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，中性粒细胞使用这些相同的机制在许多急性肾脏疾病中产生损伤，包括缺血性急性肾小管坏死、脓毒症诱导的急性肾衰竭和急性肾小球肾炎。 参与这些疾病需要从良性循环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，这一过程称为引发。我们最近发现，中性粒细胞启动依赖于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新的方法来预防和治疗几种破坏性的肾脏疾病。中性粒细胞含有四种不同类型的细胞内颗粒，每种颗粒的特征在于一组特定的腔蛋白和膜蛋白。了解胞吐作用的分子机制受到缺乏与每个颗粒相关的蛋白质知识的阻碍。这个问题是服从蛋白质组学技术的应用。本申请旨在开发 细胞内颗粒和质膜的蛋白质组图谱。将从人中性粒细胞中分离嗜天青细胞、特异性细胞和明胶酶颗粒的膜以及质膜。一项重大挑战 使用蛋白质组学方法来定义膜蛋白的一个障碍是它们由于疏水性而溶解性差。溶解度问题损害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和完整的膜蛋白。然后将使用二维电泳和质谱结合信息学来鉴定蛋白质。这些知识将使我们能够制定有关的分子机制和信号转导途径，控制中性粒细胞胞吐和启动假说。这些假设将导致NIH的应用程序，以确认这些机制，并确定方法，中断中性粒细胞参与急性肾损伤。

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.