A Proteome Map of Neutrophil Granules

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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.