Ndufaf2, a protein in mitochondrial complex I, interacts in vivo with methionine sulfoxide reductases

ABSTRACT

Background

Methionine sulfoxide reductases are found in all aerobic organisms. They function in antioxidant defense, cellular regulation by reversible oxidation of methionine in proteins, and in protein structure. However, very few in vivo binding partners or substrates of the reductases have been identified.

Methods

We implemented a proximity labeling method, TurboID, to covalently link mitochondrial methionine sulfoxide reductase A (MSRA) to its binding partners in HEK293 cells. Proteomic analyses were performed to identify putative binding partners.

Results

We show that human Ndufaf2, also called mimitin, is a binding partner of MSRA as well as all 3 MSRBs. We found that both methionine residues in Ndufaf2 were susceptible to oxidation by hydrogen peroxide and that the methionine sulfoxide reductases can reduce these methionine sulfoxide residues back to methionine.

Conclusion

Methionine sulfoxide reductases can reduce methionine sulfoxide back to methionine in Ndufaf2. In addition to a repair function, it also creates a mechanism that could regulate cellular processes by modulation of methionine oxidation in Ndufaf2.

KEYWORDS:

• Methionine sulfoxide reductase
• Ndufaf2
• mimitin
• TurboID proximity labeling
• oxidative stress
• methionine oxidation
• mass spectrometry
• mitochondrial complex I

Acknowledgements

Microscopy was performed in the Light Microscope Core of the Intramural Research Program of the National Heart, Lung, and Blood Institute. TurboID peptide analyses were performed in the Proteomics Core of the National Heart, Lung, and Blood Institute. Mass spectrometry data was acquired with instruments in our laboratory and in the Biochemistry Core Facility of the Intramural Research Program of the National Heart, Lung, and Blood Institute. Authors’ contributions: SP and RLL conceptualized the investigation and analyzed the data. SP carried out the experiments, obtained all images, and wrote the original draft. JSH-T performed the bioinformatic analyses. RLL revised the draft and provided funding.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Detailed mass spectrometry data, including the raw data files, and raw imaging files are available upon request. All other data are in the manuscript.

Additional information

Funding

This work was supported by the Intramural Research Program of the of the National Heart, Lung, and Blood Institute through grant ZIA HL000225 to RLL.