Systematic Analysis of NDUFAF6 in Complex I Assembly and Mitochondrial Disease

Background

Mitochondrial complex I (CI) is a crucial component of the respiratory chain, playing an essential role in oxidative phosphorylation (OXPHOS) by introducing electrons into the mitochondrial respiratory chain and, together with complexes II-IV, generating and maintaining the proton motive force that drives ATP synthesis. Studies suggest that isolated CI defects constitute one of the most common causes of primary mitochondrial diseases, affecting about 1 in every 5000 individuals. Approximately half of CI defects are thought to be caused by pathogenic variants that affect complex I assembly factors (CIAFs), which are not part of the holoenzyme. Although these CIAFs play a significant role in the pathogenesis of CI diseases, their precise molecular functions remain unclear, complicating not only the study of their mechanisms but also the interpretation and diagnosis of potential pathogenic gene variants.

Source Information

This study was published in the journal Nature Metabolism with the title “Systematic analysis of NDUFAF6 in Complex I Assembly and Mitochondrial Disease”, doi: https://doi.org/10.1038/s42255-024-01039-2. The authors of the paper include Andrew Y. Sung, Rachel M. Guerra, Laura H. Steenberge, among others, who are affiliated with institutions such as the University of Wisconsin School of Medicine, Washington University School of Medicine in St. Louis, Newcastle University Faculty of Medical Sciences in the UK, and the Institute of Human Genetics at the Technical University of Munich in Germany. The research commenced on September 6, 2023, and was accepted for publication on March 28, 2024.

Study Process and Methods

Deep Mutational Scanning Workflow

The research team designed a deep mutational scanning (DMS) method to systematically evaluate the function of thousands of NDUFAF6 gene variants. They followed these experimental steps:

1. Creation of NDUFAF6 Gene Knockout HEK293T Cell Lines: Researchers first used CRISPR-Cas9 technology to create two independent af6 gene knockout (KO) cell lines and verified the absence of af6 and CI in these KO cell lines through Sanger sequencing, Western blotting, and in-gel CI activity assays.

2. Generation and Transfection of Mutation Library: Researchers synthesized a mutation library covering all possible variants of NDUFAF6, cloned it into lentiviral vectors, and then transfected these into the af6 KO cell lines.

3. Screening and Selecting Functional Variants: Functional variants that could grow in media with galactose as the primary carbon source were screened by introducing the mutated NDUFAF6 variants into KO cells and selecting those able to grow in galactose-based medium.

4. Deep Sequencing Analysis: Using high-throughput sequencing technology, the frequency of each variant before and after galactose growth selection was analyzed, and fitness scores for the variants were calculated.

The study results showed that the quality of their mutation scanning data was validated through various methods, including high sequencing depth, highly correlated fitness scores, and good consistency between simulated mutation sensitivities and expected structural models.

Protein-Protein Interaction Analysis

The research team combined deep mutational scanning data with an AlphaFold predicted af6 model to conduct a series of experiments exploring the molecular function of af6. Using cross-linking mass spectrometry, they identified potential binding partners of af6, discovering that af6 specifically binds to the core CI subunit NDUFS8. Further, yeast two-hybrid experiments verified whether surface mutations on af6 affect its interaction with NDUFS8, showing that the specific binding between af6 and NDUFS8 is regulated by particular surface sites.

Blue Native Gel Electrophoresis and Western Blot

To determine the mediating role of af6 in CI assembly, researchers tracked the migration patterns of Q module subunits and assembly factors in HAP1 wild-type and CIAF knockout cell lines using blue native gel electrophoresis. They found that af6 indeed plays a critical role in transitioning NDUFS8 into the 125 kDa intermediate product by facilitating its assembly.

Main Results

af6’s Function and NDUFS8 Integration

Integrating the deep mutational scanning data with structural predictions revealed that af6 is a pseudo-enzyme that directly binds to the CI subunit NDUFS8 and guides its integration into the 125 kDa assembly intermediate. Further experiments showed that overexpressing NDUFS8 could rescue defects caused by af6 deficiency, demonstrating a potential therapeutic route for this specific interaction.

Functionally Sensitive Regions

Deep mutational scanning data highlighted some protein regions with high mutation sensitivity, suggesting functional relevance. For example, the C-terminal helical structure of af6 plays a crucial role in its membrane association, which is essential for CI assembly.

Validation of New Pathogenic Variants

This study experimentally supported the pathogenicity of seven new af6 variants related to human pathology and provided functional evidence for over 5000 unannotated af6 variants, creating a clinical resource to support the diagnosis of af6-related diseases.

Conclusion and Significance

The new findings of this research not only fill the knowledge gap regarding the molecular role of af6 in CI assembly but also provide new diagnostic resources for functional variants and disease causation. Based on these findings, the study proposes potential therapeutic approaches by modulating existing levels of NDUFS8 to compensate for af6 dysfunction. Additionally, the deep mutational scanning data improved the classification of several existing variants under the ACMG (American College of Medical Genetics and Genomics) framework and established seven new pathogenic variants.

Research Highlights

1. Function Identification of af6 as a Pseudo-Enzyme: This study confirms that af6 mediates CI assembly through protein-protein interactions rather than enzymatic activity.
2. Application of New Methods and Techniques: The systematic approach of deep mutational scanning provides a new tool for studying mitochondrial proteins with undefined functions.
3. Clinical Application Value: It provides extensive functional variant data to support more accurate clinical diagnosis of af6-related diseases and develops new therapeutic strategies.

This study successfully demonstrates the importance of understanding the function of mitochondrial protein assembly factors and provides valuable experimental frameworks and methodological insights for investigating other mitochondrial proteins with yet undefined functions.