Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA.
Department of Cell Biology and Physiology, Washington University School of Medicine in St. Louis, St. Louis, MO, USA.
Nat Metab. 2024 Jun;6(6):1128-1142. doi: 10.1038/s42255-024-01039-2. Epub 2024 May 8.
Isolated complex I (CI) deficiencies are a major cause of primary mitochondrial disease. A substantial proportion of CI deficiencies are believed to arise from defects in CI assembly factors (CIAFs) that are not part of the CI holoenzyme. The biochemistry of these CIAFs is poorly defined, making their role in CI assembly unclear, and confounding interpretation of potential disease-causing genetic variants. To address these challenges, we devised a deep mutational scanning approach to systematically assess the function of thousands of NDUFAF6 genetic variants. Guided by these data, biochemical analyses and cross-linking mass spectrometry, we discovered that the CIAF NDUFAF6 facilitates incorporation of NDUFS8 into CI and reveal that NDUFS8 overexpression rectifies NDUFAF6 deficiency. Our data further provide experimental support of pathogenicity for seven novel NDUFAF6 variants associated with human pathology and introduce functional evidence for over 5,000 additional variants. Overall, our work defines the molecular function of NDUFAF6 and provides a clinical resource for aiding diagnosis of NDUFAF6-related diseases.
孤立的复合物 I (CI) 缺陷是原发性线粒体疾病的主要原因。大量 CI 缺陷被认为是由于不属于 CI 全酶的 CI 组装因子 (CIAFs) 缺陷引起的。这些 CIAFs 的生物化学性质尚未得到很好的定义，这使得它们在 CI 组装中的作用不明确，并混淆了对潜在致病遗传变异的解释。为了解决这些挑战，我们设计了一种深度突变扫描方法来系统地评估数千种 NDUFAF6 遗传变异的功能。根据这些数据、生化分析和交联质谱分析，我们发现 CIAF NDUFAF6 有助于将 NDUFS8 掺入 CI 中，并揭示 NDUFS8 的过表达可以纠正 NDUFAF6 的缺陷。我们的数据进一步为与人类病理学相关的七个新的 NDUFAF6 变体的致病性提供了实验支持，并为 5000 多个额外变体提供了功能证据。总的来说，我们的工作定义了 NDUFAF6 的分子功能，并为辅助 NDUFAF6 相关疾病的诊断提供了临床资源。
