Sepsis afflicts approximately 500,000 Americans per year with an associated mortality of approximately 35-65 percent. The lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria, is one of the initiating stimulants in an inflammatory cascade that has been referred to as the """"""""Systemic Inflammatory Response Syndrome."""""""" During the past seven years, we have analyzed the interactions of LPS with cells of the inflammatory process that can result in mortality in Gram-negative sepsis. The long-range goal of this research program is to understand the molecular mechanisms of pathogenesis of septic shock and to identify potential therapeutic intervention strategies to reduce morbidity and mortality resulting from this collection of diseases. The goal of this application is to examine a novel pathway by which LPS can be internalized by mouse macrophages, and following interaction with cytoplasmic organelles, trigger the production of proinflammatory cytokines and other mediators of inflammation. The central hypothesis of this research project is that LPS, acting at least in part through interactions with heat shock proteins, interacts with the cytoplasmic proteasome of macrophages resulting in their proteolytic activation and resultant generation of proinflammatory cytokines. It is further hypothesized that interference with this pathway by appropriate antagonist molecules will inhibit the production of proinflammatory cytokines and provide protection against LPS-mduced septic shock. The following Specific Aims are proposed: 1. To complete the molecular characterization of specific LPS-binding proteins identified in cellular subfractions of macrophages. We will employ our newly designed photoreactive LPS probe to specifically label the murine membrane and cytosolic proteins with binding affinity for LPS, purify these crosslinked complexes by 2D-gel electrophoresis, and identify the major crosslinked proteins by MALDI/MS. 2. To define the functional role of the LPS binding proteins-proteasome/heat shock proteins. 3. To evaluate the relative contribution of the proteasome pathway to the pathogenesis of LPS-mediated septic shock. We anticipate that at the completion of these Specific Aims we will have a better understanding of the LPS-induced signal transduction pathways in macrophages. The successful completion of this research will contribute to the development of novel strategies for prevention and treatment of septic shock.
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