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

描述（由申请人提供）：红细胞成熟取决于铁摄取、转运、浓缩和血红素合成以产生血红蛋白的蛋白质表达增加。铁蛋白在红细胞和铁稳态中起着关键作用：将铁浓缩为矿物质并保护矿物质免受细胞还原剂的影响。铁蛋白mRNA，FTH和FTL与铁转运蛋白的协同调节依赖于一组mRNA结构，即结合调节蛋白IRP 1和2的铁调节元件（IRE）。铁蛋白-IRE-RNA/IRP 1复合物的晶体结构有利于IRE结合IRP肽和翻译因子的动力学/平衡研究的设计。最近的观察表明，FTH和FTL基因在转录过程中与MARE/ARE抗氧化反应基因共调节，导致Bach 1被鉴定为FTH和FTL阻遏物。铁蛋白最大限度地减少氧化应激，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的铁/O矿物的空腔（8 nm）。Fe 2+离子通过孔进入和离开铁蛋白蛋白笼。铁蛋白催化涉及其他二铁辅因子蛋白催化剂。Fe 2+进入，Fe 2 +/O2催化，矿物形成/溶解，和Fe 2+退出之间的中间步骤和机制，只有部分特征。铁2+从铁蛋白蛋白笼中退出的速率，作为矿物质还原后的螯合/结合的铁2+，取决于笼孔的局部蛋白质折叠/展开。在体外鉴定了两种调节肽，其可以模拟调节蛋白。增加溶液中铁螯合的螯合剂-肽缀合物在去除人类疾病中过量的铁方面具有潜在的用途。本补助金期间的目标是：1。了解Fe 2+进入，O2反应，运输到空腔，孔门控（折叠/解折叠）和Fe 2+释放到载体/螯合剂蛋白的Fe途径; 2.理解含IRE的mRNA的差异表达;和3.确定MafK和5-甲基胞嘧啶（5 mC）对铁蛋白DNA表达/Bach 1结合的影响。设计：1A。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推定的调节剂。2A、B.分析不同的IRE-RNA翻译和结合起始因子和/或IRP 1和来自IRP 1的设计肽的动力学，2. 3A.分析Bach 11 MafK与MARE/ARE-DNAs 15 mC的结合; 3B.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、紫外可见荧光MCD/CD、NMR光谱和X射线晶体学）、分子生物学（体外翻译、诱变、蛋白质表达、EMSA、DNA-5 mC分析）和细胞生物学（RT-PCR、免疫印迹）。铁稳态，铁蛋白功能，蛋白质催化，蛋白质孔，mRNA功能和蛋白质合成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。公共卫生相关性：铁稳态、基因和mRNA调节以及铁蛋白中的宿主/病原体二铁催化的基础研究可以转化为药物靶向，以及连接的肽螯合剂，镰状细胞病、地中海贫血、遗传性血色病中的铁螯合，以及对铁在疟疾和糖尿病中的新兴认识。

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.