Mutations in NDUFS4, which encodes an accessory subunit of mitochondrial oxidative phosphorylation (OXPHOS) complex I (CI), induce Leigh syndrome (LS). LS is a poorly understood pediatric disorder featuring brain-specific anomalies and early death. To study the LS pathomechanism, we here compared OXPHOS proteomes between various Ndufs4-/- mouse tissues. Ndufs4-/- animals displayed significantly lower CI subunit levels in brain/diaphragm relative to other tissues (liver/heart/kidney/skeletal muscle), whereas other OXPHOS subunit levels were not reduced. Absence of NDUFS4 induced near complete absence of the NDUFA12 accessory subunit, a 50% reduction in other CI subunit levels, and an increase in specific CI assembly factors. Among the latter, NDUFAF2 was most highly increased. Regarding NDUFS4, NDUFA12 and NDUFAF2, identical results were obtained in Ndufs4-/- mouse embryonic fibroblasts (MEFs) and NDUFS4-mutated LS patient cells. Ndufs4-/- MEFs contained active CI in situ but blue-native-PAGE highlighted that NDUFAF2 attached to an inactive CI subcomplex (CI-830) and inactive assemblies of higher MW. In NDUFA12-mutated LS patient cells, NDUFA12 absence did not reduce NDUFS4 levels but triggered NDUFAF2 association to active CI. BN-PAGE revealed no such association in LS patient fibroblasts with mutations in other CI subunit-encoding genes where NDUFAF2 was attached to CI-830 (NDUFS1, NDUFV1 mutation) or not detected (NDUFS7 mutation). Supported by enzymological and CI in silico structural analysis, we conclude that absence of NDUFS4 induces near complete absence of NDUFA12 but not vice versa, and that NDUFAF2 stabilizes active CI in Ndufs4-/- mice and LS patient cells, perhaps in concert with mitochondrial inner membrane lipids.

中文翻译：

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NDUFS4缺失触发Ndufs4-/-小鼠和Leigh综合征患者中NDUFA12的丢失：NDUFAF2的稳定作用。

NDUFS4中的突变编码线粒体氧化磷酸化（OXPHOS）复合体I（CI）的辅助亚基，诱发了Leigh综合征（LS）。LS是一种鲜为人知的小儿疾病，具有特定于大脑的异常和早期死亡。为了研究LS的发病机理，我们在这里比较了各种Ndufs4-/-小鼠组织之间的OXPHOS蛋白质组。Ndufs4-/-动物相对于其他组织（肝脏/心脏/肾脏/骨骼肌）在大脑/隔膜中的CI亚基水平明显降低，而其他OXPHOS亚基水平并未降低。NDUFS4的缺乏导致NDUFA12辅助亚基几乎完全不存在，其他CI亚基水平降低了50％，并且特定CI组装因子增加。在后者中，NDUFAF2增加最多。关于NDUFS4，NDUFA12和NDUFAF2，在Ndufs4-/-小鼠胚胎成纤维细胞（MEF）和NDUFS4突变的LS患者细胞中获得了相同的结果。Ndufs4-/-MEF包含原位活性CI，但蓝色本机PAGE突出显示NDUFAF2附着于无活性CI亚复合物（CI-830）和分子量较高的无活性组装体。在NDUFA12突变的LS患者细胞中，NDUFA12的缺失不会降低NDUFS4的水平，但会触发NDUFAF2与活动CI的关联。BN-PAGE显示在LS患者成纤维细胞中，与NDUFAF2附着于CI-830的其他CI亚基编码基因的突变（NDUFS1，NDUFV1突变）或未被检测到（NDUFS7突变）没有这种关联。在酶学和CI硅计算机结构分析的支持下，我们得出结论，不存在NDUFS4会诱导NDUFA12几乎完全不存在，反之亦然，