CORNIFIED ENVELOPE ASSOCIATED PROTEINS AND PERMEABILITY BARRIER FUNCTION

虽然结构蛋白在表皮机械屏障中的作用是明确的，但它们在通透性屏障中的作用尚不清楚。我们假设，所选择的结构和酶蛋白，即那些既受钙调控并与角化包膜(CE)相关的蛋白：总蛋白、氯化蛋白、转谷氨酰胺酶1(TG1)、填充素原和K1/10，是屏障稳态调节/所需的。这项提案将评估这些蛋白质中的哪些受到通透性屏障要求的调节，以及这些CE相关蛋白质如何与脂质结合包膜(LBE)和富含脂质的细胞外膜一起，从通透性屏障中分离出来。具体地说，在目标1中，我们将确定哪些CE相关蛋白受到改变的屏障要求的调节，以及Ca是否调节它们的表达，方法是：a)测量无毛小鼠急性屏障破坏(丙酮、磁带剥离)和必需脂肪酸缺乏症(EFAD)后CE相关蛋白的mRNA/蛋白质表达的时间进程。B)确定细胞外钙的变化(体内通过超声导入和浸泡)是否调节受调控的CE蛋白(S)的表达；以及钙受体(CAR)和电压敏感钙通道在这些变化中的参与。C)确定CE相关蛋白表达的一过性降低是否有助于屏障的恢复。使用三种转基因过表达的小鼠模型(总蛋白、K6/16和微丝蛋白原)，其中一种蛋白质干扰通透性屏障动态平衡和板层小体分泌。在目标2中，我们将确定哪些CE相关蛋白是正常屏障动态平衡所必需的，以及这些蛋白的改变导致屏障异常的机制，方法是：a)通过CE相关蛋白的特定缺失或突变来评估人类和小鼠模型的动态平衡；即，在板层状鱼鳞病(LI；TG1缺失)、表皮松解性角化过度(EHK；K1或10突变)、西门子大疱性痤疮(IBS；K2E突变)、掌足部角化病(NSPPK；K1突变)、鱼鳞病、角化病(NSPPK；K1突变)、寻常型鱼鳞病(原微丝蛋白缺乏症)、Vohwinkle病(氯蛋白突变)以及总蛋白和K10小鼠基因敲除动物。B)评估CE相关蛋白影响通透性屏障功能的机制。

Although the role of structural proteins in the epidermal mechanical barrier is clear, their role in the permeability barrier is unknown. We hypothesize that selected structural and enzymatic proteins; i.e., those that are both regulated by Ca++ and associated with the cornified envelope (CE): involucrin, loricrin, transglutaminase 1 (TG1), pro-fillaggrin, and K1/10, are regulated/required for barrier homeostasis. This proposal will assess which of these proteins are regulated by permeability barrier requirements, and how these CE-associated proteins, in partnership with the lipid bound envelope (LBE) and the lipid-enriched extracellular lamellae, from the permeability barrier. Specifically, in Aim #1 we will determine which CE- associated proteins are regulated by altered barrier requirements, and whether Ca++ regulates their expression, by: a) measuring the time course of mRNA/protein expression for the CE-associated proteins after acute barrier disruption (acetone, tape stripping) and in essential fatty acid deficiency (EFAD) in hairless mice. b) Ascertaining whether changes in extracellular Ca++ (in vivo by sonophoresis and by immersion) modulate expression of the regulated CE protein(s); and the participation of the calcium receptor (CaR) and voltage-sensitive Ca++ channels in these changes. c) Determining whether the transient decrease in CE-associated protein expression facilitates barrier recovery. Using three transgenic over- expressing (involucrin, K6/16, and pro-filaggrin) murine models, and one of these proteins interferes with permeability barrier homeostasis and lamellar body secretion. In Aim #2, we will determine which CE-associated proteins are required for normal barrier homeostasis, and the mechanisms by which alterations in these proteins lead to barrier abnormalities, by: a) Assessing homeostasis in human and murine models with specific deletions or mutations of CE-associated proteins; i.e., in patients with lamellar ichthyosis (LI; TG1 deletion), epidermolytic hyperkeratoses (EHK;K1 or 10 mutation), ichethyosis bullosa of Siemens (IBS; K2e mutation), palmo-plantar keratoderma (NSPPK; K1 mutation), ichthyosis, Keratoderma (NSPPK; K1 mutation), ichthyosis vulgaris (pro-filaggrin deficiency), Vohwinkle's disease (loricrin mutation), plus involucrin and K10 murine knockout animals. b) Assessing the mechanisms whereby CE-associated proteins influence permeability barrier function.