ROLE OF ARGININE TRANSPORT IN NITRIC OXIDE PRODUCTION

活化的巨噬细胞表现出精氨酸依赖的细胞毒作用 细胞内病原体(单核细胞增生性李斯特氏菌、分枝杆菌、 利什曼病)由一氧化氮(NO)介导。前情提要，坎宁安博士 和他的同事们已经证实，细胞因子依赖的增加 在精氨酸的摄取中，精氨酸是合成一氧化氮的底物，通过 一个相关转运蛋白家族成员(MCAT-2B)的表达 在与iNOS协调调节的阳离子氨基酸中， 巨噬细胞中催化合成一氧化氮的酶。在最近 实验中，他们观察到MCAT-2B专门驻留在 细胞内膜，可能是溶酶体，一个不同于 MCAT-1和MCAT-2A。他们的发现表明，MCAT-2B具有特定的 为国产化生产NO提供L精氨酸的作用。它是 他们的目标是确定MCAT-2B和MCAT-2B的确切细胞内位置 确定非依赖型的正确本地化的重要性 细胞毒性。 特异性靶点1验证MCAT-2B在溶酶体上的定位 膜。A.验证MCAT-2B在溶酶体上的定位 异构体特异性抗体印迹纯化的膜组分 巨噬细胞来源的细胞系RAW264.7。B.确认本地化 MCAT-2B对小鼠活化巨噬细胞溶酶体的影响。 特异性靶点2研究MCAT-2B的靶向机制 到溶酶体。A.通过研究本地化确定目标领域(S) 嵌合转运体。B.确定结合到的细胞蛋白 这些域(S)经过两次杂交筛选。 特异性靶点3决定MCAT蛋白在NO-1中的作用 依赖性细胞毒性。A.重新研究精氨酸转运的动力学 用纯化的MCAT蛋白重组成脂质双层。胡麻B. 确定MCAT-2B在NO依赖杀灭细菌中的重要性 利用基因敲除获得的巨噬细胞系在吞噬溶酶体中 老鼠。

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.