Activated macrophages demonstrate arginine-dependent cytotoxicity for intracellular pathogens (Listeria monocytogenes, mycobacteria, Leishmania) mediated by nitric oxide (NO). Previously, Dr. Cunningham and his colleagues have established that the cytokine-dependent increase in uptake of arginine, the substrate for NO synthesis, is mediated by expression of one member (MCAT-2B) of a family of related transporters of cationic amino acids that is coordinately regulated with iNOS, the enzyme that catalyzes NO synthesis in macrophages. In recent experiments, they have observed that MCAT-2B resides exclusively on intracellular membranes, likely lysosomes, a location distinct from MCAT-1 and MCAT-2A. Their findings suggest that MCAT-2B has a specific role in providing L-arginine for localized production of NO. It is their goal to identify the extact intracellular location of MCAT-2B and determine the importance of correct localization for NO-dependent cytotoxicity.
Specific Aim number 1 Verify the localization of MCAT-2B on lysosomal membranes. A. Verify the localization of MCAT-2B on lysosomes using isoform specific antibodies to blot purified membrane fractions from the macrophage-derived cell line, RAW264.7. B. Confirm the localization of MCAT-2B on lysosomes of activated macrophages from mice.
Specific Aim number 2 Investigate the mechanism of targeting of MCAT-2B to lysosomes. A. Identify targeting domain(s) by studying localization of chimeric transporters. B. Identify cellular proteins that bind to these domain(s) by two hybrid screening.
Specific Aim number 3 Determine the role of MCAT proteins in NO- dependent cytotoxicity. A. Reexamine the kinetics of arginine transport using purified MCAT proteins reconstituted into lipid bilayers. B. Determine the importance of MCAT-2B for NO-dependent killing of bacteria in phagolysosomes using macrophage cell lines obtained from knockout mice.
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