Mutations of ferritin H chain C-terminus produced by nucleotide insertions have altered stability and functional properties
Mutations of ferritin H chain C-terminus produced by nucleotide insertions have altered stability and functional properties
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DOI：
10.1093/jb/mvj101
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发表时间：
2006-05-01
影响因子：
2.7
通讯作者：
Arosio, Paolo
中科院分区：
文献类型：
--
作者：
Ingrassia, Rosaria;Gerardi, Gianmario;Arosio, Paolo
铁蛋白是一种由24个亚基组成的储铁蛋白。先前的突变分析表明，铁蛋白c端区域在蛋白质的稳定性和组装中起主要作用，但在铁的掺入机制中只起很小的作用。然而，最近有研究表明，携带由核苷酸插入引起的铁蛋白c端序列改变的患者表现出可能与蛋白质功能改变和细胞铁失调有关的神经系统疾病。为了重新评估该区域的作用，通过插入2个核苷酸，修饰了最后6-29个残基，延长了14个氨基酸的序列，产生了5个小鼠h -铁蛋白突变体。突变体在大肠杆菌中表达，并分析其溶解度、稳定性和含铁能力。最后6个残基非螺旋延伸的改变对铁蛋白的性质没有明显影响，但随着修饰区域的延长，铁蛋白的溶解度和在铁蛋白壳内的组装能力逐渐降低。结果还表明，即使是末端e -螺旋的一部分修饰也会破坏铁蛋白在细胞表达过程中吸收铁的能力，这可能是由疏水通道的构象修饰引起的。这些数据支持致病突变改变细胞铁稳态的假设。
Ferritin is an iron storage protein made of 24 subunits. Previous mutational analyses showed that ferritin C-terminal region has a major role in protein stability and assembly but is only marginally involved in the mechanism of iron incorporation. However, it has recently been shown that patients who carry alterations of ferritin C-terminal sequence caused by nucleotide insertions show neurological disorders possibly related to altered protein functionality and cellular iron deregulation. To re-evaluate the role of this region, five mutants of mouse H-ferritin were produced by 2-nucleotide insertions that modified the last 6-29 residues and extended the sequence of 14 amino acids. The mutants were expressed in Escherichia coli and analysed for solubility, stability and capacity to incorporate iron. The alteration of the last 6-residue non-helical extension had no evident effect on the properties of ferritin, while solubility and capacity to assemble in ferritin shells decreased progressively with the extension of the modified region. The results also showed that the modification of even a part of the terminal E-helix abolished the capacity of ferritin to incorporate iron during expression in the cells, probably caused by conformational modification of the hydrophobic channels. The data support the hypothesis that the pathogenic mutations alter cellular iron homeostasis.