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Cell-specific modulation of mitochondrial respiration and metabolism by the pro-apoptotic Bcl-2 family members Bax and Bak

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Abstract

Proteins from the Bcl-2 family play an essential role in the regulation of apoptosis. However, they also possess cell death-unrelated activities that are less well understood. This prompted us to study apoptosis-unrelated activities of the Bax and Bak, pro-apoptotic members of the Bcl-2 family. We prepared Bax/Bak-deficient human cancer cells of different origin and found that while respiration in the glioblastoma U87 Bax/Bak-deficient cells was greatly enhanced, respiration of Bax/Bak-deficient B lymphoma HBL-2 cells was slightly suppressed. Bax/Bak-deficient U87 cells also proliferated faster in culture, formed tumours more rapidly in mice, and showed modulation of metabolism with a considerably increased NAD+/NADH ratio. Follow-up analyses documented increased/decreased expression of mitochondria-encoded subunits of respiratory complexes and stabilization/destabilization of the mitochondrial transcription elongation factor TEFM in Bax/Bak-deficient U87 and HBL-2 cells, respectively. TEFM downregulation using shRNAs attenuated mitochondrial respiration in Bax/Bak-deficient U87 as well as in parental HBL-2 cells. We propose that (post)translational regulation of TEFM levels in Bax/Bak-deficient cells modulates levels of subunits of mitochondrial respiratory complexes that, in turn, contribute to respiration and the accompanying changes in metabolism and proliferation in these cells.

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Data availability

Metabolomic and proteomic analyses will be publicly available on the IBT CAS/BIOCEV website, and on the Zenodo website - https://zenodo.org/, the links will be specified. All other materials and cells will be available upon request.

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Acknowledgements

The work on the project was supported by the Czech Science Foundation grants GA19-08772 S (LA), GX21-04607X (JN) and by Ministry of Health of the Czech Republic grant AZV NU21-03-00386 (PK) and NU23-03-00226 (SD). We also acknowledge the BIOCEV European Regional Development Fund CZ.1.05/1.100/02.0109 and Institute of Biotechnology funding (RVO: 86652036). Electron Microscopy Core Facility, IMG AS CR was supported by the Czech-BioImaging large RI project (LM2023050 funded by MEYS CR) and by OP RDE (Project No. CZ.02.1.01/0.0/0.0/18_046/0016045 “Modernization and support of research activities of the national infrastructure for biological and medical imaging Czech-BioImaging”). Metabolomic and proteomic LC-MS analyses were partly supported by Czech Science Foundation grant number 20-05942 S. Calcium imaging microscopy was supported by Czech-BioImaging large RI project (LM2023050). JK was supported by project L200392251 from the Czech Academy of Sciences.

Funding

This work was supported by Czech Science Foundation grants GA19-08772 S (L.A.), GX21-04607X (J.N.), and 20-05942 S (Metabolomics’ and proteomics’ LC-MS analyses), Ministry of Health of the Czech Republic grant AZV NU21-03-00386 (P.K.) and NU23-03-00226 (S.D.), BIOCEV European Regional Development Fund CZ.1.05/1.100/02.0109 and Institute of Biotechnology funding (RVO: 86652036) (L.A., J.N.). We also acknowledge support by the Czech-BioImaging large RI project (LM2023050 funded by the Ministry of Education, Youth and Sports of Czech Republic (H.R., V.F., J.K., Z.H., T.K., M.A.) and by OP RDE (Project No. CZ.02.1.01/0.0/0.0/18_046/0016045 “Modernization and support of research activities of the national infrastructure for biological and medical imaging Czech-BioImaging”; H.R., V.F.). J.K. was supported by project L200392251 from the Czech Academy of Sciences.

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Authors and Affiliations

  1. Institute of Biotechnology, Czech Academy of Sciences, Vestec, Prague, Czech Republic

    Dana Sovilj, Cristina Daniela Kelemen, Sarka Dvorakova, Renata Zobalova, Jiri Neuzil & Ladislav Andera

  2. Faculty of Science, Charles University, Prague, Czech Republic

    Cristina Daniela Kelemen & Jiri Neuzil

  3. Institute of Molecular Genetics, Czech Academy of Sciences, Prague, Czech Republic

    Helena Raabova, Vlada Filimonenko & Ladislav Andera

  4. Institute of Experimental Medicine, Czech Academy of Sciences, Prague, Czech Republic

    Jan Kriska, Zuzana Hermanova, Tomas Knotek & Miroslava Anderova

  5. First Faculty of Medicine, Institute of Pathological Physiology, Charles University, Prague, Czech Republic

    Pavel Klener

  6. First Faculty of Medicine, Charles University, Prague, Czech Republic

    Jiri Neuzil

  7. School of Pharmacy and Medical Science, Griffith University, Southport, QLD, Australia

    Jiri Neuzil

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  1. Dana Sovilj
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Contributions

All authors contributed to the study’s conception and design. LA supervised and coordinated the study. DS, CDK, SD performed molecular and cell biological experiments. DS, RZ, PK realized in vivo experiments. HR, VF run EM experiments. JK, ZH, TK, MA performed and discussed calcium imaging experiment. DS performed the statistical analyses for all the data. DS, LA, JN drafted the manuscript. All authors reviewed the manuscript.

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Correspondence to Ladislav Andera.

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Conflict of interest

Jiri Neuzil is co-owner of MitoTax s.r.o., receives no compensation, and has no competing or financial interest. The other authors have no relevant financial or non-financial interests to disclose

Ethical approval

Subcutaneous xenotransplantations into NSG mice and follow-up surveillance were performed in accordance with the European Communities Council Directive November 24, 1986 (86/609/EEC) and animal care guidelines approved by the Animal Care Committee of the Institute of Molecular Genetics CAS, approval number 74/2020 and by the Animal Welfare Committee of the Charles University, approval number MSMT-26148/2022-4. We aimed to minimize the suffering and the number of animals. The mice were kept on a 12-h light/dark cycle with access to food and water ad libitum.

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Sovilj, D., Kelemen, C.D., Dvorakova, S. et al. Cell-specific modulation of mitochondrial respiration and metabolism by the pro-apoptotic Bcl-2 family members Bax and Bak. Apoptosis 29, 424–438 (2024). https://doi.org/10.1007/s10495-023-01917-2

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