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: 10582061
• 负责人: ALAN R. BRASH
• 金额: $ 8.05万
• 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 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10582061
• 关键词: Acute; Affect; Amino Acids; Atopic Dermatitis; Binding; Biochemical; Ceramides; Chemicals; Congenital ichthyosis; Coupling; Data; Dehydration; Dermatitis; Disease; Enzymes; Epidermis; Essential Fatty Acids; Family; Family suidae; Genes; Goals; Human; Hydroxyl Radical; Ichthyoses; Inherited; LOX gene; Link; Lipids; Lipoxygenase; Metabolism; Modeling; Mus; Neonatal; Orphan; Oxides; Oxidoreductase; Pathway interactions; Peptides; Permeability; Physiology; Property; Proteins; Psoriasis; Reaction; Recombinants; Role; Series; Skin; Structure; Syndrome; Therapeutic; Water; Work; adduct; keratinocyte differentiation; knockout gene; linoleates; microorganism toxin; oxidation; rational design; skin barrier; skin disorder; social

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.

皮肤屏障形成中脂氧合酶-肝素途径的解构 GM-134548总结/摘要 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都具有破坏性的生物学效应。 结果，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）， 对受影响家庭的社会挑战。皮肤屏障功能障碍也牵连在共同的皮肤 特应性皮炎和牛皮癣的疾病。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，它们串联作用，使必需脂肪酸亚油酸酯酯化到ω- 独特的表皮酰基神经酰胺Cer-EOS的羟基[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）。由于至今尚未解决的原因， LOX基因（或神经酰胺代谢途径中较早的其它鱼鳞病基因）的失活破坏了 神经酰胺与蛋白质角质细胞包膜的共价连接，通常形成关键的结构 屏障的特征，“角质细胞脂质包膜”，CLE。我们建议研究一个新的假设， Cer-EOS的LOX途径氧化和神经酰胺的共价偶联之间的联系，这是 屏障形成的多个步骤的最终结果。特别重要的是最近发现的一个 孤儿鱼鳞病基因SDR 9 C7，我们的初步数据确定为NAD依赖性脱氢酶， 将Cer-EOS-Hepoxilin氧化成Cer-EOS-酮-Hepoxilin。这种酮基-Hepoxilin亚结构（9，10-环氧- 11 E-13-酮）从化学先例和生物化学研究中已知自发地和特异性地结合 共价连接至蛋白质的氨基酸残基，并且因此也实现EOS-1的共价偶联。 神经酰胺因此，这一假设合理化了LOX催化氧化的需要，其最终目标是 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sdr 9 c7基因的作用 敲除小鼠皮肤中的脂氧合酶产物和神经酰胺，（ii）将分析扩展到人类和猪 （iii）确定重组SDR 9 C7的反应， LOX途径的产物。在具体目标2中，我们将（i）制备以下氨基酸加合物的真实标准品： 酮-海泊西林与氨基酸和模型肽，（ii）定性检查表皮蛋白， 定量测定共价结合的神经酰胺及其与小鼠中氨基酸残基的结合模式 表皮以及（iii）人和猪皮肤，最终通过LC-MS鉴定加合蛋白 分析回收的肽。在具体目标3中，我们将使用培养中的分化角质形成细胞来操作 并剖析这些途径，以帮助描述神经酰胺与蛋白质结合的化学机制， LOX/SDR 9 C7通路的作用。这项研究的结果将揭示潜在的机制， 表皮水屏障建设的重要方面。了解生理学允许理性的 设计的治疗，它是合理化的作用，表皮水屏障， 这个项目的最终目标。

项目成果

Analysis of 12/15-lipoxygenase metabolism of EPA and DHA with special attention to authentication of docosatrienes.

• DOI: 10.1016/j.jlr.2021.100088
• 发表时间: 2021
• 期刊: Journal of lipid research
• 影响因子: 6.5
• 作者: Jin J;Boeglin WE;Brash AR
• 通讯作者: Brash AR

Lipidomic and transcriptional analysis of the linoleoyl-omega-hydroxyceramide biosynthetic pathway in human psoriatic lesions.

• DOI: 10.1016/j.jlr.2021.100094
• 发表时间: 2021
• 期刊: Journal of lipid research
• 影响因子: 6.5
• 作者: Tyrrell VJ;Ali F;Boeglin WE;Andrews R;Burston J;Birchall JC;Ingram JR;Murphy RC;Piguet V;Brash AR;O'Donnell VB;Thomas CP
• 通讯作者: Thomas CP

Challenging the evidence for hepoxilin A3 being a mediator of neutrophil epithelial transmigration.

质疑赫泊西林 A3 是中性粒细胞上皮迁移介质的证据。

• DOI: 10.1152/ajplung.00349.2020
• 发表时间: 2020
• 期刊: American journal of physiology. Lung cellular and molecular physiology
• 作者: Brash,AlanR

Epoxide hydrolase 3 (Ephx3) gene disruption reduces ceramide linoleate epoxide hydrolysis and impairs skin barrier function.

• DOI: 10.1074/jbc.ra120.016570
• 发表时间: 2021-01
• 期刊: The Journal of biological chemistry
• 作者: Edin ML;Yamanashi H;Boeglin WE;Graves JP;DeGraff LM;Lih FB;Zeldin DC;Brash AR
• 通讯作者: Brash AR