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.

解构皮肤屏障形成中的脂氧合酶-Hepoxilin 通路 GM-134548 的摘要/摘要 与哺乳动物皮肤渗透屏障有关的任何一个基因的缺陷都会造成毁灭性的后果。 后果，对小鼠和人类的新生儿致命，导致先天性鱼鳞病（鳞状皮肤）， 对于受影响的家庭来说，皮肤屏障功能障碍也与普通皮肤有关。 脂氧合酶 12R- 是特应性皮炎和牛皮癣等疾病中对屏障形成至关重要的两个基因。 LOX 和 eLOX3，串联作用，氧化与 omega-酯化的必需脂肪酸亚油酸酯 独特的表皮酰基神经酰胺 Cer-EOS 的羟基 [E = 酯化，O = omega-羟基]。 产品是亚油酸酯-Hepoxilin（“hep”表示羟基-环氧结构）。由于迄今尚未解决的原因， LOX 基因（或神经酰胺代谢途径早期的其他鱼鳞病基因）失活会破坏 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来识别。 Cer-EOS 的 LOX 途径氧化与神经酰胺的共价偶联之间的联系，即 屏障形成过程中多个步骤的最终结果特别重要的是最近发现的一种活动。 孤儿鱼鳞病基因 SDR9C7，我们的初步数据将其确定为 NAD 依赖性脱氢酶， 将 Cer-EOS-Hepoxilin 氧化为 Cer-EOS-keto-Hepoxilin，该酮基-Hepoxilin 子结构（9,10-环氧-） 11E-13-酮）从化学先例和生化研究中已知，可以自发地、特异性地结合 与蛋白质的氨基酸残基共价结合，因此也实现了 EOS- 的共价偶联 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 sdr9c7 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) 将分析扩展到人和猪 皮肤进行等效的 SDR9C7 催化转化，(iii) 确定重组 SDR9C7 的反应 在具体目标 2 中，我们将 (i) 准备氨基酸加合物的真实标准品。 酮-Hepoxilin 与氨基酸和模型肽，(ii) 检查表皮定性蛋白质和 定量测定共价结合的神经酰胺及其与小鼠氨基酸残基的结合模式 表皮以及 (iii) 人皮和猪皮，最终通过 LC-MS 鉴定加合蛋白 在特定目标 3 中，我们将使用培养物中的分化角质形成细胞来分析回收的肽。 并剖析这些途径，以帮助表征神经酰胺与蛋白质结合的化学机制 LOX/SDR9C7 通路的作用 这项研究的结果将揭示其背后的机制。 了解表皮水屏障结构的重要方面可以进行合理的研究。 疗法的设计，是为了合理化表皮水屏障的多种关键酶的作用 这个项目的最终目标。

项目成果

Evaluation of ω-alkynyl-labeled linoleic and arachidonic acids as substrates for recombinant lipoxygenase pathway enzymes.

评估α-炔基标记的亚油酸和花生四烯酸作为重组脂氧合酶途径酶的底物。

• 发表时间: 2023-09
• 作者: Noguchi, Saori;Tallman, Keri A;Porter, Ned A;Stec, Donald F;Calcutt, M Wade;Boeglin, William E;Brash, Alan R
• 通讯作者: Brash, Alan R

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

环氧化物水解酶 3 (Ephx3) 基因破坏会减少神经酰胺亚油酸酯环氧化物水解并损害皮肤屏障功能。

• 发表时间: 2021-01
• 作者: Edin, Matthew L;Yamanashi, Haruto;Boeglin, William E;Graves, Joan P;DeGraff, Laura M;Lih, Fred B;Zeldin, Darryl C;Brash, Alan R
• 通讯作者: Brash, Alan R

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

分析 EPA 和 DHA 的 12/15-脂氧合酶代谢，特别关注二十二碳三烯的认证。

• 发表时间: 2021
• 影响因子: 6.5
• 作者: Jin, Jing;Boeglin, William E;Brash, Alan R
• 通讯作者: Brash, Alan R

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。

• 发表时间: 2020
• 作者: Brash; Alan R
• 通讯作者: Alan R

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

人类银屑病病变中亚油酰-omega-羟基神经酰胺生物合成途径的脂质组学和转录分析。

• 发表时间: 2021
• 影响因子: 6.5
• 作者: Tyrrell, Victoria J;Ali, Faraz;Boeglin, William E;Andrews, Robert;Burston, James;Birchall, James C;Ingram, John R;Murphy, Robert C;Piguet, Vincent;Brash, Alan R;O'Donnell, Valerie B;Thomas, Christopher P
• 通讯作者: Thomas, Christopher P