Disease Variants of the Human Mitochondrial DNA Helicase Encoded by C10orf2 Differentially Alter Protein Stability, Nucleotide Hydrolysis, and Helicase Activity
Disease Variants of the Human Mitochondrial DNA Helicase Encoded by C10orf2 Differentially Alter Protein Stability, Nucleotide Hydrolysis, and Helicase Activity
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DOI：
10.1074/jbc.m110.151795
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发表时间：
2010-09-24
影响因子：
4.8
通讯作者：
Copeland, William C.
中科院分区：
文献类型：
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作者：
Longley, Matthew J.;Humble, Margaret M.;Copeland, William C.
人类C10orf2基因的错义突变编码线粒体DNA(MtDNA)解旋酶，与线粒体疾病共同分离，如成人起病的进行性外眼肌麻痹、肝脑综合征伴mtDNA耗竭综合征和婴儿起病的脊髓小脑性共济失调。为了了解C10orf2突变的生化后果，我们在大肠杆菌中过度生产了野生型和20种突变形式的人mtDNA解旋酶，并开发了新的方案来纯化重组酶，使其接近均一。需要结合分子拥挤、非离子洗涤剂、镁离子和离子强度的提高来对抗某些突变变种的不溶性和内在不稳定性。对这些酶的系统生化评估包括DNA结合亲和力、DNA解旋酶活性、核苷酸水解动力学和热稳定性评估。与其他研究相比，我们发现，在优化的体外条件下，所有20个突变体都保持了解旋酶的功能，尽管一些突变体的DNA结合亲和力、核苷酸水解性或热稳定性有所降低。这种部分缺陷与C10orf2突变相关的线粒体疾病的延迟表现是一致的。
Missense mutations in the human C10orf2 gene, encoding the mitochondrial DNA (mtDNA) helicase, co-segregate with mitochondrial diseases such as adult-onset progressive external ophthalmoplegia, hepatocerebral syndrome with mtDNA depletion syndrome, and infantile-onset spinocerebellar ataxia. To understand the biochemical consequences of C10orf2 mutations, we overproduced wild type and 20 mutant forms of human mtDNA helicase in Escherichia coli and developed novel schemes to purify the recombinant enzymes to near homogeneity. A combination of molecular crowding, non-ionic detergents, Mg2+ ions, and elevated ionic strength was required to combat insolubility and intrinsic instability of certain mutant variants. Asystematic biochemical assessment of the enzymes included analysis of DNA binding affinity, DNA helicase activity, the kinetics of nucleotide hydrolysis, and estimates of thermal stability. In contrast to other studies, we found that all 20 mutant variants retain helicase function under optimized in vitro conditions despite partial reductions in DNA binding affinity, nucleotide hydrolysis, or thermal stability for some mutants. Such partial defects are consistent with the delayed presentation of mitochondrial diseases associated with mutation of C10orf2.