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Molecular and Functional Effects of Loss of Cytochrome c Oxidase Subunit 8A

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Abstract

In this work we studied molecular and functional effects of the loss of the smallest nuclear encoded subunit of cytochrome c oxidase COX8A in fibroblasts from a patient with a homozygous splice site mutation and in CRISPR/Cas9 genome-edited HEK293T cells. In both cellular model systems, between 20 to 30% of the residual enzymatic activity of cytochrome c oxidase (COX) was detectable. In immunoblots of BN-PAGE separated mitochondria from both cellular models almost no monomers and dimers of the fully assembled COX could be visualized. Interestingly, supercomplexes of COX formed with complex III and also with complexes I and III retained considerable immunoreactivity, while nearly no immunoreactivity attributable to subassemblies was found. That indicates that COX lacking subunit 8A is stabilized in supercomplexes, while monomers and dimers are rapidly degraded. With transcriptome analysis by 3′-RNA sequencing we failed to detect in our cellular models of COX8A deficiency transcriptional changes of genes involved in the mitochondrial unfolded protein response (mtUPR) and the integrated stress response (ISR). Thus, our data strongly suggest that the smallest subunit of cytochrome c oxidase COX8A is required for maintenance of the structural stability of COX monomers and dimers.

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Abbreviations

COX:

cytochrome c oxidase

IMM:

inner mitochondrial membrane

OXPHOS:

oxidative phosphorylation

RC:

respiratory chain

References

  1. Hackenbrock, C. R., Chazotte, B., and Gupte, S. S. (1986) The random collision model and a critical assessment of diffusion and collision in mitochondrial electron transport, J. Bioenerg. Biomembr., 18, 331-368, doi: https://doi.org/10.1007/BF00743010.

    Article  CAS  PubMed  Google Scholar 

  2. Schägger, H., and Pfeiffer, K. (2000) Supercomplexes in the respiratory chains of yeast and mammalian mitochondria, EMBO J., 19, 1777-1783, doi: https://doi.org/10.1093/emboj/19.8.1777.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Acín-Pérez, R., Fernández-Silva, P., Peleato, M. L., Pérez-Martos, A., and Enriquez, J. A. (2008) Respiratory active mitochondrial supercomplexes, Mol. Cell, 32, 529-539, doi: https://doi.org/10.1016/j.molcel.2008.10.021.

    Article  CAS  PubMed  Google Scholar 

  4. Schägger, H., and Pfeiffer, K. (2001) The ratio of oxidative phosphorylation complexes I-V in bovine heart mitochondria and the composition of respiratory chain supercomplexes, J. Biol. Chem., 276, 37861-37867, doi: https://doi.org/10.1074/jbc.M106474200.

    Article  CAS  PubMed  Google Scholar 

  5. Greggio, C., Jha, P., Kulkarni, S. S., Lagarrigue, S., Broskey, N. T., et al. (2017) Enhanced respiratory chain supercomplex formation in response to exercise in human skeletalm, Cell Metab., 25, 301-311, doi: https://doi.org/10.1016/j.cmet.2016.11.004.

    Article  CAS  PubMed  Google Scholar 

  6. Lapuente-Brun, E., Moreno-Loshuertos, R., Aciń-Pérez, R., Latorre-Pellicer, A., Colaś, C., et al. (2013) Supercomplex assembly determines electron flux in the mitochondrial electron transport chain, Science, 340, 1567-1570, doi: https://doi.org/10.1126/science.1230381.

    Article  CAS  PubMed  Google Scholar 

  7. Fedor, J. G., and Hirst, J. (2018) Mitochondrial supercomplexes do not enhance catalysis by quinone channeling, Cell Metab., 28, 525-531.e4, doi: https://doi.org/10.1016/j.cmet.2018.05.024.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Calvaruso, M. A., Willems, P., Van den Brand, M., Valsecchi, F., Kruse, S., et al. (2012) Mitochondrial complex III stabilizes complex I in the absence of NDUFS4 to provide partial activity, Hum. Mol. Genet., 21, 115-120, doi: https://doi.org/10.1093/hmg/ddr446.

