The Structural Basis for Control of 5-Lipoxgenease Activity

控制 5-脂氧酶活性的结构基础

• 批准号: 8082429
• 负责人: MARCIA E. NEWCOMER
• 金额: $ 35.78万
• 依托单位: LOUISIANA STATE UNIV A&M COL BATON ROUGE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-08 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8082429
• 关键词: Achievement; Address; Anabolism; Animals; Antigens; Arachidonate 5-Lipoxygenase; Arachidonic Acids; Asthma; Binding; Binding Proteins; Binding Sites; Biochemical; Biological Process; Carbon; Catalytic Domain; Chemistry; Data; Development; Drug Delivery Systems; Eicosanoids; Engineering; Enzymes; Event; Foundations; Hand; Human; Hydrogen; Inflammatory; Inflammatory Response; Integral Membrane Protein; LOX gene; Leukotriene A4; Leukotriene C4; Leukotrienes; Life; Lipoxygenase; Mediating; Medicine; Membrane; Modeling; Molecular; Nuclear Envelope; Pharmaceutical Preparations; Phospholipase A2; Production; Protein Isoforms; Proteins; Reaction; Regulation; Relative (related person); Resolution; Role; Site; Specificity; Structure; System; Therapeutic; Tissues; Work; base; constriction; engineering design; enzyme activity; enzyme biosynthesis; insight; lipid mediator; mutant; peroxidation; prevent; programs; research study; suicide inactivation

DESCRIPTION (provided by applicant): The enzyme 5-lipoxygenase (5-LOX) initiates the synthesis of pro-inflammatory leukotrienes. These lipid mediators are synthesized from arachidonic acid (AA) released from the bilayer by the action of Ca2+-dependent phospholipase A2. 5-LOX activity is short-lived, and temporal control appears in part due to an intrinsic instability of the enzyme. This instability provides a mechanism for auto-regulation, preventing an over-production of pro-inflammatory leukotrienes. However, "programmed obsolescence" is not common to all lipoxygenases, and stable isoforms have been identified. We propose to address three critical aspects of control of 5-LOX activity: (1) Product specificity: The substrate for 5-LOX is the polyunsaturated eicosanoid arachidonic acid. The first step of the reaction is the abstraction of hydrogen from the central carbon of a pentadiene. AA has three pentadiene moieties (and six possible sites of peroxidation, each with either R- or S- chirality). Yet animal lipoxygenases generally produce a single, regio- and stereo- specific product. We will develop a model for 5-LOX specificity that is consistent with its product specificty. We have a 2.86E resolution structure of an engineered 5-LOX that establishes the foundation for these biochemical and structural studies. (2) Programmed obsolescence "Programmed obsolescence" in 5-LOX appears to have two components: structural instability and turnover-based suicide inhibition. Our data, including our stable mutant form of 5-LOX, suggest that features unique to 5-LOX result in a tenuously restrained C- terminus that contributes to 5-LOX instability. Experiments to define the molecular basis for non- turnover and turnover-based inactivation are proposed. (3) Compartmentalization Ca2+- dependent membrane binding of 5-LOX targets the enzyme to substrate reservoirs and promotes proximity to downstream enzyme activities. Experimental data support a model in which specific Ca2+ binding sites stabilize "insertion loops" in the C2-like domain of 5-LOX. Others have suggested that 5-LOX binds to its helper protein FLAP, an integral membrane protein. We propose experiments to define the interaction of 5-LOX with the bilayer and determine whether the catalytic domain interacts with the membrane as well, and whether FLAP hands off the substrate to the enzyme, or simply concentrates the AA in the membrane. PUBLIC HEALTH RELEVANCE: Effective therapeutic strategies require that drugs be specific for their protein targets, and therefore the structures of these targets, as well as an understanding of their molecular mechanisms, are essential to guide the development of new medicines. Because of its pivotal role in the biosynthesis of inflammatory leukotrienes, the enzyme 5-lipoxygenase is a target for drugs to treat asthma. The proposed studies will provide both structural and mechanistic information for this key enzyme.

描述（由申请人提供）：5-脂氧合酶（5-LOX）启动促炎白三烯的合成。这些脂质介质是由通过 Ca2+ 依赖性磷脂酶 A2 的作用从双层释放的花生四烯酸 (AA) 合成的。 5-LOX 活性是短暂的，并且时间控制的出现部分是由于该酶的内在不稳定性。这种不稳定性提供了一种自动调节机制，防止促炎性白三烯的过度产生。然而，“程序性报废”并非所有脂氧合酶都常见，并且已经鉴定出稳定的亚型。我们建议解决控制 5-LOX 活性的三个关键方面： (1) 产品特异性：5-LOX 的底物是多不饱和类二十烷酸花生四烯酸。反应的第一步是从戊二烯的中心碳中提取氢。 AA 具有三个戊二烯部分（以及六个可能的过氧化位点，每个位点具有 R- 或 S- 手性）。然而动物脂氧合酶通常产生单一的、区域和立体特异性的产物。我们将开发一个与其产品特异性一致的 5-LOX 特异性模型。我们拥有工程化 5-LOX 的 2.86E 分辨率结构，为这些生化和结构研究奠定了基础。 (2) 程序性报废 5-LOX 中的“程序性报废”似乎有两个组成部分：结构不稳定和基于更新的自杀抑制。我们的数据，包括 5-LOX 的稳定突变体形式，表明 5-LOX 独特的特征导致 C 末端受到轻微限制，从而导致 5-LOX 不稳定。提出了定义非周转失活和基于周转失活的分子基础的实验。 (3) 5-LOX 的区室化 Ca2+ 依赖性膜结合将酶靶向底物库并促进接近下游酶活性。实验数据支持一个模型，其中特定的 Ca2+ 结合位点稳定 5-LOX 的 C2 样结构域中的“插入环”。其他人认为 5-LOX 与其辅助蛋白 FLAP（一种完整的膜蛋白）结合。我们提出实验来定义 5-LOX 与双层的相互作用，并确定催化结构域是否也与膜相互作用，以及 FLAP 是否将底物交给酶，或者只是将 AA 浓缩在膜中。 公共健康相关性：有效的治疗策略要求药物针对其蛋白质靶点具有特异性，因此这些靶点的结构以及对其分子机制的了解对于指导新药的开发至关重要。由于其在炎症性白三烯生物合成中的关键作用，5-脂氧合酶是治疗哮喘药物的靶标。拟议的研究将为这种关键酶提供结构和机制信息。