Protease-Mediated Events in Epidermal Differentiation

表皮角质形成细胞的终末分化导致角质层结构的形成，角质层为生物体与其环境之间提供了保护屏障。从活的表皮颗粒细胞到无核锥形鳞状细胞的快速转变是由多种信号分子和途径调控的，这些信号分子和途径尚不清楚。参与末梢分化过程的一组重要分子是细胞内蛋白酶，它切割表皮蛋白，导致细胞器的破坏和角质层的形成。该项目的总体目标是确定caspase 14的功能，caspase 14是一种表皮特异性的半胱氨酸天冬氨酸蛋白酶，在末端分化过程中被激活，并检查在钙结合蛋白聚丝蛋白的蛋白水解过程中释放的游离前聚丝蛋白末端肽的作用。需要验证的假设是：(1)caspase-14在角化过程中发挥重要的生物学作用，通过切割对终端分化起始和/或执行至关重要的结构和/或调节蛋白；(2)游离侧聚蛋白末端通过与其他角质细胞蛋白（包括膜联蛋白和14-3-3家族成员）相互作用，在调节侧聚蛋白加工和其他钙依赖性细胞质或核事件中具有特定作用，这些事件对角化至关重要。为了解决这些问题，提出的具体目标是：(1)在大肠杆菌中表达caspase-14并纯化其活性酶；(2)确定caspase-14的底物特异性和天然角质形成细胞靶点；(3)确定caspase-14的底物特异性和天然角质形成细胞靶点；(3)通过靶向破坏小鼠体内caspase-14的功能；(4)鉴定结合游离前聚丝蛋白末端肽的蛋白质，并确定这些相互作用如何影响其细胞内分布和可能的生物学功能。这些研究将为表皮分化的生物学和常染色体显性和隐性鱼鳞病（如寻常型鱼鳞病和板层性鱼鳞病）的分子基础提供见解，这些鱼鳞病表现为角质层结构和表皮屏障功能的缺陷。

The terminal differentiation of epidermal keratinocytes results in the formation of a structure, the stratum corneum, which provides a protective barrier between an organism and its environment. The rapid transition from a living epidermal granular cell to a anucleate cornified squame is regulated by multiple signaling molecules and pathways that are poorly understood. One important group of molecules involved in the terminal differentiation process are intracellular proteases that cleave epidermal proteins leading to destruction of organelles and formation of the stratum corneum. The overall goal of this project is to determine the function of caspase 14, an epidermal-specific cysteine aspartate protease activated during terminal differentiation, and to examine the role of the free pro-filaggrin terminal peptide which is liberated during the proteolytic processing of the calcium binding protein profilaggrin. Hypotheses to be tested are (1) that caspase-14 plays important biological roles in the keratinization process by cleaving structural and/or regulatory proteins that are critical for initiation and/or execution of terminal differentiation; and (2) that the free profilaggrin terminal, by interacting with other keratinocyte proteins including members of the annexin and 14-3-3 family, has a specific role in regulating profilaggrin processing and other calcium-dependent cytoplasmic or nuclear events that are essential for keratinization. To address these questions, the specific aims proposed are (1) to express caspase-14 in E. coli and purify the active enzyme; (2) to determine the substrate specificity and natural keratinocyte targets of caspase-14; (3) To determine the substrate specificity and natural keratinocyte targets of caspase-14; (3) to determine the function of caspase-14 in vivo by targeted disruption in mice; and (4) to identify proteins that bind the free pro-filaggrin terminal peptide and determine how these interactions affect its intracellular distribution and possible biological function(s). These studies will provide insight into both the biology of epidermal differentiation and the molecular basis of autosomal dominant and recessive ichthyosis such as ichthyosis vulgaris and lamellar ichthyosis that display defects in stratum corneum structure and function of the epidermal barrier.