    Article  CAS  PubMed  Google Scholar 

  9. Schägger, H., De Coo, R., Bauer, M. F., Hofmann, S., Godino, C., and Brandt, U. (2004) Significance of respirasomes for the assembly/stability of human respiratory chain complex I, J. Biol. Chem., 279, 36349-36353, doi: https://doi.org/10.1074/jbc.M404033200.

    Article  CAS  PubMed  Google Scholar 

  10. Tropeano, C. V., Aleo, S. J., Zanna, C., Roberti, M., Scandiffio, L., et al. (2020) Fine-tuning of the respiratory complexes stability and supercomplexes assembly in cells defective of complex III, Biochim. Biophys. Acta Bioenerg., 1861, 148133, doi: https://doi.org/10.1016/j.bbabio.2019.148133.

    Article  CAS  PubMed  Google Scholar 

  11. Hirst, J. (2018) Open questions: respiratory chain supercomplexes-why are they there and what do they do? BMC Biol., 16, 5-8, doi: https://doi.org/10.1186/s12915-018-0577-5.

    Article  CAS  Google Scholar 

  12. Maranzana, E., Barbero, G., Falasca, A. I., Lenaz, G., and Genova, M. L. (2013) Mitochondrial respiratory supercomplex association limits production of reactive oxygen species from complex I, Antioxid. Redox Signal., 19, 1469-1480, doi: https://doi.org/10.1089/ars.2012.4845.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Zong, S., Wu, M., Gu, J., Liu, T., Guo, R., and Yang, M. (2018) Structure of the intact 14-subunit human cytochrome c oxidase, Cell Res., 28, 1026-1034, doi: https://doi.org/10.1038/s41422-018-0071-1.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Sinkler, C. A., Kalpage, H., Shay, J., Lee, I., Malek, M. H., Grossman, L. I., and Hüttemann, M. (2017) Tissue- and condition-specific isoforms of mammalian cytochrome c oxidase subunits: from function to human disease, Oxid. Med. Cell. Longev., 2017, 1534056, doi: https://doi.org/10.1155/2017/1534056.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Goldberg, A., Wildman, D. E., Schmidt, T. R., Hüttemann, M., Goodman, M., et al. (2003) Adaptive evolution of cytochrome c oxidase subunit VIII in anthropoid primates, Proc. Natl. Acad. Sci. USA, 100, 5873-5878, doi: https://doi.org/10.1073/pnas.0931463100.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Hallmann, K., Kudin, A. P., Zsurka, G., Kornblum, C., Reimann, J., et al. (2016) Loss of the smallest subunit of cytochrome c oxidase, COX8A, causes Leigh-like syndrome and epilepsy, Brain, 139, 338-345, doi: https://doi.org/10.1093/brain/awv357.

    Article  PubMed  Google Scholar 

  17. Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A., and Zhang, F. (2013) Genome engineering using the CRISPR-Cas9 system, Nat. Protoc., 8, 2281-2308, doi: https://doi.org/10.1038/nprot.2013.143.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Bednarczyk, P., Wieckowski, M. R., Broszkiewicz, M., Skowronek, K., Siemen, D., and Szewczyk, A. (2013) Putative structural and functional coupling of the mitochondrial BKCa channel to the respiratory chain, PLoS One, 8, e68125, doi: https://doi.org/10.1371/journal.pone.0068125.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Wiedemann, F. R., Vielhaber, S., Schröder, R., Elger, C. E., and Kunz, W. S. (2000) Evaluation of methods for the determination of mitochondrial respiratory chain enzyme activities in human skeletal muscle samples, Anal. Biochem., 279, 55-60, doi: https://doi.org/10.1006/abio.1999.4434.

