Protease-Mediated Events in Epidermal Differentiation

表皮角质形成细胞的终末分化导致形成一种结构，即角质层，其在生物体与其环境之间提供保护屏障。从一个活的表皮颗粒细胞到一个无核的皮质鳞片的快速转变是由多个信号分子和途径，目前还知之甚少。参与终末分化过程的一组重要分子是细胞内蛋白酶，其切割表皮蛋白，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是一种在终末分化过程中激活的表皮特异性半胱氨酸天冬氨酸蛋白酶，并研究游离的前聚丝蛋白末端肽的作用，该肽在钙结合蛋白前聚丝蛋白的蛋白水解加工过程中释放。待检验的假设是（1）胱天蛋白酶-14通过切割对终末分化的起始和/或执行至关重要的结构和/或调节蛋白在角化过程中起重要的生物学作用;和（2）游离的聚丝蛋白原末端，通过与包括膜联蛋白和14-3-3家族成员在内的其它角质形成细胞蛋白相互作用，在调节纤丝聚集蛋白原加工和其它钙依赖性细胞质或细胞核事件中具有特异性作用，所述细胞质或细胞核事件对于角化是必需的。为了解决这些问题，我们提出的具体目标是：（1）在大肠杆菌中表达caspase-14;（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;和（4）鉴定结合游离聚丝蛋白原末端肽的蛋白质，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。这些研究将提供深入了解表皮分化的生物学和常染色体显性和隐性鱼鳞病的分子基础，如寻常型鱼鳞病和层状鱼鳞病，显示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.