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

描述(申请人提供)：铁蛋白通过铁和氧敏感的信使核糖核酸调节和蛋白纳米笼中的铁和氧催化底物，将铁稳态和抗氧化保护联系起来。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如下：1)增加了对Fe2+进入、离开和清除细胞质和线粒体铁蛋白的了解，导致了治疗铁超载疾病(SCD，2-THL)的新型铁络合剂：Fe2+进入：我们确定了催化(DFP形成)所需的Fe2+的活性位点配体。Fe2+退出：我们确定了控制门控蛋白孔的小分子和多肽。当与Desferal结合时，这些多肽将铁的螯合作用增加8倍。2)对IRE基因功能的上下文依赖性调控作用的确定：我们在下游发现了选择性控制H和L铁蛋白(FTH和FTL)基因的5‘UTR序列，以及由血红素/Bach1)/DNA-mare/ARE介导的铁蛋白调控协同作用，补充了血红素/irp/RNA-ire。铁蛋白DNA通过Bach1/DNA阻遏与2-珠蛋白、血红素加氧酶、苯醌还原酶和硫氧还蛋白还原酶连接。3)体内操纵3DmRNA功能的化合物的鉴定。一种小型的天然产物育亨宾与IRE-RNA结合，增加了mRNA的翻译。对铁蛋白-IRE/Irp1复合体的X射线结晶学研究表明，RNA和蛋白质都被诱导匹配，蛋白质-RNA接触面，apo-IRP在调节中的可能作用，以及eIF-相互作用的可能位置。我们现在建议进行以下实验：1)通过分析蛋白质中控制的多个Fe/O2反应，通过铁蛋白连接Fe2+循环中的各个步骤；2)表征改变毛孔的多肽与铁蛋白的相互作用；3)在铁超载的小鼠模型中分析新型铁络合剂；以及4)确定细胞中的铁蛋白相互作用。我们还建议从以下几个方面来研究铁蛋白基因的调控机制：1)测定“弱”或“强”IRE-IRP1-eIF(S)结合/释放1多聚体结合蛋白的动力学；2)测定PABP和EIF对人IRE-mRNAs(FTH、FTL、mt-ACON)翻译率的影响；3)寻找IRE-RNA结合肽；以及4)DNA甲基化。 公共卫生相关性：这些结果将提供对IRE-mRNA翻译在铁稳态和疾病中重要的机械性理解，例如eIF4F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，对铁蛋白结构/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-地中海贫血和镰刀细胞疾病的小鼠模型中识别以铁蛋白为靶点的新型螯合剂。

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.