Structure-function studies of endotoxin receptor CD14

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DESCRIPTION (provided by applicant): Endotoxins such as LPS and PGN, components of outer cell walls from Gram-negative and Gram-positive bacteria are potent immunostimulators of monocytes and macrophages in humans and have been implicated in the pathogenesis of sepsis and septic shock, a clinical progression that results in 250,000 deaths annually in the United States alone. Inflammatory responses as part of innate immunity are initiated via high-affinity binding of these endotoxins to a key host cell receptor CD 14, triggering release of a variety of pro inflammatory mediators, including cytokines, through a signal transduction pathway involving Toll-like receptors. An overactivation of this cellular response may, however, result in excessive release of the proinflammatory mediators, inducing septic shock and death. CD 14 is known to play a central role in the innate system, by modulating the inflammatory response and enhancing the sensitivity of endotoxininduced activation. However, the molecular mechanisms by which it accomplishes this are not clear. The proposed studies are focused on understanding the molecular details of CD 14-endotoxin interactions responsible for cellular activation by structure/function studies of CD14. Our central hypothesis is that CD14 functions as a pattern recognition receptor by recognition of characteristic molecular patterns on various endotoxins and binding them at structurally similar but distinct sites. We will test our hypothesis with the following specific aims: 1) High-resolution structure determination of a soluble form of CD 14 by solution NMR spectroscopy and mapping functional epitopes on CD 14 necessary for endotoxin recognition, 2) Structural characterization of CD14-ligand complexes to unravel shared ligand structural features required for CD14 binding, 3) Mutagenesis and biophysical studies to identify key CD14 residues involved in interaction with different endotoxins and their role in pattern recognition by CD14. Completion of these studies will provide the structural basis for the deleterious effects of LPS, PGN and rational design of potentially novel LPS, PGN antagonists for treatment of sepsis.