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: 10355508
• 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 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10355508
• 关键词: Acute; Affect; Amino Acids; Appearance; Atopic Dermatitis; Binding; Binding Proteins; Biochemical; Cells; Ceramides; Chemicals; Congenital ichthyosis; Coupling; Data; Dehydration; Dermatitis; Disease; Enzymes; Epidermis; Essential Fatty Acids; Family; Family suidae; Functional disorder; Gene Deletion; Genes; Goals; Human; Hydroxyl Radical; Ichthyoses; Impairment; Inherited; Kinetics; Knock-out; LOX gene; Link; Linoleic Acids; Lipids; Lipoxygenase; Metabolism; Modeling; Modification; Mus; Neonatal; Orphan; Oxides; Oxidoreductase; Pathogenesis; Pathway interactions; Peptides; Permeability; Physiology; Play; Process; Production; Property; Proteins; Psoriasis; Reaction; Recombinants; Role; Series; Skin; Sphingosine; Structure; Syndrome; System; Therapeutic; Tissues; Water; Work; adduct; insight; keratinocyte; keratinocyte differentiation; knockout gene; linoleates; microorganism toxin; 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Lipoxygoganase-Hepoxilin途径 概括 涉及形成哺乳动物皮肤渗透性屏障的任何一个基因的缺乏 毁灭性后果，在小鼠和人类中是新生儿致死的，导致先天性鱼性病（scaly） 皮肤），对受苦家庭的社会挑战条件。皮肤屏障故障也在 特应性皮炎和牛皮癣的常见皮肤疾病。对屏障形成至关重要的两个基因是 Lipoxygyass 12R-Lox和Elox3，它们串联起作以氧化为脂肪酸linoleate 酯化为独特表皮酰亚胺疗法的欧米茄 - 羟基[e =酯化，o = omega-- 羟基]。氧化的产物是一种Linoleate-Hepoxilin（“ H​​EP”，表明羟基 - 环氧结构）。为了 迄今未解决的原因，lox基因的失活（或神经酰胺早期的其他精神病基因 代谢途径）破坏神经酰胺在蛋白质角膜膜上的共价附着， 通常形成屏障的关键结构特征，即“角膜细胞脂质信封”。我们建议 研究一个新的假设，可以识别Cer-Eos的Lox途径氧化与 神经酰胺的共价耦合，这是屏障形成多个步骤的顶点。特别 重要性是最近确定的孤儿虫病基因SDR9C7的活性，即我们的初步数据 鉴定为NAD依赖性脱氢酶，将Cer-eos-Hepoxilin氧化为Cer-eos-eTo-keto- 肝素。从化学先例中知道，这种酮 - 霍普氧蛋白的子结构（9,10-Epoxy-11e-13-酮）是知道的 生化研究以赞助商，并特别与蛋白质的氨基酸残基共价结合，并 结果还达到了EOS-陶瓷的共价耦合。因此，这个假设合理化了 需要结合神经酰胺与蛋白质并形成CLE的最终目标的Lox催化氧化。 在特定目标1中，我们将（i）定义SDR9C7基因敲除对脂氧合酶产物和 小鼠皮肤中的神经酰胺，（ii）将分析扩展到人类和猪皮肤的等效sdr9c7催化 转化，（iii）确定重组SDR9C7与LOX途径产品的反应。具体 AIM 2我们将（i）用氨基酸和 pepides模型，（ii）在定性和定量上检查表皮蛋白的共价神经酰胺 它们与氨基酸的结合模式保留在小鼠表皮中，在人和猪皮中也保留（iii）， 最终，通过对回收肽的LC-MS分析来鉴定添加的蛋白质。在特定目标中 3我们将在培养中使用差异化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 路径。这项研究的结果将揭示表皮重要方面的基础机制 水壁构建。了解生理学允许治疗的理性设计，这是 为了使该项目的最终目标的表皮水屏障多种键酶合理化。