基因突变后ATP2C1剪切体异常在HHD发病机制中的关键作用

ATP2C1 is the pathogenic gene of familial chronic benign pemphigus (also called Hailey–Hailey disease, HHD)，encoding SPCA1 protein. So far, there are more than 170 SNPs have been found, but the effect of such mutations on the transcription and translation levels as well as the mechanism of acantholysis are still unknown. Previous literatures showed that ATP2C1 has different splicing variants, meanwhile, the SPCA1 isoforms can regulate the expression of a variety of downstream proteins. Recently, we demonstrated that the SPCA1 expression is haploinsufficiency in skin lesion of HHD, leading to abnormal expression of several proteins associated with keratinocyte differentiation. Furthermore, SPCA1 silence can induce the cellular microstructural changes. Therefore, It suggested that the mutations interfere the expression of SPCA1 isoforms in keratinocytes, lead to the abnormal expression of proteins associated with intercellular junction, and then destroy the adhesion and connection between cells. The aim of this study is to explore the specific mutations in ATP2C1 gene, and to illustrate the pathogenic mechanism of HHD by focusing on the influence of abnormal SPCA1 isoforms on intercellular junction. The techniques of transcriptomics, immunological and molecular biological approaches will be applied in the experiments. Moreover, the mRNA compensation intervention will be used to reduce the effect of abnormal SPCA1 isoforms on acantholysis.

ATP2C1是家族性慢性良性天疱疮（又称Hailey–Hailey disease, HHD）的致病基因，其编码蛋白为SPCA1。现已发现SNP超过170个，但突变对基因在转录和翻译水平的影响，及导致棘层松解的机制仍不清楚。文献证实ATP2C1存在不同的剪切体，而SPCA1亚体可调控下游多种蛋白表达。我们前期研究发现HHD皮损内SPCA1表达不足，造成角质形成细胞成熟相关蛋白异常；预实验发现干扰SPCA1表达，能诱导细胞微观结构改变。因此推测：基因突变影响角质形成细胞内SPCA1亚体表达，造成细胞连接相关蛋白表达异常，破坏细胞的粘附和连接，最终导致HHD发病。本课题拟运用转录组学、免疫学和分子生物学技术，探索ATP2C1基因突变特殊位点和形式，并揭示SPCA1亚体紊乱对细胞间连接的作用，阐明基因突变导致HHD的机制。此外，将采用mRNA补偿干预方法，以图纠正剪切体异常参与棘层松解的作用。

ATP2C1是家族性慢性良性天疱疮（又称Hailey–Hailey disease, HHD）的致病基因，其编码蛋白为SPCA1。现已发现SNP超过170个，但突变对基因在转录和翻译水平的影响，及导致棘层松解的机制仍不清楚。本项目收集HHD患者及对照正常皮肤组织，进行iTRAQ定量蛋白质组学分析。收集皮肤组织或ATP2C1 siRNA转染的人原代角化细胞，使用Western blot以及RT-PCR对iTRAQ分析的差异蛋白进行表达验证。结果显示，iTRAQ共鉴定到134个在HHD患者组织中发生显著差异表达的蛋白质。对这些差异蛋白进行GO分析以及KEGG分析，123个显著匹配的注释蛋白参与187个已知的KEGG代谢或信号通路。HHD患者组织中差异表达的蛋白主要参与PI3K-Akt信号通路，黏着斑，细胞外基质-受体相互作用，蛋白质降解与吸收等。使用shRNA慢病毒建立了稳定的体外ATP2C1基因敲减的HHD细胞模型。利用细胞模型，对紧密连接蛋白（Tight junction protein，TJ protein）和丝状/纤维状肌动蛋白（Filamentous actin/Fibrous actin，F-ACTIN）在HHD中的表达情况进行了研究。结果显示，ATP2C1基因异常导致SPCA1单倍剂量不足，通过SPCA1的P区减少COFILIN磷酸化，导致纤维肌动蛋白F-ACTIN聚合减少，通过下游相关信号传导从而致TJ蛋白CLAUDIN1、CLAUDIN4、OCCLUDIN和ZO1表达减少，因而影响细胞间的连接和粘附，可能为HHD发病的主要原因。