Deconstructing the lipoxygenase-hepoxilin pathway in skin barrier formation

皮肤屏障形成中脂氧合酶-肝素途径的解构 总结 参与形成哺乳动物皮肤渗透性屏障的任何一种基因的缺陷， 毁灭性的后果，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），这对患病家庭来说是一种具有社会挑战性的状况。皮肤屏障功能障碍也与 常见的皮肤病有特应性皮炎和银屑病。对屏障形成至关重要的两个基因是 脂氧合酶12 R-LOX和eLOX 3，其串联作用以使必需脂肪酸亚油酸酯水解 酯化至独特的表皮酰基神经酰胺Cer-EOS的ω-羟基[E =酯化，O = ω- 羟基]。氧化产物是亚油酸酯-Hepoxilin（“hep”表示羟基-环氧结构）。为 原因迄今尚未解决，LOX基因（或其他鱼鳞病基因）的失活在神经酰胺 代谢途径）破坏神经酰胺与蛋白质性角质细胞包膜的共价连接， 通常形成屏障的关键结构特征，“角质细胞脂质包膜”，CLE。我们建议 研究一个新的假设，确定铈-EOS的LOX途径氧化和 神经酰胺的共价偶联，这是屏障形成中多个步骤的顶点。特殊 重要的是最近发现的孤儿鱼鳞病基因SDR 9 C7的活性，我们的初步数据 鉴定为NAD依赖性脱氢酶，其将Cer-EOS-Hepoxilin氧化为Cer-EOS-酮- 肝素。这种酮基-海泊昔林亚结构（9，10-环氧基-11 E-13-酮基）从化学先例中已知， 生物化学研究自发地和特异性地共价结合蛋白质的氨基酸残基， 结果也实现了EOS-神经酰胺的共价偶联。因此，这一假设合理化了 需要LOX催化的氧化，最终目的是将神经酰胺结合到蛋白质上并形成CLE。 在具体目标1中，我们将（i）定义sdr 9 c7基因敲除对脂氧合酶产物的影响， 神经酰胺在小鼠皮肤，（ii）扩展分析人类和猪皮肤的等效SDR 9 C7催化 （iii）确定重组SDR 9 C7与LOX途径产物的反应。在特定 目的2：（i）制备酮-海泊西林与氨基酸的氨基酸加合物的真实标准品， 模型肽，（ii）定性和定量地检查表皮蛋白质中共价结合的神经酰胺 以及它们与小鼠表皮以及（iii）人和猪皮肤中氨基酸残基的结合模式， 最终通过回收的肽的LC-MS分析鉴定加合蛋白。具体目标 3我们将使用分化的角质形成细胞在文化中操纵和解剖这些途径，以帮助 表征神经酰胺与蛋白质结合的化学机制以及LOX/SDR 9 C7的作用 通路这项研究的结果将揭示表皮细胞的一个重要方面的机制， 水屏障建设。了解生理学允许合理设计治疗方法，这是 合理的表皮水屏障的多个关键酶的作用，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.