    Article  CAS  PubMed  Google Scholar 

  20. Pellegrino, M. W., Nargund, A. M., and Haynes, C. M. (2013) Signaling the mitochondrial unfolded protein response, Biochim. Biophys. Acta, 1833, 410-416, doi: https://doi.org/10.1016/j.bbamcr.2012.02.019.

    Article  CAS  PubMed  Google Scholar 

  21. Quirós, P. M., Langer, T., and López-Otín, C. (2015) New roles for mitochondrial proteases in health, ageing and disease, Nat. Rev. Mol. Cell Biol., 16, 345-359, doi: https://doi.org/10.1038/nrm3984.

    Article  CAS  PubMed  Google Scholar 

  22. Fiorese, C. J., and Haynes, C. M. (2017) Integrating the UPRmt into the mitochondrial maintenance network, Crit. Rev. Biochem. Mol. Biol., 52, 304-313, doi: https://doi.org/10.1080/10409238.2017.1291577.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Wang, Y. T., Lim, Y., McCall, M. N., Huang, K. T., Haynes, C. M., Nehrke, K., and Brookes, P. S. (2019) Cardioprotection by the mitochondrial unfolded protein response requires ATF5, Am. J. Physiol., 317, H472-H478, doi: https://doi.org/10.1152/ajpheart.00244.2019.

    Article  CAS  Google Scholar 

  24. Fiorese, C. J., Schulz, A. M., Lin, Y. F., Rosin, N., Pellegrino, M. W., and Haynes, C. M. (2016) The transcription factor ATF5 mediates a mammalian mitochondrial UPR, Curr. Biol., 26, 2037-2043, doi: https://doi.org/10.1016/j.cub.2016.06.002.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Harding, H. P., Zhang, Y., Zeng, H., Novoa, I., Lu, P. D., et al. (2003) An integrated stress response regulates amino acid metabolism and resistance to oxidative stress, Mol. Cell, 11, 619-633, doi: https://doi.org/10.1016/S1097-2765(03)00105-9.

    Article  CAS  PubMed  Google Scholar 

  26. Taniuchi, S., Miyake, M., Tsugawa, K., Oyadomari, M., and Oyadomari, S. (2016) Integrated stress response of vertebrates is regulated by four eIF2α kinases, Sci. Rep., 6, 32886, doi: https://doi.org/10.1038/srep32886.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Palam, L. R., Baird, T. D., and Wek, R. C. (2011) Phosphorylation of eIF2 facilitates ribosomal bypass of an inhibitory upstream ORF to enhance CHOP translation, J. Biol. Chem., 286, 10939-10949, doi: https://doi.org/10.1074/jbc.M110.216093.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Fessler, E., Eckl, E. M., Schmitt, S., Mancilla, I. A., Meyer-Bender, M. F., et al. (2020) A pathway coordinated by DELE1 relays mitochondrial stress to the cytosol, Nature, 579, 433-437, doi: https://doi.org/10.1038/s41586-020-2076-4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Timón-Gómez, A., Nývltová, E., Abriata, L. A., Vila, A. J., Hosler, J., and Barrientos, A. (2018) Mitochondrial cytochrome c oxidase biogenesis: recent developments, Semin. Cell Dev. Biol., 76, 163-178, doi: https://doi.org/10.1016/j.semcdb.2017.08.055.

    Article  CAS  PubMed  Google Scholar 

  30. Bourens, M., Boulet, A., Leary, S. C., and Barrientos, A. (2014) Human COX20 cooperates with SCO1 and SCO2 to mature COX2 and promote the assembly of cytochrome c oxidase, Hum. Mol. Genet., 23, 2901-2913, doi: https://doi.org/10.1093/hmg/ddu003.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Lobo-Jarne, T., Pérez-Pérez, R., Fontanesi, F., Timón-Gómez, A., Wittig, I., et al. (2020) Multiple pathways coordinate assembly of human mitochondrial complex IV and stabilization of respiratory supercomplexes, EMBO J., 39, e103912, doi: https://doi.org/10.15252/embj.2019103912.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Kovářová, N., Čížková Vrbacká, A., Pecina, P., Stránecký, V., et al. (2012) Adaptation of respiratory chain biogenesis to cytochrome c oxidase deficiency caused by SURF1 gene mutations, Biochim. Biophys. Acta, 1822, 1114-1124, doi: https://doi.org/10.1016/j.bbadis.2012.03.007.

