Reactive Intermediates in Lipoxygenase Pathways

脂氧合酶途径中的反应中间体

• 批准号: 7925055
• 负责人: BETTY J GAFFNEY
• 金额: $ 7.83万
• 依托单位: FLORIDA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7925055
• 关键词: Active Sites; Arachidonic Acids; Binding; Binding Sites; Carbon; Cell physiology; Cells; Chronic; Cysteine; Dimensions; Docosahexaenoic Acids; Electron Spin Resonance Spectroscopy; Enzymes; Esters; Excision; Family; Fatty Acids; Health; Housing; Human; Infection; Inflammation Mediators; Inflammatory Response; Iron; LOX gene; Label; Left; Leukocytes; Ligand Binding; Ligands; Linoleic Acids; Lipid Peroxides; Lipids; Lipoxygenase; Lipoxygenase 2; Lipoxygenase Inhibitors; Location; Lung; Maps; Measurement; Medical; Methods; Modeling; Molecular Conformation; Mutation; N-terminal; Organism; Oxygen; Pathway interactions; Pharmacologic Substance; Physiologic pulse; Positioning Attribute; Protein Isoforms; Proteins; Pseudomonas aeruginosa; Relaxation; Research; Resolution; Shapes; Side; Signal Pathway; Signaling Molecule; Site; Soybeans; Spectrum Analysis; Spin Labels; Staging; Stearates; Structure; Substrate Interaction; Tissues; Trees; Work; allene oxide synthase; analog; base; coral; design; fatty acid-binding proteins; inhibitor/antagonist; injured; interest; member; migration; mutant; pathogen; protein structure; public health relevance; research study; response

DESCRIPTION (provided by applicant): Lipoxygenase enzymes catalyze the first step in one of the major pathways to signalling molecules derived from arachidonic acid. A theme emerging from the medical perspective on different human lipoxygenases is one of both positive and negative influences on human health from these molecules. The overall focus of this proposal is to understand how the active site cavity of lipoxygenase domains interacts with fatty acids in catalytically relevant states. The approaches developed will have broader applications to inhibition of lipoxygenase and other pharmaceutical target enzymes having lipid substrates, and to design of NMR structural studies based on paramagnetic relaxation. Here, electron paramagnetic resonance (EPR) spectroscopy experiments are proposed to map interactions of substrate/product analogs with the active site cavity of a lipoxygenase. The prototypical lipoxygenase, soybean isoform 1, LOX-1, is the subject of most experiments. The specific aims are: Specific Aim 1- A grid of distances between spins in doubly-spin labeled LOX-1 will be established by pulsed dipolar EPR spectroscopy (PDS). Positions of introduced spin labels within the structure will be determined by triangulation and modeling, to facilitate subsequent protein-ligand distance determinations. Specific Aim 2- The location of the doxyl spin of spin label stearates (DSAs), inhibitors of lipoxygenase, will be determined by EPR distance measurements between DSAs and covalently spin labeled LOX-1 grid sites. Specific Aim 3- A hypothesis that fatty acid hydroperoxides (activators) and the substrate interact differently with the lipoxygenase cavity will be examined using spin label nitroxide esters at the polar ends of substrate and product analogs. Each of the spin label esters proposed in this section will be prepared in two versions: with an oleic acyl chain for EPR distance measurements and with a linoleoyl chain for analysis of enzymatic products. Specific Aim 4- Fatty acid binding to the LOX-1 mutant, A542T, that is inactive for turnover, will be characterized with EPR studies of ferric iron in the mutant and spin-labeled fatty acid localization. Specific Aim 5- Similar approaches will be taken to map fatty acid interactions with the coral allene oxide synthase (cAOS) cavity. PUBLIC HEALTH RELEVANCE: In humans, lipoxygenase isoforms are tissue specific and are related to pathological and house-keeping cell functions and inhibitors must be selective. The work proposed will develop substrate analogs that include a versatile spectroscopic functionality that can be used to examine how and where an inhibitor resides in the protein structure. Lipoxygenases are important in other organisms also and the methods could be widely applied to understand non-mammalian signalling pathways as well. The type of medium-resolution EPR structure obtained here can provide essential background for higher resolution NMR structure studies.

描述（由申请人提供）：脂氧合酶酶催化了源自蛛网膜酸的信号分子的主要途径之一。从医学角度出现的主题是对不同人类脂氧酶的主题，是对这些分子对人类健康的积极和负面影响之一。该提案的总体重点是了解脂氧合酶结构域的活性位点腔与催化相关状态中的脂肪酸相互作用。开发的方法将在抑制具有脂质底物的脂氧合酶和其他药物靶酶以及基于顺磁性弛豫的NMR结构研究设计方面具有更广泛的应用。在这里，提出了电子顺磁共振（EPR）光谱实验，以绘制底物/产物类似物与脂氧酶的活性位点腔的相互作用。典型的lipoxygogy酶，大豆同工型1，LOX-1是大多数实验的主题。具体目的是：特定的目标1-将通过脉冲型偶极EPR光谱（PDS）建立双旋转标记的LOX-1的自旋之间的距离网格。引入的自旋标记在结构内的位置将由三角测量和建模确定，以促进随后的蛋白质配体距离测定。具体目标2-脂氧酶抑制剂的自旋标记硬脂酸酯（DSAS）的紫xy旋旋转的位置将由DSA和共价旋转标记的LOX-1网格位点之间的EPR距离测量值确定。特定目的3-一种假设，即将使用自旋标记硝基氧化酯在底物和产物类似物的极端使用自旋标记硝基氧化酯，将与脂氧合酶腔的相互作用不同。本节中提出的每个自旋标记酯都将以两个版本制备：具有乙酰酰基链，用于EPR距离测量，并带有lieneoyoyl链，用于分析酶产物。特定的目标4-脂肪酸与LOX-1突变体A542T结合（无活性）将以EPR研究在突变体和自旋标记的脂肪酸定位中的EPR研究。特定的目标5-将采用类似的方法来绘制脂肪酸相互作用与珊瑚Allene氧化物合酶（CAOS）腔。公共卫生相关性：在人类中，脂氧合酶同工型是组织特异性的，并且与病理和保管细胞功能有关，并且抑制剂必须具有选择性。提出的工作将开发底物类似物，其中包括多功能光谱功能，该功能可用于检查抑制剂在蛋白质结构中的含量和地点。脂氧酶在其他生物体中也很重要，并且这些方法也可以广泛应用于了解非哺乳动物信号通路。此处获得的中分辨率EPR结构的类型可以为高分辨率NMR结构研究提供必不可少的背景。