STRUCTURE/FUNCTION STUDIES OF ANTIHEMOSTATIC LIPOCALINS

吸血昆虫的唾液中含有多种抗止血剂 有助于获得不间断的血液流向 口器这些药物包括血管扩张剂，血小板聚集抑制剂， 和抗凝血剂。 这些物质大多是蛋白质， 已经确定了一些目标。 然而，分子 它们的作用机制和结构特征， 它们的活动基本上是未知的。 这一建议主要针对两种类型 从锥蝽的唾液中提取的抗止血剂 prolixus，抗凝血剂nitrophorin 2和三种蛋白质， 唾液抗血小板脂质运载蛋白（SAPLs）。Nitrophorins是一组 四种多功能血红素蛋白，其转运一氧化氮并结合 组胺 Nitrophorin 2是唯一一个 抗凝血活性也是如此。SAPLs 1-3抑制胶原介导的 血小板聚集和凝血酶活性。 令人惊讶的是， 硝化蛋白和SAPL属于脂质运载蛋白家族， 完全不同的活动。 这些测试中的假设 研究表明，硝酸转运蛋白2的抗凝活性和 SAPLs 1-3的抗血小板活性是由于它们之间的相互作用， 具有特异性蛋白质靶点的分子或结合必需的 配体。 本研究的具体目的是分析这些机制 和硝化蛋白2和SAPLs 1-3活性的结构决定因素 使用功能分析、X射线晶体学和定点 诱变

The saliva of blood-feeding insects contains a variety of antihemostatic agents that aid in obtaining an uninterrupted flow of blood to the mouthparts. These include vasodilators, platelet aggregation inhibitors, and anticoagulants. Most of these substances are proteinaceous, and a number of their targets have been identified. However, the molecular mechanisms for their actions and the structural features responsible for their activity are largely unknown. This proposal focuses on two types of antihemostatic agents from the saliva of the triatomine bug Rhodnius prolixus, the anticoagulant nitrophorin 2 and three proteins known as salivary antiplatelet lipocalins (SAPLs). Nitrophorins are a group of four multifunctional hemeproteins which transport nitric oxide and bind histamine. Nitrophorin 2 is the only member of the group possessing anticoagulant activity as well. SAPLs 1-3 inhibit collagen-mediated platelet aggregation and thrombin activity. Surprisingly, both the nitrophorins and SAPLs belong to the lipocalin protein family despite their completely different activities. The hypotheses tested in these studies are that the anticoagulant activity of nitrophorin 2 and the antiplatelet activity of SAPLs 1-3 are due to interaction of these molecules with specific proteinaceous targets or binding of essential ligands. The specific aims of the study are to analyze the mechanisms and structural determinants of activity of nitrophorin 2 and SAPLs 1-3 using functional assays, X-ray crystallography, and site-directed mutagenesis.