ROLE OF ARGININE TRANSPORT IN NITRIC OXIDE PRODUCTION

活化的巨噬细胞表现出对肿瘤细胞的依赖性细胞毒性， 细胞内病原体（单核细胞增生李斯特菌，分枝杆菌， 利什曼原虫）介导的一氧化氮（NO）。之前坎宁安医生 和他的同事们已经证实， 在摄取精氨酸（NO合成的底物）中， 相关转运蛋白家族的一个成员（MCAT-2B）表达 与iNOS协同调节的阳离子氨基酸， 在巨噬细胞中催化NO合成的酶。 近几 实验中，他们观察到MCAT-2B只存在于 细胞内膜，可能是溶酶体， MCAT-1和MCAT-2A。 他们的研究结果表明，MCAT-2B具有特定的 在提供L-精氨酸用于NO的局部产生中的作用。 他们的目标是确定MCAT-2B的完整细胞内位置， 确定NO依赖的正确定位的重要性 细胞毒 具体目标1验证MCAT-2B在溶酶体上的定位 膜。A.使用以下方法验证MCAT-2B在溶酶体上的定位 同种型特异性抗体，以从细胞中印迹纯化的膜级分。 巨噬细胞来源的细胞系RAW264.7。B。确认本地化 小鼠活化巨噬细胞溶酶体上的MCAT-2B。 具体目标2研究MCAT-2B的靶向机制 到溶酶体。 A.通过研究定位确定靶向结构域 嵌合体转运蛋白 B。识别细胞蛋白质， 通过两个杂交筛选这些结构域。 具体目标3确定MCAT蛋白在NO- 依赖性细胞毒性。 A.重新检查精氨酸转运的动力学 使用重组到脂质双层中的纯化MCAT蛋白。 B。 确定MCAT-2B对于NO依赖性杀死细菌的重要性 在吞噬溶酶体中，使用从敲除获得的巨噬细胞系 小鼠

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.