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，角质层在生物体和环境之间提供了一种保护性屏障。从活的表皮颗粒细胞到无核角化鳞片的快速转变受到多个信号分子和通路的调控，而这些信号分子和通路对此知之甚少。参与终末分化过程的一组重要分子是细胞内蛋白酶，它能裂解表皮蛋白，导致细胞器的破坏和角质层的形成。这个项目的总体目标是确定caspase 14的功能，这是一种在末端分化过程中被激活的表皮特异性半胱氨酸天冬氨酸蛋白酶，并检测在钙结合蛋白profilgrin的蛋白分解过程中释放的游离前微丝蛋白末端多肽的作用。有待检验的假设是：(1)caspase-14通过切割对启动和/或执行终末分化至关重要的结构和/或调节蛋白，在角化过程中发挥重要的生物学作用；(2)游离profilaggrin末端通过与包括Annexin和14-3-3家族成员在内的其他角质形成细胞蛋白相互作用，在调节Profilaggrin加工和其他依赖钙的细胞质或核事件中具有特定的作用，这些事件对角化至关重要。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并纯化活性酶；(2)测定caspase-14的底物特异性和天然角质形成细胞靶点；(3)确定caspase-14的底物专一性和天然角质形成细胞靶点；(3)通过靶向干扰确定caspase-14在小鼠体内的功能；以及(4)鉴定与游离微丝蛋白前体末端肽结合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(S)。这些研究将深入了解表皮分化的生物学以及常染色体显性和隐性鱼鳞病的分子基础，如普通型鱼鳞病和板层型鱼鳞病，这些鱼鳞病表现出表皮屏障角质层结构和功能的缺陷。

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.