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 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导致 毁灭性的后果，在小鼠和人类中是新生儿致死的，导致先天性鱼鳞病(鳞片 皮肤)，这对受影响的家庭来说是一种具有社会挑战性的疾病。皮肤屏障功能障碍也与 特应性皮炎和牛皮癣的常见皮肤病。两个对屏障形成至关重要的基因是 脂氧合酶12R-LOX和eLOX3串联作用，氧化必需脂肪酸亚油酸 酯化为唯一的表皮酰神经酰胺Cer-EOS的欧米伽-羟基[E=酯化，O=欧米伽- 羟基]。氧化产物是亚油酸酯-海泊西林(“HEP”，表示羟基-环氧基结构)。为 迄今为止，LOX基因(或神经酰胺中较早的其他鱼鳞病基因)失活的原因尚未解决 代谢途径)破坏神经酰胺与蛋白质质角质细胞被膜的共价附着， 正常情况下，形成屏障的一个关键结构特征是“角膜细胞脂膜”，即CLE。我们建议 研究一种新的假说，确定Cer-EOS的LOX途径氧化与 神经酰胺的共价偶联，这是屏障形成过程中多个步骤的结果。特别之处 重要的是最近发现的孤儿鱼鳞病基因SDR9C7的活性，我们的初步数据 鉴定为一种依赖NAD的脱氢酶，可将Cer-EOS-Hepoxlin氧化为Cer-EOS-keto- 贺波西林。这种酮-海泊西林亚结构(9，10-环氧基-11E-13-酮)是从化学先例和 自发和特异地与蛋白质的氨基酸残基结合的生化研究，以及AS 结果还实现了EOS-神经酰胺的共价偶联。因此，这一假设使 需要LOX催化的氧化，最终目标是将神经酰胺结合到蛋白质上，形成CLE。 在特定的目标1中，我们将(I)确定sdr9c7基因敲除对脂氧合酶产物和 小鼠皮肤中的神经酰胺，(Ii)将SDR9C7催化的等效神经酰胺的分析扩展到人和猪的皮肤 转化，(Iii)确定重组SDR9C7与LOX途径产物的反应。具体而言 目的：(1)制备酮类海泊西林氨基酸加合物的标准品。 模型多肽，(Ii)定性和定量检测共价结合神经酰胺的表皮蛋白 以及它们与小鼠表皮中氨基酸残基的结合方式，以及(Iii)在人和猪皮肤中的结合方式， 最终通过LC-MS分析回收的多肽对加合蛋白进行鉴定。以特定的目标 3我们将使用培养中分化的角质形成细胞来操纵和剖析这些途径，以帮助 神经酰胺与蛋白质结合的化学机制及LOX/SDR9C7的作用 路径。这项研究的结果将揭开表皮一个重要方面的潜在机制。 防水层施工。了解生理学有助于合理设计治疗学，而且它是 合理发挥多种关键酶的作用，是本项目的最终目标。

Deconstructing the Lipoxygenase-Hepoxilin Pathway in Skin Barrier Formation SUMMARY 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.