Iron Trafficking to Ribonucleotide Reductases

描述（由申请人提供）：核糖核苷酸还原酶（RR）对于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 蛋白由包含活性位点的 R1 亚基和包含二铁簇和空间相邻的酪氨酸自由基中心的 R2 亚基组成。虽然不直接参与核糖核苷酸还原为脱氧核糖核苷酸，但该辅基对于酶活性至关重要。尽管已经对二铁核心组装的体外化学进行了一些详细研究，但对于铁对 apoR2 蛋白的细胞靶向作用知之甚少。此外，无论铁转运至 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该过程效率的一个因素。 112 的铁可能来源是细胞质不稳定铁池 (LIP)。该池通过膜渗透螯合剂、铁反应元件结合蛋白 (IRP1) 和脱铁铁蛋白进行采样。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可以抑制病毒 RR 的激活，阻止病毒 DNA 的复制并抑制 DNA 病毒的毒力。这表明铁的营养；铁运输和铁螯合都会影响病毒的发病机制，因为它们会影响 RR 激活。该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；确定对这种可与螯合剂接触的细胞内池进行营养和遗传操作是否可以抑制培养细胞中的有效病毒感染；并确定对小鼠和大鼠的补充营养和遗传操作是否可以在病毒发病机制的体内模型中抑制 DNA 病毒的毒力。这项工作应该为体内关键铁假体基团的组装提供新的见解，并证明该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.