Iron Trafficking and Regulation in Biological Systems

项目总结/摘要 PI请求支持他的研究计划铁贩运和生物调节 系统.利用强大的生物物理，生物分析和数学工具，PI和他的 同事们提出表征和定量分离的细胞器中的主要铁物质， 从多种多样的生物系统中获取完整的细胞和器官。穆斯堡尔光谱学将 是主要的生物物理工具，EPR和紫外可见光谱起辅助作用。 芽殖酵母和人类细胞的各种遗传菌株将富含57 Fe。线粒体、 将分离胞质溶胶、空泡和其它细胞器。这些细胞中的主要铁物种 组件将被探测。基于常微分方程的数学模型将 开发的分析所获得的结果，并产生一个系统的水平描述铁 贩运和监管。世界上没有其他组织以这种方式研究Fe。酵母菌株 Mrs 3/4是一种高亲和力的铁转运蛋白，位于线粒体上， 内膜，已经被删除和过度表达。其他铁相关酵母菌株，如 以及其中线粒体铁蛋白（Mrs 3/4的同源物）和铁蛋白被敲除的人Jurkat细胞- 下来，也会受到调查。大肠杆菌和其他原核生物的铁含量将 类似的探索。中心生物分析工具将是位于 在连接到在线电感耦合等离子体的冷冻厌氧手套箱中 质谱仪和在线电喷雾电离质谱仪。本信用证- （ICP）-ESI-MS系统将是全球独一无二的。它将被用来描述几十个不稳定的 PI和他的团队在1999年发现的低分子量（LMM）金属络合物， 细胞器、细胞质、血浆中，E.杆菌线粒体中的LMM Fe复合物， 质量CA。580 Da（称为Fe 580）正在积极研究中。Fe 580最有可能用作 用于铁-硫簇（ISC）组装和用于血红素的铁插入步骤的原料 生物合成称为X-S的LMM含硫物质的组成也将是 研究了X-S从线粒体中输出，与ISC组装相关，并且它可能是 用于组装胞质ISCs和调节Fe运输。血液中的LMM Fe物质 将使用猪研究称为“非转铁蛋白结合铁”或NTBI的血浆， 饲管和取样导管已通过外科手术植入。NTBI损害器官 铁超负荷疾病如血色病患者。不同品系的小鼠与铁- 相关疾病将进行调查，以确定铁的形式，积累在他们的 机关缺氧对铁积累的影响也将被检查。

Project Summary/Abstract The PI requests support of his research program in Iron Trafficking and Regulation in Biological Systems. Using powerful biophysical, bioanalytical, and mathematical tools, the PI and his coworkers propose to characterize and quantify the major iron species in isolated organelles, whole cells and organs from a wide diversity of biological systems. Mössbauer spectroscopy will be the primary biophysical tool, with EPR and UV-vis spectroscopies playing supporting roles. Various genetic strains of budding yeast and human cells will be enriched in 57Fe. Mitochondria, cytosol, vacuoles and other organelles will be isolated. The major Fe species in these cellular components will be probed. Ordinary-differential-equations-based mathematical models will be developed to analyze the results obtained and to generate a system’s level description of Fe trafficking and regulation. No other group worldwide investigates Fe in this way. Yeast strains are being investigated in which Mrs3/4, high-affinity Fe importers located on the mitochondrial inner membrane, have been deleted and overexpressed. Other Fe-associated yeast strains, as well as human Jurkat cells in which mitoferrins (homologs of Mrs3/4) and ferritin are knocked- down, will also be investigated. The Fe content of Escherichia coli and other prokaryotes will be similarly explored. The central bioanalytical tool will be a liquid chromatography system located in a refrigerated anaerobic glove box that is linked to an on-line inductively coupled plasma mass spectrometer AND to an on-line electrospray ionization mass spectrometer. This LC- (ICP)-ESI-MS system will be unique worldwide. It will be used to characterize dozens of labile low-molecular-mass (LMM) metal complexes that the PI and his group have discovered in organelles, cytosol, blood plasma, and in E. coli. A LMM Fe complex in mitochondria that has a mass of ca. 580 Da (called Fe580) is actively under investigation. Fe580 is most probably used as feedstock for iron-sulfur cluster (ISC) assembly and for the iron-insertion step of heme biosynthesis. The composition of a LMM sulfur-containing species called X-S will also be investigated. X-S is exported from mitochondria in association with ISC assembly, and it may be used to assemble cytosolic ISCs and to regulate Fe trafficking. A LMM Fe species in blood plasma called “non-transferrin-bound iron” or NTBI will be investigated using pigs into which a feeding tube and sampling catheters have been surgically implanted. NTBI damages organs in patients with Fe-overload diseases such as hemochromatosis. Various strains of mice with Fe- associated diseases will be investigated to determine the form of Fe that accumulates in their organs. The effect of hypoxia on Fe accumulation will also be examined.