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。哺乳动物和病毒的RR蛋白由含有活性位点的R1亚基和含有异源簇和空间相邻的酪氨酸自由基中心的R2亚基组成。虽然不直接参与核糖核苷酸还原为脱氧核糖核苷酸，但这个假基对酶的活性是必不可少的。尽管已经详细研究了异铁核组装的体外化学，但对铁靶向apoR2蛋白的细胞作用知之甚少。此外，无论铁转运到apoR2的机制如何，RR对细胞铁的依赖性表明，细胞的铁营养状况将是这一过程效率的一个因素。112可能的铁来源是胞质不稳定铁池（LIP）。该池由膜渗透螯合剂、铁反应元件结合蛋白（IRP1）和载铁蛋白取样。我们已经证明，通过螯合或过量生产铁蛋白从这个池中去除铁可以抑制病毒RR的激活，阻断病毒DNA的复制并抑制DNA病毒的毒力。这表明铁营养；铁转运和铁螯合都通过对RR激活的影响而影响病毒的发病机制。这个应用程序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apoR2的传递；确定这种螯合剂可及的细胞内池的营养和遗传操作是否抑制培养细胞中的多产病毒感染；以及确定小鼠和大鼠的补充营养和遗传操作是否在病毒发病的体内模型中抑制DNA病毒的毒力。这项工作应该为体内关键铁假体群的组装提供新的见解，并证明这一过程可以成为干预病毒感染的目标。

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.