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

描述(由申请人提供)：红细胞的成熟依赖于铁摄取、运输、浓缩和血红素合成所需蛋白质表达的增加，以产生血红蛋白。铁蛋白在红细胞和铁稳态中起着关键作用：将铁作为一种矿物质浓缩，并保护矿物质不受细胞还原剂的影响。铁蛋白mRNAs、FTH和FTL与铁转运蛋白的共同调节依赖于一组mRNA结构，即与调节蛋白Irp1和2结合的铁调节元件(IRE)。铁蛋白-IRE-RNA/Irp1复合体的晶体结构有助于设计IRE与IRP肽和翻译因子结合的动力学/平衡研究。最近观察到FTH和FTL基因在转录过程中与mare/are抗氧化反应基因共同调节，这使得Bach1被确定为FTH和FTL抑制因子。铁蛋白将氧化压力降至最低，并为蛋白质储存铁。铁蛋白的结构和功能非常复杂。蛋白质纳米笼自组装形成一个空腔(8 Nm)，形成Fe/O矿物质。Fe2离子通过毛孔进入和离开铁蛋白笼。铁蛋白催化涉及到其他双铁辅因子蛋白催化剂。Fe2进入、Fe2/O2催化、矿物形成/溶解和Fe2退出之间的中间步骤和机制仅有部分描述。在矿物还原后，Fe2以螯合/结合Fe2的形式从铁蛋白笼子中退出的速度取决于笼孔的局部蛋白质折叠/展开。在体外鉴定了两个可能模拟调节蛋白的调节多肽。这种螯合剂-多肽结合物在溶液中增加了铁的螯合作用，在去除人类疾病中多余的铁方面具有潜在的用途。本授权期的目的是：1.了解Fe2进入、O2反应、转运到空洞、孔门(折叠/去折叠)和Fe2向载体/螯合剂蛋白释放的途径；2.了解含有IRE的mRNAs的差异表达；3.确定MafK和5-甲基胞嘧啶(5mC)对铁蛋白DNA表达/Bach1结合的影响。设计：1.通过突变共变残基鉴定铁蛋白中间体；1B。表征溶液中的结合肽和体内可能的调节剂。分析不同的IRE-RNAs翻译和与起始因子和/或Irp1结合的动力学，并从Irp1、2.3a设计多肽。分析Bach1 1 MafK与mare/are-DNA 1 5mC；3B的结合。分析铁对FTL/FTH DNA 5 mC的影响。技术：生物化学(结合动力学/平衡、UV-Vis荧光MCD/CD、核磁共振光谱和X射线结晶学)、分子生物学(体外翻译、突变、蛋白质表达、EMSA、DNA-5mC分析)和细胞生物学(RT-PCR、免疫印迹)。在铁稳态、铁蛋白功能、蛋白质催化、蛋白质毛孔、信使核糖核酸功能和蛋白质合成方面的结果也可以转化为疾病中的铁，例如HH、SCD、地中海贫血和疟疾。与公众健康相关：铁蛋白中铁稳态、基因和信使核糖核酸调控以及宿主/病原体双铁催化的基础研究可以转化为药物靶向，对于连接的多肽-螯合剂，可以转化为镰刀细胞疾病、地中海贫血、遗传性血色素沉着症中的铁螯合，以及对疟疾和糖尿病中铁的新认识。

DESCRIPTION (provided by applicant): Red cell maturation depends on increased expression of proteins for iron uptake, transport, concentration, and heme synthesis to produce hemoglobin. Ferritin plays a key role in red cells and iron homeostasis: concentrating iron as a mineral and protecting the mineral from cell reductants. Coregulation of ferritin mRNAs, FTH and FTL, with iron trafficking proteins depends on a group of mRNA structures, the iron regulatory element (IRE) that binds regulatory proteins IRP1 and 2. The crystal structure of a ferritin-IRE-RNA/IRP1 complex facilitates designs for kinetic/equilibrium studies of IRE binding to IRP peptide and translation factors. Recent observations that FTH and FTL genes are coregulated during transcription with MARE/ARE antioxidant response genes led to identification of Bach1 as the FTH and FTL repressor. Ferritin minimizes oxidative stress and stores iron for proteins. Ferritin protein structure and function are very complex. The protein nanocages self-assemble with a cavity (8 nm) for Fe/O minerals. Fe2+ ions enter and leave the ferritin protein cage through pores. Ferritin catalysis relates to other di-iron cofactor protein catalysts. Intermediate steps and mechanisms between Fe2+ entry, Fe2+/O2 catalysis, mineral formation/dissolution, and Fe2+ exit are only partly characterized. Rates of Fe2+ exit from the ferritin protein cage, as chelated/bound Fe2+ after reduction of the mineral, depend on localized protein folding/unfolding of the cage pores. Two regulating peptides were identified in vitro that may model regulatory proteins. The chelator-peptide conjugate that increased iron chelation in solution has potential uses in removing excess iron in human disease. The Aims for this grant period, are to: 1. understand Fe pathways for Fe2+ entry, O2 reactions, transit to the cavity, pore gating (folding/unfolding) and Fe2+ release to carriers/chelators protein; 2. understand the differential expression of IRE-containing mRNAs; and 3. determine the effect of MafK and 5-methylcytosine (5mC) on ferritin DNA expression/Bach1 binding. Design: 1A. Identify Fe-protein intermediates by mutating covarying residues; 1B. Characterize binding peptides in solution and putative regulators in vivo. 2A,B. Analyze different IRE-RNAs translation and kinetics of binding to initiation factors and/or IRP1 and designed peptides from IRP1, 2. 3A. Analyze Bach1 1 MafK binding to MARE/ARE-DNAs 1 5mC; 3B. Analyze iron effects on FTL/FTH DNA 5mC. Techniques: Biochemistry (binding kinetics/equilibria, UV-vis fluorescence MCD/CD, NMR spectroscopies, and X-ray crystallography), Molecular Biology (in vitro translation, mutagenesis, protein expression, EMSA, DNA-5mC analysis), and Cell Biology (RT-PCR, immunoblotting). The results on iron homeostasis, ferritin function, protein catalysis, protein pores, mRNA function, and protein synthesis can also translate to iron in diseases, e.g., HH, SCD, Thalassemia and malaria. PUBLIC HEALTH RELEVANCE: Basic studies of iron homeostasis, gene and mRNA regulation, and host/pathogen diiron catalysis in ferritin can be translated to drug targeting, and for the linked peptide-chelator, to iron chelation in Sickle Cell Disease, Thalassemia, Hereditary Hemochromatosis, and to the emerging awareness of iron in malaria and diabetes.