类聚丝蛋白亚家族在皮肤屏障功能中的作用机制

Skin barrier homeostasis is essential for human survival. Impaired epidermal barrier function often leads to serious skin diseases such as atopic dermatitis (AD). Due to the fact that the etiopathogenesis of AD is complex and not yet fully elucidated, there is no cure for AD. Therefore, AD has brought a heavy burden on carriers, their families and society as a whole and is becoming a serious social problem. Recently loss-of-function mutations in the filaggrin (FLG) gene, encoding an epidermal scaffold protein, were uncovered to be major predisposing factors for AD. The new question is that, however, not all patients with FLG mutations develop AD while only less than 30% of AD patients carry FLG mutations. To explore the cause-and-effect relationships underlying this contradiction, the applicant has identified, in the previous study, two FLG-like proteins, namely Hornerin (HRNR) and Filaggrin-2 (FLG-2), which might have overlapping functions together with FLG and could possibly compensate for each other. Further, both of them were uncovered to generate active compounds against bacteria challenge. Herein, the project aims to elucidate molecular function and biological significance of the FLG-like proteins subfamily in skin barrier function at the molecular, cellular, tissue and intact small animal levels, as well as the key role in the occurrence and development of AD. We will also explore HRNR and FLG-2 as new players for diagnosis and potential therapeutic targets of AD patients.

皮肤屏障稳态是人体赖以生存的第一道屏障。表皮屏障功能平衡的失调常导致严重皮肤疾病的发生, 如异位性皮炎（AD）。近年研究发现，角化细胞支架蛋白基因filaggrin（FLG）的异常突变是早期触发AD发病的主要因素。然而,只有不到30%的AD患者携带FLG的突变，而携带FLG突变的某些群体又不表现AD症状。为探索这个问题的根源，申请者在前期研究中发现了两个类似FLG的姐妹成员，即Hornerin和Filaggrin-2，二者在功能上可能与FLG重叠和互补，并且可以通过分解产生抗菌混合多肽。因此，本项目将在此基础上，综合运用分子生物学、细胞生物学和生物化学手段对Hornerin和Filaggrin-2的分子功能和生物学意义进行完整的鉴定，以期在细胞和个体动物水平上阐明它们在皮肤屏障中的关键作用及其与AD发生、发展的相关性，为人类皮肤复杂疾病的诊治提供新思路。

皮肤屏障功能是维护人体健康抵御病菌侵染的第一道屏障，屏障功能的失调常常引发多种疾病。近年来，通过对特应性皮炎与丝聚蛋白(filaggrin)相关性研究的积累，启发大家高度重视皮肤屏障功能对皮肤复杂性疾病发生发展的重要性。本项目旨在对皮肤屏障功能的两个要素，即皮肤天然抗菌蛋白和类丝聚蛋白），进行深层次的功能分析。1）我们首先从皮肤中分离纯化得到一个新的抗菌蛋白，即SPINK9,。SPINK9通过与大肠杆菌伴侣蛋白SKP的互作，破坏膜的完整性进入细菌细胞质选择性杀灭大肠杆菌。这是我国在人体上发现的第一个天然抗菌肽,人源抗菌肽在替代抗生素的应用上具有很大的潜力。2) 通过类聚丝蛋白基因的起源和系谱树分析，揭示了它们作为哺乳动物特有基因的分子证据。3）对多年来国际上对类聚丝蛋白基因hornerin和filaggrin 2的基因敲除模型构建都失败了。作为这两个基因的发现者，我们采用改良的CRISPR技术进行敲入替代敲除的方式，成功构建了这两个基因的小鼠KO模型，并获得纯合子。进一步发现，hornerin基因KO小鼠发生脱毛症，而filaggrin 2基因KO小鼠可能发生皮肤剥脱综合症。这两个KO模型将会对今后深入研究皮肤屏障功能与皮肤复杂疾病的相关性产生重要的影响。