Bites from the Loxosceles genus (brown spiders) cause several clinical manifestations in mammals, especially necrotic skin degeneration, hematological disturbances, and renal failure. These spiders have a world-wide distribution and accidents have been described in America, Europe, Asia, Africa and Oceania. The venoms of Loxosceles comprise a toxic component with a rare enzymatic activity, termed sphingomyelinase D (SMD). This enzyme catalyzes the conversion of sphingomyelin (SM) into ceramide-1-phosphate (Cer-1-P). While the enzymatic substrate SM is an integral constituent of many cell membranes, especially in the vascular epithelium and red blood cells, the reaction product Cer-1-P occurs in very low concentrations. Cer-1-P is suggested to be a novel lipid second messenger in cellular signal transduction events. At present, the precise mechanism of venom action is incompletely understood. This proposal describes a multidisciplinary strategy to explore the mechanism of SMD action at different molecular and supramolecular levels. To evaluate the effect of the interaction between SMD and biological membranes, model membrane systems of different compositional complexity will be used. This involves giant unilamellar vesicles as well as planar bilayers composed of relevant lipid mixtures and native biological membranes. Among others, state-of-the-art fluorescence techniques will be applied to directly visualize and spatially correlate the different events including biochemical (enzyme kinetics) and biophysical (membrane structure) aspects of enzyme action. This combinatorial approach will also explore enzyme/membrane interactions in relevant cellular systems, e.g., red blood cells and adipocytes. The proposed project implies extensive training of the fellow in a large variety of fluorescence techniques and membrane model systems. It emphasizes a comprehensive vision of scientific topics that will advance the professional maturity of the applicant.
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