Iron Trafficking to Ribonucleotide Reductases

描述(申请人提供)：核糖核苷酸还原酶(RR)对于哺乳动物及其携带的大型DNA病毒(痘、疱疹和腺病毒)的基因组复制是必不可少的。哺乳动物和病毒RR蛋白由包含活性部位的R1亚基和包含差异铁簇和空间相邻的酪氨酸自由基中心的R2亚基组成。虽然不直接参与将核糖核苷酸还原为脱氧核糖核苷酸，但这个修复基团对酶的活性是必不可少的。虽然对二铁核组装的体外化学已经进行了一些详细的研究，但对于铁对apoR2蛋白的细胞靶向知之甚少。此外，RR对细胞铁的依赖，无论铁被运输到apoR2的机制如何，都表明细胞的铁营养状态将是这一过程效率的一个因素。112可能的铁来源是胞质中的不稳定铁池(LIP)。这一池是由膜永久螯合剂，铁反应元件结合蛋白(Irp1)和载铁蛋白采样。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，阻止病毒DNA的复制，并抑制DNA病毒的毒力。这表明铁营养、铁转运和铁螯合都将通过影响RR的激活而影响病毒的致病。这项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嘴唇向apoR2的输送；确定对这种可通过螯合剂访问的细胞内池的营养和基因操作是否能抑制培养细胞中的生产性病毒感染；以及确定在病毒致病的体内模型中，对小鼠和大鼠的互补营养和基因操作是否能抑制DNA病毒的毒力。这项工作应该为在体内组装关键的铁修复基团提供新的见解，并提供证据表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.