Protease-Mediated Events in Epidermal Differentiation

表皮分化中蛋白酶介导的事件

基本信息

批准号：
6612752
负责人：
RICHARD B. PRESLAND
金额：
$ 33.09万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-07-15 至 2007-05-31
项目状态：
已结题

项目摘要

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.

表皮角质形成细胞的末端分化导致形成结构，即角质层，该结构在生物体及其环境之间提供了保护屏障。从活着的表皮颗粒细胞到Anuleclete的巨型蹲下的快速过渡受多种信号分子和途径的调节。终端分化过程中涉及的一组重要分子是细胞内蛋白酶，裂解表皮蛋白会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的功能，这是一种在末端分化过程中激活的表皮特异性半胱氨酸天冬氨酸蛋白酶，并检查了在钙结合蛋白蛋白胶原蛋白的蛋白水解过程中释放的游离pro-Filaggrin末端肽的作用。要测试的假设是（1）caspase-14通过切割结构和/或调节蛋白在角化过程中起重要的生物学作用，这对于启动和/或执行终末分化至关重要；（2）通过与包括膜联蛋白和14-3-3家族在内的其他角质细胞蛋白相互作用，自由profilaggrin末端在调节叶子蛋白依赖性的细胞质或核事件中具有特定的作用。为了解决这些问题，提出的具体目的是（1）在大肠杆菌中表达caspase-14并纯化活性酶；（2）确定caspase-14的底物特异性和天然角质形成靶；（3）确定caspase-14的底物特异性和天然角质形成靶标；（3）通过小鼠的靶向破坏来确定caspase-14在体内的功能；（4）鉴定结合游离前纤维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。这些研究将洞悉表皮分化的生物学以及常染色体显性和隐性鱼质病的分子基础，例如紫罗兰鱼裂和层状鱼质病，在表皮屏障的角质层结构和功能中表现出缺陷。