化降解，导致皮肤脆性增加。近两年研究显示KLHL24功能增强型突变可以介导毛囊干细胞中角蛋白K15的过度泛素化降解，使得毛囊干细胞的衰竭和毛囊退化而导致脱发。另外，功能增强型KLHL24在心肌组织和成纤维细胞中可分别介导结构蛋白Desmin及Vimentin的过度降解，导致扩张型心肌病及萎缩性瘢痕。本项目基于KLHL24突变的遗传性大疱性表皮松解症患者的临床样本，开展细胞水平及动物水平的研究，围绕KLHL24在皮肤角质形成细胞中的新底物进行探索，发现与健康对照相比，KLHL24突变患者皮肤角蛋白K5、K15、K17蛋白水平显著降低；在HaCaT细胞中过表达KLHL24突变体可导致角蛋白K5、K15、K17蛋白量降低，通过泛素化试验发现KLHL24突变体介导了角蛋白K5、K15、K17过度泛素化及降解，该过程可被蛋白酶体抑制剂阻断，在转基因小鼠的组织样品中验证了该发现。这些结果提示角蛋白K5、K15、K17为泛素连接酶KLHL24的新底物，KLHL24可能通过调节这些结构蛋白的降解对皮肤脆性产生影响，参与皮肤疾病的发生发展，可能为遗传性大疱病等皮肤脆性相关疾病的机制阐明和靶点开发提供新的思路。