A Proteome Map of Neutrophil Granules

中性粒细胞颗粒的蛋白质组图谱

• 批准号: 6739106
• 负责人: Kenneth R MCLEISH
• 金额: $ 14.7万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2005-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6739106
• 关键词: biological signal transduction; cell membrane; clinical research; collagenase; cytotoxicity; electrospray ionization mass spectrometry; exocytosis; granule; high performance liquid chromatography; human tissue; kidney disorder; matrix assisted laser desorption ionization; membrane proteins; neutrophil; protein structure; proteomics

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.

描述（由申请人提供）：中性粒细胞通过杀死入侵微生物的能力为健康做出了贡献，但是，中性粒细胞使用这些相同的机制来在许多急性肾脏疾病中造成损伤，包括缺血性急性管状肾上腺炎，症状，急性肾脏肾衰竭，急性肾衰竭，肌肌张力肌肌张力肌炎。 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，这是一种称为启动的过程。我们最近表明，中性粒细胞启动取决于细胞内颗粒的胞吐作用。因此，定义胞吐作用的机制可能会导致预防和治疗几种毁灭性肾脏疾病的新方法。中性粒细胞包含四种不同类型的细胞内颗粒，每种颗粒的特征是一组特定的腔和膜蛋白。由于缺乏与每个颗粒相关的蛋白质的知识，因此阻碍了胞吐作用的分子机制。这个问题适合应用蛋白质组学技术。该应用建议开发细胞内颗粒和质膜的蛋白质组图。来自硫醇，特异性和明胶酶颗粒和质膜的膜将从人嗜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主要挑战是由于疏水性而导致其溶解度差。溶解度问题会损害这些蛋白质的提取，分离和鉴定。我们建议通过应用顺序溶解技术提取与膜相关和积分膜蛋白。然后，将使用二维电泳和质谱法结合信息学鉴定蛋白质。这些知识将使我们能够制定与控制中性粒细胞增多症和启动的分子机制和信号转导途径有关的假设。这些假设将导致NIH应用以确认这些机制，并确定中性粒细胞参与急性肾脏损伤的方法。