Deconstructing the lipoxygenase-hepoxilin pathway in skin barrier formation

解构皮肤屏障形成中的脂氧合酶-海泊西林途径

• 批准号: 10576839
• 负责人: ALAN R. BRASH
• 金额: $ 40.29万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10576839
• 关键词: Acute; Affect; Amino Acids; Appearance; Atopic Dermatitis; Binding; Binding Proteins; Biochemical; Bypass; Cells; Ceramides; Chemicals; Congenital ichthyosis; Coupling; Data; Dehydration; Dermatitis; Disease; Enzymes; Epidermis; Essential Fatty Acids; Esterification; Family; Family suidae; Functional disorder; Gene Deletion; Genes; Goals; Human; Hydroxyl Radical; Hydroxylation; Ichthyoses; Impairment; Inherited; Invaded; Kinetics; Knock-out; LOX gene; Link; Linoleic Acids; Lipids; Lipoxygenase; Metabolism; Modeling; Modification; Mus; Neonatal; Orphan; Oxidoreductase; Pathogenesis; Pathway interactions; Peptides; Permeability; Physiology; Play; Process; Production; Property; Proteins; Psoriasis; Rationalization; Reaction; Recombinants; Role; Series; Skin; Sphingosine; Structure; Syndrome; System; Therapeutic; Tissues; Toxin; Water; Work; adduct; autosome; insight; keratinocyte; keratinocyte differentiation; knockout gene; linoleates; microorganism; oxidation; oxidized lipid; rational design; skin barrier; skin disorder; social; three dimensional cell culture

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.

解构皮肤屏障形成中的脂氧合酶-Hepoxilin 通路 概括 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，对小鼠和人类的新生儿来说是致命的，导致先天性鱼鳞病（鳞状鱼鳞病） 皮肤），这对于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 常见的皮肤病有特应性皮炎和牛皮癣。对屏障形成至关重要的两个基因是 脂氧合酶 12R-LOX 和 eLOX3，串联作用以氧化必需脂肪酸亚油酸酯 与独特的表皮酰基神经酰胺 Cer-EOS 的 omega-羟基酯化 [E = 酯化，O = omega- 羟基]。氧化产物是亚油酸酯-Hepoxilin（“hep”表示羟基-环氧结构）。为了 迄今为止尚未解决的原因，LOX基因（或神经酰胺早期的其他鱼鳞病基因）的失活 代谢途径）破坏神经酰胺与蛋白质角质细胞包膜的共价附着， 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，该假设确定了 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最终结果。特殊的 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 被鉴定为 NAD 依赖性脱氢酶，可将 Cer-EOS-Hepoxilin 氧化为 Cer-EOS-keto- 赫泊西林。这种酮-Hepoxilin 子结构（9,10-环氧-11E-13-酮）是从化学先例中已知的，并且 自发地、特异性地与蛋白质的氨基酸残基共价结合的生化研究，以及 结果还实现了 EOS-神经酰胺的共价偶联。因此，这个假设合理化了 需要 LOX 催化的氧化反应，最终目标是将神经酰胺与蛋白质结合并形成 CLE。 在具体目标 1 中，我们将 (i) 定义 sdr9c7 基因敲除对脂氧合酶产物的影响，以及 小鼠皮肤中的神经酰胺，(ii) 将分析扩展到人类和猪皮肤，以获得等效的 SDR9C7 催化 转化，(iii)确定重组SDR9C7与LOX途径产物的反应。具体来说 目标 2 我们将 (i) 制备酮-Hepoxilin 与氨基酸的氨基酸加合物的真实标准品，以及 模型肽，(ii) 定性和定量检查表皮蛋白中的共价结合神经酰胺 以及它们与小鼠表皮以及（iii）人和猪皮肤中的氨基酸残基的结合模式， 最终通过回收肽的 LC-MS 分析来鉴定加合蛋白。特定目标 3 我们将在培养物中使用分化的角质形成细胞来操纵和剖析这些途径，以帮助 表征神经酰胺与蛋白质结合的化学机制以及 LOX/SDR9C7 的作用 途径。这项研究的结果将揭示表皮重要方面的潜在机制。 防水屏障建设。了解生理学有助于合理设计治疗方法，而且 理顺表皮水屏障多种关键酶的作用是该项目的最终目标。