Ferritin: Protein/mRNA/DNA in Fe/O Regulation/Metabolism

描述（由申请人提供）：铁蛋白通过蛋白质纳米笼中的Fe和O2敏感性mRNA调节以及Fe和O2催化底物连接Fe稳态和抗氧化保护。复杂的基因调控和保守的蛋白质结构强调了铁蛋白的生物学重要性。审查期间的成果和结果是：1）增加了对Fe 2+进入，退出和从细胞质和线粒体铁蛋白中去除的理解，从而产生用于铁过载疾病的新型Fe螯合剂（SCD，2-THL）：Fe 2+进入：我们确定了催化（DFP形成）所需的Fe 2+的活性位点配体。Fe 2+退出：我们鉴定了控制门控蛋白孔的小分子和肽。当偶联去铁，肽增加铁螯合8倍。2)确定IRE-mRNA功能的背景依赖性调节作用：我们鉴定了选择性控制H和L铁蛋白（FTH和FTL）mRNA的下游5 'UTR序列，以及由血红素/Bach 1）/DNA-MARE/ARE介导的铁蛋白调节协同作用，其补充血红素/IRP/RNA-IRE。铁蛋白DNA通过Bach 1/DNA阻遏与2-珠蛋白、血红素加氧酶、醌还原酶和硫氧还蛋白还原酶连接。3)鉴定在体内操纵3D mRNA特征的化合物。一种小的天然产物育亨宾结合IRE-RNA并增加mRNA翻译。铁蛋白-IRE/IRP 1复合物的X射线晶体学显示了RNA和蛋白质的诱导拟合、蛋白质-RNA接触表面、apo-IRP在调节中的可能作用以及eIF相互作用的可能位点。我们现在提出实验：1）通过分析蛋白质中控制的多个Fe/O2反应，连接Fe 2+循环中通过铁蛋白的步骤; 2）表征孔改变肽与铁蛋白的相互作用; 3）分析铁过载小鼠模型中的新型铁螯合剂;以及4）鉴定细胞中的铁蛋白相互作用。我们还建议通过以下方式了解铁蛋白mRNA和DNA（基因）调节的机制：1）测量“弱”或“强”IRE-IRP 1-eIF（s）结合/释放1 PolyA结合蛋白（PABP）的动力学; 2）确定PABP和eIF对人IRE-mRNA（FTH、FTL、mt-acon）翻译速率的影响; 3）搜索IRE-RNA结合肽;以及4）检查DNA甲基化。 公共卫生相关性：这些结果将提供IRE-mRNA翻译在铁稳态和疾病中重要的机制理解，如eIF 4F介导的细胞凋亡和选择性mRNA翻译，并确定DNA甲基化在组织特异性铁蛋白表达中的作用。此外，铁蛋白结构/功能的研究将确定铁蛋白纳米笼内的铁途径（进入、催化和退出），并在2-地中海贫血和镰状细胞病铁过载的小鼠模型中鉴定新型铁蛋白靶向螯合剂。

DESCRIPTION (provided by applicant): Ferritin links Fe homeostasis and antioxidant protection though Fe and O2 sensitive mRNA regulation and Fe and O2 catalytic substrates in the protein nanocage. Complex gene regulation and conserved protein structure emphasize the biological importance of ferritin. Outcomes in the grant period under review and results are: 1) Increased understanding of Fe2+ entry, exit and removal from cytoplasmic and mitochondrial ferritin leading to novel Fe chelators for iron overload diseases (SCD, 2-THL): Fe2+ entry: We identified the active site ligands for Fe2+ required for catalysis (DFP formation). Fe2+ exit: We identified small molecules and peptides that control the gated protein pores. When coupled to desferal, the peptides increase Fe chelation 8-fold. 2) Determination of the role of context-dependent regulation of IRE-mRNA function: We identified downstream, 5'UTR sequences selectively controlling H and L ferritin (FTH and FTL) mRNA, and a ferritin regulatory synergy mediated by heme /Bach1)/DNA-MARE/ARE that complements heme/IRP/RNA-IRE. Ferritin DNA is linked to 2-globin, heme oxygenase, quinone reductase, and thioredoxin reductase through Bach1/DNA repression. 3) Identification of compounds to manipulate 3D mRNA features in vivo. A small natural product, yohimbine, bound the IRE-RNA and increased mRNA translation. X-ray crystallography of the ferritin-IRE/IRP1 complex revealed induced fitting of both RNA and protein, the protein-RNA contact surface, a possible role for of apo-IRP in regulation, and possible sites of eIF-interactions. We now propose experiments to: 1) Connect the steps in the Fe2+ cycle through ferritin by analysis of the multiple Fe/O2 reactions controlled in the protein; 2) Characterize pore-altering-peptide interactions with ferritin; 3) Analyze novel iron chelators in mouse models of iron overload; and 4) Identify ferritin interactions in cells. We also propose to learn mechanisms of ferritin mRNA and DNA (gene) regulation by: 1) Measuring kinetics of "weak" or "strong" IRE-IRP1-eIF(s) binding/release 1 PolyA Binding Protein (PABP); 2) Determining effects of PABP and eIFs on translation rates of human IRE-mRNAs (FTH, FTL, mt-acon); 3) Searching for IRE-RNA binding peptides; and 4) Examining DNA methylation. PUBLIC HEALTH RELEVANCE: The results will provide mechanistic understanding of IRE-mRNA translation important in iron homeostasis and in disease, such as eIF4F mediated apoptosis and selective mRNA translation, and determine the role of DNA methylation in tissue-specific ferritin expression. In addition, studies of ferritin protein structure/function will determine Fe pathways (entry, catalysis, and exit) within the ferritin protein nanocages and identify novel, ferritin-targeted chelators in mouse models for iron overload in 2-Thalassemia and Sickle cell disease.