The Structural Basis for Control of 5-Lipoxgenease Activity

描述(申请人提供)：酶5-脂氧合酶(5-LOX)启动促炎症白三烯的合成。这些脂质介质是由双层释放的花生四烯酸(AA)在钙离子依赖的磷脂酶A2的作用下合成的。5-LOX的活性是短暂的，时间控制出现的部分原因是酶的内在不稳定性。这种不稳定性提供了一种自动调节机制，防止促炎白三烯的过度产生。然而，并不是所有的脂氧合酶都有“程序性陈旧”，而且已经鉴定出稳定的异构体。我们提出了控制5-LOX活性的三个关键方面：(1)产品特异性：5-LOX的底物是多不饱和二十烷类花生四烯酸。反应的第一步是从戊二烯的中心碳中提取氢。AA有三个戊二烯部分(和六个可能的过氧化部位，每个都有R手性或S手性)。然而，动物脂肪氧合酶通常产生单一的、区域和立体特异的产物。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2.86E分辨率的工程5-LOX结构，为这些生化和结构研究奠定了基础。(2)5-LOX中的程序性陈旧“程序性陈旧”似乎有两个组成部分：结构不稳定和基于周转的自杀抑制。我们的数据，包括我们稳定的5-LOX突变形式，表明5-LOX特有的特征导致了一个微小的C-末端被抑制，这导致了5-LOX的不稳定性。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依赖于钙离子的膜区划结合靶向底物库，促进下游酶活性的接近。实验数据支持一个模型，在该模型中，特定的钙结合位点稳定了5-LOX的C2样结构域中的“插入环”。其他人则认为5-LOX与其辅助蛋白Flop结合，这是一种完整的膜蛋白。我们建议通过实验来确定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Flip是将底物递给酶，还是简单地将AA浓缩在膜中。 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点，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-脂氧合酶成为治疗哮喘药物的靶点。拟议的研究将提供这种关键酶的结构和机制信息。

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.