ATP6 homoplasmic mutations inhibit and destabilize the human F1F0-ATP synthase without preventing enzyme assembly and oligomerization
ATP6 homoplasmic mutations inhibit and destabilize the human F1F0-ATP synthase without preventing enzyme assembly and oligomerization
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DOI：
10.1074/jbc.m606828200
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发表时间：
2007-01-12
影响因子：
4.8
通讯作者：
Garcia, Jose J.
中科院分区：
文献类型：
--
作者：
Cortes-Hernandez, Paulina;Vazquez-Memije, Martha E.;Garcia, Jose J.
在线粒体 ATP6 基因（编码 F1F0-ATP 合酶的 6 亚基）中含有同质 T8993G/T8993C 点突变的培养人类细胞中，确定了人类线粒体疾病神经源性共济失调、色素性视网膜炎和母系遗传 Leigh 综合征的分子致病机制。免疫沉淀和蓝色原生电泳表明 F1F0-ATP 合酶在同质突变体线粒体中正确组装。突变体表现出对亚饱和量的寡霉素敏感性增加的趋势；这为 F-1 和 F-0 部门之间的完整组装和紧密耦合提供了进一步的证据。此外，首次观察到人类ATP合酶二聚体和更高的同源寡聚体，并证明突变酶保留了足够的结构完整性以进行寡聚化。 T8993G 突变体发现寡聚酶与单体酶的比例可重复增加，表明 F1F0 寡聚化在体内受到调节，并且可以在病理条件下进行修饰。尽管组装正确，T8993G 突变仍对 ATP 合成周转产生 60% 的抑制。体外变性条件显示突变赋予 F1F0 不稳定性，尽管这种不稳定性在用于测定 ATP 合成的条件下不会产生酶分解。总而言之，数据表明这些有害的人类线粒体突变的主要分子致病机制是正确组装的 ATP 合酶的功能抑制。正如所讨论的，在潜在的变性条件下，结构不稳定性可能在疾病的进展中发挥作用。
The molecular pathogenic mechanism of the human mitochondrial diseases neurogenic ataxia and retinitis pigmentosa and maternally inherited Leigh syndrome was determined in cultured human cells harboring homoplasmic T8993G/T8993C point mutations in the mitochondrial ATP6 gene, which encodes subunit 6 of the F1F0-ATP synthase. Immunoprecipitation and blue native electrophoresis showed that F1F0-ATP synthase assembles correctly in homoplasmic mutant mitochondria. The mutants exhibited a tendency to have an increased sensitivity to subsaturating amounts of oligomycin; this provided further evidence for complete assembly and tight coupling between the F-1 and F-0 sectors. Furthermore, human ATP synthase dimers and higher homo-oligomers were observed for the first time, and it was demonstrated that the mutant enzymes retain enough structural integrity to oligomerize. A reproducible increase in the proportion of oligomeric-to-monomeric enzyme was found for the T8993G mutant suggesting that F1F0 oligomerization is regulated in vivo and that it can be modified in pathological conditions. Despite correct assembly, the T8993G mutation produced a 60% inhibition in ATP synthesis turnover. In vitro denaturing conditions showed F1F0 instability conferred by the mutations, although this instability did not produce enzyme disassembly in the conditions used for determination of ATP synthesis. Taken together, the data show that the primary molecular pathogenic mechanism of these deleterious human mitochondrial mutations is functional inhibition in a correctly assembled ATP synthase. Structural instability may play a role in the progression of the disease under potentially denaturing conditions, as discussed.