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; 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）：酶5-脂氧酶（5-lox）启动了促炎白细胞的合成。这些脂质介质是通过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，时间控制部分是由于酶的内在不稳定。这种不稳定性提供了一种自动调节的机制，防止了促炎白细胞的过度生产。然而，“程序化过时”并不是所有脂氧酶的共同点，并且已经鉴定出稳定的同工型。我们建议解决5-lox活性控制的三个关键方面：（1）产品特异性：5-lox的底物是多不饱和的eicosananoid arachidonic Acid。反应的第一步是从戊二烯的中央碳中氢气的抽象。 AA具有三个五烯部分（以及六个可能的过氧化位点，每个都有R-或S-手性）。然而，动物Lipoxygyass通常会产生单一的，区域和立体的产品。我们将为5-LOX特异性开发模型，该模型符合其产品的特殊性。我们具有2.86E工程的5-LOX分辨率结构，为这些生化和结构研究奠定了基础。 （2）5-LOX中编程的过时的“编程过时”似乎具有两个组成部分：结构不稳定性和基于营业额的自杀抑制。我们的数据，包括我们稳定的5-lox突变体形式，表明5-lox独有的特征导致了严格约束的c-末端，这导致了5-lox不稳定性。提出了定义非离职和基于离职的灭活的分子基础的实验。 （3）5-lox的隔室化Ca2+ - 依赖性膜结合靶向酶以底物储层，并促进与下游酶活性的接近度。实验数据支持一个模型，其中特定的Ca2+结合位点在5-LOX的C2样域中稳定“插入环”。其他人则建议5-lox与其辅助蛋白皮瓣（一种整体膜蛋白）结合。我们提出的实验是定义5-LOX与双层的相互作用，并确定催化域是否也与膜相互作用，以及是否将皮瓣移开底物转移到酶上，还是简单地将AA浓缩在膜上。 公共卫生相关性：有效的治疗策略要求药物针对其蛋白质靶标具有特异性，因此这些靶标的结构以及对它们的分子机制的理解对于指导新药物的开发至关重要。由于其在炎症白细胞的生物合成中的关键作用，酶5-脂氧合酶是治疗哮喘的药物的靶标。拟议的研究将为此关键酶提供结构和机械信息。