Deconstructing the lipoxygenase-hepoxilin pathway in skin barrier formation

解构皮肤屏障形成中的脂氧合酶-海波西林途径 GM-134548摘要/摘要 参与形成哺乳动物皮肤通透性屏障的任何一个基因的缺陷都会造成毁灭性的后果 后果，在小鼠和人类中是新生儿致命的，导致先天性鱼鳞病(鳞状皮肤)， 对于受苦家庭来说，这是一种具有社会挑战性的状况。皮肤屏障功能障碍也与常见皮肤有关 特应性皮炎和牛皮癣。两个对屏障形成至关重要的基因是脂氧合酶12R- LOX和eLOX3，它们串联作用，将必需的脂肪酸亚油酸酯氧化成欧米茄-亚油酸酯- 唯一的表皮酰神经酰胺Cer-EOS的羟基[E=酯化，O=欧米伽-羟基]。被氧化的 产品是亚油酸酯-海泊西林(“HEP”表示羟基-环氧基结构)。由于到目前为止尚未解决的原因， LOX基因(或神经酰胺代谢途径早期的其他鱼鳞病基因)的失活会中断 神经酰胺共价附着在蛋白质类角质细胞被膜上，通常形成一个关键的结构 该屏障的特征为“角膜细胞脂膜”，简称CLE。我们建议研究一种新的假说，它可以确定 Cer-EOS的LOX途径氧化与神经酰胺的共价偶联之间的联系，即 障碍形成的多个步骤达到顶峰。特别重要的是最近发现的一项活动 孤儿鱼鳞病基因SDR9C7，我们的初步数据确定为依赖NAD的脱氢酶 将Cer-EOS-Hepoxlin氧化为Cer-EOS-keto-Hepoxlin。该酮-海泊西林亚结构(9，10-环氧基- 11E-13-keto)是从化学先例和生化研究中得知的，可以自发地和特定地结合 与蛋白质的氨基酸残基共价，因此也实现了EOS的共价偶联- 神经酰胺。因此，这一假设使LOX催化氧化的需要合理化，最终目标是 神经酰胺与蛋白质结合形成CLE。在特定目标1中，我们将(I)定义sdr9c7基因的作用 小鼠皮肤中脂肪氧合酶产物和神经酰胺的基因敲除，(Ii)将分析扩展到人和猪 皮肤为等价的SDR9C7催化的转化，(III)确定重组SDR9C7的反应 使用LOX途径产品。在具体目标2中，我们将(I)制定氨基酸加合物的真实标准。 酮-海泊西林与氨基酸和模型多肽，(Ii)定性检测表皮蛋白，并 小鼠共价结合神经酰胺及其与氨基酸残基结合方式的定量研究 人和猪皮肤中的(III)，最终用LC-MS鉴定加合物蛋白 回收的多肽的分析。在特定的目标3中，我们将使用培养中分化的角质形成细胞来操纵 并剖析这些途径，以帮助表征神经酰胺与蛋白质和 LOX/SDR9C7途径的作用。这项研究的结果将揭开 表皮水屏障构建的重要方面。理解生理学允许理性的 治疗方法的设计，它是为了使表皮水屏障的多种关键酶的作用合理化 这个项目的最终目标。

Deconstructing the Lipoxygenase-Hepoxilin Pathway in Skin Barrier Formation SUMMARY/ABSTRACT of GM-134548 A deficiency in any one of the genes involved in forming the mammalian skin permeability barrier has devastating consequences, being neonatal lethal in mice and in humans leading to congenital ichthyosis (scaly skin), a socially challenging condition for afflicted families. Skin barrier malfunction is also implicated in the common skin diseases of atopic dermatitis and psoriasis. Two genes critical to barrier formation are the lipoxygenases 12R- LOX and eLOX3, which act in series to oxygenate the essential fatty acid linoleate esterified to the omega- hydroxyl of the unique epidermal acylceramide Cer-EOS [E = esterified, O = omega-hydroxy]. The oxidized product is a linoleate-Hepoxilin (“hep” indicating a hydroxy-epoxy structure). For reasons heretofore unresolved, inactivation of the LOX genes (or other ichthyosis genes earlier in the ceramide metabolism pathway) disrupts the covalent attachment of ceramide to the proteinaceous corneocyte envelope, normally forming a key structural feature of the barrier, the “corneocyte lipid envelope”, CLE. We propose to study a new hypothesis that identifies the link between the LOX pathway oxidations of Cer-EOS and the covalent coupling of ceramides, which is the culmination of multiple steps in barrier formation. Of special importance is the activity of a recently identified orphan ichthyosis gene SDR9C7, that our preliminary data identifies as a NAD-dependent dehydrogenase that oxidizes the Cer-EOS-Hepoxilin to a Cer-EOS-keto-Hepoxilin. This keto-Hepoxilin sub-structure (9,10-epoxy- 11E-13-keto) is known from chemical precedent and biochemical studies to spontaneously and specifically bind covalently to amino acid residues of protein, and as a consequence also achieve covalent coupling of the EOS- ceramide. This hypothesis thus rationalizes the need for LOX-catalyzed oxidations with the ultimate goal of binding ceramide to protein and forming the CLE. In Specific Aim 1 we will (i) define the effects of sdr9c7 gene knockout on the lipoxygenase products and ceramides in mouse skin, (ii) extend the analyses to human and pig skin for the equivalent SDR9C7-catalyzed transformations, (iii) determine the reactions of recombinant SDR9C7 with LOX pathway products. In Specific Aim 2 we will (i) prepare authentic standards of amino acid adducts of keto-Hepoxilin with amino acids and model peptides, (ii) examine epidermal proteins qualitatively and quantitatively for covalently bound ceramides and their mode of binding to amino acid residues in mouse epidermis and also (iii) in human and pig skin, ultimately with identification of the adducted proteins by LC-MS analysis of recovered peptides. In Specific Aim 3 we will use differentiated keratinocytes in culture to manipulate and dissect these pathways to help characterize the chemical mechanisms of ceramide binding to protein and the role of the LOX/SDR9C7 pathway. The results of this study will unravel the mechanisms underlying an important facet of epidermal water barrier construction. Understanding the physiology allows for the rational design of therapeutics, and it is to rationalize the role of multiple key enzymes of the epidermal water barrier that this project’s ultimate goal.