    Article  CAS  PubMed  Google Scholar 

  33. Baertling, F., van den Brand, M. A. M., Hertecant, J. L., Al-Shamsi, A., van den Heuvel, L. P., et al. (2015) Mutations in COA6 cause cytochrome c oxidase deficiency and neonatal hypertrophic cardiomyopathy, Hum. Mutat., 36, 34-38, doi: https://doi.org/10.1002/humu.22715.

    Article  CAS  PubMed  Google Scholar 

  34. Baertling, F., Al-Murshedi, F., Sánchez-Caballero, L., Al-Senaidi, K., Joshi, N. P., et al. (2017) Mutation in mitochondrial complex IV subunit COX5A causes pulmonary arterial hypertension, lactic acidemia, and failure to thrive, Hum. Mutat., 38, 692-703, doi: https://doi.org/10.1002/humu.2321035.

    Article  CAS  PubMed  Google Scholar 

  35. Schäfer, E., Seelert, H., Reifschneider, N. H., Krause, F., Dencher, N. A., and Vonck, J. (2006) Architecture of active mammalian respiratory chain supercomplexes, J. Biol. Chem., 281, 15370-15375, doi: https://doi.org/10.1074/jbc.M513525200.

    Article  CAS  PubMed  Google Scholar 

  36. Qureshi, M. A., Haynes, C. M., and Pellegrino, M. W. (2017) The mitochondrial unfolded protein response: signaling from the powerhouse, J. Biol. Chem., 292, 13500-13506, doi: https://doi.org/10.1074/jbc.R117.791061.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work is dedicated to Dr. Alexander A. Konstantinov who passed away in May 2020. The authors wish to thank Prof. Mike Ryan and Dr. David Stroud for providing the HEK293T cell line.

Funding

This work was financially supported by the DFG – Deutsche Forschungsgemeinschaft (grants KU 911/21-2 and KU 911/22-1 to W. S. K.; ZS 99/3-2 and ZS 99/4-1 to GZ), by the Polish National Science Center (grants No. 2019/34/A/NZ1/00352 to AS and 2015/18/E/NZ1/00737 to BK), and the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska–Curie grant agreement No. 665735 (Bio4Med).

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Authors and Affiliations

  1. Institute of Experimental Epileptology and Cognition Research, Life & Brain Center, University of Bonn, Venusberg-Campus 1, 53127, Bonn, Germany

    Daria Rotko, Alexei P. Kudin, Gábor Zsurka & Wolfram S. Kunz

  2. Laboratory of Intracellular Ion Channels, Nencki Institute of Experimental Biology, Polish Academy of Sciences, 02-093, Warsaw, Poland

    Daria Rotko, Bogusz Kulawiak & Adam Szewczyk

  3. Department of Epileptology, University Bonn Medical Center, Venusberg-Campus 1, 53127, Bonn, Germany

    Gábor Zsurka & Wolfram S. Kunz

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  1. Daria Rotko
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  2. Alexei P. Kudin
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Correspondence to Wolfram S. Kunz.

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The authors declare no conflicts of interest in financial or any other sphere. All the procedures carried out in the research with participation of humans were in compliance with the ethical standards of the National Research Ethics Committee and with the Helsinki Declaration of 1964 and its subsequent changes or with comparable ethics standards. Informed voluntary consent was obtained from the participants of the study.

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Rotko, D., Kudin, A.P., Zsurka, G. et al. Molecular and Functional Effects of Loss of Cytochrome c Oxidase Subunit 8A. Biochemistry Moscow 86, 33–43 (2021). https://doi.org/10.1134/S0006297921010041

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