A Proteome Map of Neutrophil Granules

描述(由申请人提供)：中性粒细胞通过其杀死入侵微生物的能力对健康做出贡献，然而，中性粒细胞在许多急性肾脏疾病中使用同样的机制来产生损伤，包括缺血性急性肾小管坏死、败血症引起的急性肾功能衰竭和急性肾小球肾炎。参与这些疾病需要从良性循环的中性粒细胞到能够广泛释放有毒氧自由基和蛋白水解酶的细胞的表型变化，这一过程被称为启动。我们最近发现，中性粒细胞的启动依赖于细胞内颗粒的胞吐作用。因此，确定胞吐作用的机制可能会导致一些毁灭性的肾脏疾病的预防和治疗的新方法。中性粒细胞包含四种不同类型的细胞内颗粒，每种颗粒都具有一组特定的管腔和膜蛋白。由于缺乏与每个颗粒相关的蛋白质的知识，阻碍了对胞吐作用的分子机制的理解。这一问题是蛋白质组学技术应用的必然结果。这项应用建议开发细胞内颗粒和质膜的蛋白质组图。从嗜天青、特异性和明胶酶颗粒中分离的膜和质膜将从人的中性粒细胞中分离出来。使用蛋白质组学方法来定义膜蛋白的一个主要挑战是由于疏水性导致它们的溶解性很差。溶解度问题影响了这些蛋白质的提取、分离和鉴定。我们建议通过应用连续增溶技术来提取膜相关的和完整的膜蛋白。然后，将使用双向电泳和质谱学结合信息学来鉴定这些蛋白质。这一知识将使我们能够提出与控制中性粒细胞胞吐和启动的分子机制和信号转导途径有关的假设。这些假说将导致美国国立卫生研究院应用来证实这些机制，并确定在急性肾损伤中阻断中性粒细胞参与的方法。

DESCRIPTION (provided by applicant): Neutrophils contribute to health through their ability to kill invading microorganisms, however, these same mechanisms are used by neutrophils to produce injury in a number of acute renal diseases, including ischemic acute tubular necrosis, sepsis-induced acute renal failure, and acute glomerulonephritis. Participation in these diseases requires a phenotypic change from benign circulating neutrophils to cells capable of extensive release of toxic oxygen radicals and proteolytic enzymes, a process termed priming. We showed recently that neutrophil priming is dependent on exocytosis of intracellular granules. Thus, defining the mechanisms of exocytosis will likely lead to new approaches to prevention and treatment of several devastating renal diseases. Neutrophils contain four different types of intracellular granules, each of which is characterized by a particular set of luminal and membrane proteins. Understanding the molecular mechanisms of exocytosis is hindered by the lack of knowledge of the proteins associated with each granule. This problem is amenable to the application of proteomic techniques. This application proposes to develop proteome maps of the intracellular granule and plasma membranes. Membranes from azurophil, specific, and gelatinase granules and plasma membranes will be isolated from human neutrophils. A major challenge to using a proteomic approach to define membrane proteins is their poor solubility due to hydrophobicity. The solubility problem impairs extraction, separation, and identification of these proteins. We propose to extract membrane-associated and integral membrane proteins through application of sequential solubilization techniques. The proteins will then be identified using two-dimensional electrophoresis and mass spectrometry, combined with informatics. This knowledge will allow us to formulate hypotheses related to the molecular mechanisms and signal transduction pathways that control neutrophil exocytosis and priming. These hypotheses will lead to NIH applications to confirm these mechanisms and to identify methods to interrupt neutrophil participation in acute renal injury.