PROFILAGGRIN IN EPIDERMAL DIFFERENTIATION AND FUNCTION

描述：(改编自申请人的摘要)-Profilaggrin是一种 丰富的、高度磷酸化的钙结合蛋白在 角质形成前表皮角质形成细胞的分化 细胞。Profilaggrin包含一个N-末端钙结合结构域和 多个细丝研磨单元。建议每个地区都有特定的、 一旦profilaggrin被切割，在体内的不同功能 N-末端和微丝蛋白多肽。这项提议的目标是 通过表达来理解Profilaggrin这些结构域的功能(S) 无功能钙结合突变蛋白的角质形成细胞 并通过靶向破坏胚胎干细胞来创造 Profilaggrin缺陷小鼠。新证据表明，衍生的微丝蛋白 Profilaggrin通过特定的蛋白降解，在培养的 角质形成细胞作为角蛋白聚集蛋白，导致角质形成细胞崩溃 角蛋白细胞骨架、细胞形状变化和细胞周期停滞。 微丝蛋白在体内进一步分解为游离氨基酸， 结合水分，被认为能维持角膜细胞的正常渗透压。 要检验的假设是：(1)钙结合结构域 Profilaggrin在调节钙依赖中的特定功能(S) 表皮角化的事件，包括Profilaggrin的表达和 对微丝蛋白的蛋白质水解性加工，以及(2)微丝蛋白，通过其作为 一种角蛋白相关蛋白，是吸湿性氨基酸的主要来源 酸，在调节正常角膜细胞中起关键作用 成熟和功能。为了解决这些问题，具体目标是 提出：(1)确定N-末端钙结合的功能 Profilaggrin的结构域，首先取消其结合钙和 然后在稳定的角质形成细胞系和转基因小鼠中表达 (2)靶向测定Profilaggrin在体内的功能(S) 对小鼠的破坏。Profilaggrin缺陷小鼠将允许评估 Profilaggrin在体内的功能意义。这项工作也应该 为理解生物化学的意义提供了坚实的基础 Profilagrin在皮肤病中表达降低或缺失，如 寻常型鱼鳞病和银屑病与Profilaggrin加工失败 在严重的鳞屑障碍小丑鱼鳞病中使用微丝凝集素。

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.