PROFILAGGRIN IN EPIDERMAL DIFFERENTIATION AND FUNCTION

描述：（改编自申请人的摘要）-聚丝蛋白原是一种 丰富的，高度磷酸化的，钙结合蛋白表达， 在形成角质形成细胞之前分化表皮角质形成细胞 细胞 聚丝蛋白原含有N-末端钙结合结构域， 多个丝聚蛋白单位。 建议每个区域都有具体的， 一旦聚丝蛋白原被切割产生聚丝蛋白， N-末端和聚丝蛋白肽。 本提案的目的是 理解这些前丝聚蛋白结构域的功能， 具有非功能性钙结合的突变蛋白质的角质形成细胞 结构域，并通过有针对性地破坏胚胎干细胞， 原丝聚集蛋白缺陷小鼠。 新的证据表明， 从原丝聚蛋白通过特异性蛋白水解，在培养的 角化细胞作为角蛋白聚集蛋白，导致 角蛋白细胞骨架、细胞形状变化和细胞周期停滞。 聚丝蛋白在体内进一步蛋白水解成游离氨基酸， 结合水并被认为维持角质细胞的正常渗透压。 待检验的假设是：（1） 聚丝蛋白原在调节钙依赖性细胞内钙离子浓度方面具有特异性功能， 表皮角化事件，包括聚丝蛋白原表达和 蛋白水解加工成丝聚蛋白，和（2）丝聚蛋白，通过其作为 一种角蛋白相关蛋白，作为吸湿氨基的主要来源， 酸，在调节正常角质细胞中起关键作用 成熟和功能。 为了解决这些问题， 提出的是（1）确定N端钙结合的功能， 通过首先消除其结合钙的能力， 然后在稳定的角质形成细胞系和转基因小鼠中表达 和（2）通过靶向的免疫细胞化学方法测定聚丝蛋白原在体内的功能 在小鼠中的破坏。 前丝聚集蛋白缺陷小鼠将允许评估 原丝聚蛋白在体内的功能意义。 这项工作还应 为理解生物化学意义提供了坚实的基础。 在皮肤病中丝聚蛋白原表达减少或缺失， 寻常性鱼鳞病和银屑病与聚丝蛋白原加工失败 与丝聚蛋白在严重的鱼鳞病中的作用

DESCRIPTION: (Adapted from the applicant's abstract) - Profilaggrin is an abundant, highly phosphorylated, calcium-binding protein expressed in differentiating epidermal keratinocytes just prior to formation of cornified cells. Profilaggrin contains an N-terminal calcium-binding domain and multiple filaggrin units. Each region is proposed to have specific, distinct functions in vivo once profilaggrin is cleaved to yield the N-terminal and filaggrin peptides. The goal of this proposal is to understand the function(s) of these domains of profilaggrin by expression in keratinocytes of mutant proteins with a non-functional calcium-binding domain and by targeted disruption in embryonic stem cells to create profilaggrin-deficient mice. New evidence suggests that filaggrin, derived from profilaggrin by specific proteolysis, functions in cultured keratinocytes as a keratin-aggregating protein, leading to collapse of the keratin cytoskeleton, changes in cell shape, and cell cycle arrest. Filaggrin undergoes further proteolysis in vivo to free amino acids which bind water and are thought to maintain normal osmolarity of corneocytes. The hypotheses to be tested are (1) The calcium-binding domain of profilaggrin has a specific function(s) in regulating the calcium-dependent events of epidermal cornification, including profilaggrin expression and proteolytic processing to filaggrin, and (2) filaggrin, through its roles as a keratin-associated protein and as the major source of hygroscopic amino acids, performs critical functions in regulating normal corneocyte maturation and function. To address these questions, the specific aims proposed are to (1) Determine the function of the N-terminal calcium-binding domain of profilaggrin by first abolishing its ability to bind calcium and then expressing it in stable keratinocyte cell lines and in transgenic mice and (2) determine the function(s) of profilaggrin in vivo by targeted disruption in mice. Profilaggrin-deficient mice will allow assessment of the functional significance of profilaggrin in vivo. This work should also provide a firm basis for understanding the biochemical significance of reduced or absent profilaggrin expression in skin diseases such as ichthyosis vulgaris and psoriasis and the failure of profilaggrin processing to filaggrin in the severe scaling disorder Harlequin ichthyosis.