Iron Trafficking to Ribonucleotide Reductases

描述（由申请人提供）：核糖核苷酸还原酶（RR）对哺乳动物及其宿主的大型DNA病毒（痘病毒、疱疹病毒和腺病毒）的基因组复制至关重要。哺乳动物和病毒RR蛋白由含有活性位点的R1亚基和含有二铁簇和空间相邻的酪氨酸自由基中心的R2亚基组成。虽然不直接参与还原核糖核苷酸脱氧核糖核苷酸，这个辅基是必不可少的酶活性。尽管已经详细研究了二铁核心组装的体外化学，但关于铁对apoR 2蛋白的细胞靶向知之甚少。此外，RR对细胞铁的依赖性，无论铁被贩运到apoR 2的机制如何，都表明细胞的铁营养状况将是该过程效率的一个因素。112的铁可能来源于细胞溶质不稳定铁库（LIP）。该池通过膜渗透螯合剂、铁反应元件结合蛋白（IRP 1）和脱铁铁蛋白采样。我们已经证明，通过螯合或过量产生铁蛋白从该池中去除铁抑制病毒RR的活化，阻断病毒DNA的复制并抑制DNA病毒的毒力。这表明铁营养、铁运输和铁螯合都将影响病毒的发病机制，因为它们对RR激活有影响。本申请描述了从试管到培养细胞再到啮齿动物的实验。这些实验将评估铁从LIP到apoR 2的递送;以确定该螯合剂可接近的细胞内池的营养和遗传操作是否抑制培养细胞中的生产性病毒感染;并确定小鼠和大鼠的补充营养和遗传操作是否抑制病毒发病机理的体内模型中的DNA病毒的毒力。这项工作应该提供新的见解组装的一个关键的铁辅基在体内和证据表明，这一过程可以是一个目标，在病毒感染的干预。

DESCRIPTION (provided by applicant): Ribonucleotide reductase (RR) is essential to genome replication of mammals and the large DNA viruses that they host (pox, herpes, and adenoviruses). The mammalian and viral RR proteins are composed of an R1 subunit that contains the active site, and an R2 subunit that contains a diferric cluster and a spatially adjacent tyrosinate radical center. While not directly involved in the reduction of ribonucleotides to deoxyribonucleotides, this prosthetic group is essential to enzyme activity. Although the in vitro chemistry of the assembly of the diferric core has been studied in some detail, little is known about the cellular targeting of iron to the apoR2 protein. Furthermore, the dependence that RR has on cell iron, irrespective of the mechanism by which iron is trafficked to apoR2, suggests that the iron nutritional status of the cell will be a factor in the efficiency of this process. The likely source of iron for 112 is the cytosolic labile iron pool (LIP). This pool is sampled by membrane permeant chelators, the iron response element binding protein (IRP1), and apoferritin. We have shown that removing iron from this pool by either chelation or over-production of ferritin inhibits activation of viral RR, blocks the replication of viral DNA and suppresses the virulence of a DNA virus. This suggests that iron nutrition; iron trafficking and iron chelation will all impact on viral pathogenesis due to their effects on RR activation. This application describes experiments that evolve from test-tube to cultured cells to rodents. These experiments will evaluate the delivery of iron from the LIP to apoR2; to determine whether nutritional and genetic manipulation of this chelator-accessible intracellular pool suppresses a productive viral infection in cultured cells; and to determine whether complementary nutritional and genetic manipulation of mice and rats suppresses the virulence of a DNA virus in an in vivo model of viral pathogenesis. This work should provide new insight into the assembly of a critical iron prosthetic group in vivo and evidence that this process can be a target for intervention in a viral infection.