bims-resufa 2025-04-06 papers

Mitochondrion. 2025 Apr 01. pii: S1567-7249(25)00032-7. [Epub ahead of print]83 102035

Kinetic characterization of respirasomes and free complex I from Yarrowia lipolytica.

Giovanni García-Cruz, Mercedes Esparza-Perusquía, Alejandro Cruz-Cárdenas, Diana Cruz-Vilchis, Oscar Flores-Herrera.

The mitochondrion is a highly dynamic organelle capable of adapting to external stimuli and the energetic demands of the cell. As the primary source of cellular ATP, generating approximately 90 % of the total, mitochondrion facilitates the association of respiratory complexes I, III2, and IV into supramolecular structures called respirasomes. This supramolecular organization enhances protein density within the mitochondrial inner membrane, enabling homogenous energy production. In this study, we investigate the subunits composition and the kinetic characterization of digitonin-solubilized respirasomes and the free complex I from Yarrowia lipolytica as well as their role in reactive oxygen species (ROS) production. The NADH:DBQ oxido reductase activity of respirasome and free complex I was similar. Respiration by respirasome was inhibited with rotenone, antimycin A, or cyanide, simultaneously to an increase in the ROS production. A value of 1.6 ± 0.2 for the NADH oxidized/oxygen reduced ratio was determined for the respirasome activity. The role of interaction between complexes in the function of the respirasome is discussed.

Keywords: Complex I activity; Mitochondrial supercomplexes; ROS production; Respirasome; Yarrowia lipolytica mitochondria

DOI: https://doi.org/10.1016/j.mito.2025.102035

Organelle. 2025 ;1(2):

Robust assembly of electron transfer chain in the brain of naked mole-rats during aging.

Ting Liang, Belinda Bernal, Wenbo Qi, Yuji Ikeno, Adam B Salmon, Daisy Kwok, Joel Michalek, Mai Zhou, Yidong Bai.

Naked mole-rats (NMR, Heterocephalus glaber) are the longest-lived rodent species, with a maximum life span of more than 30 years. These long-lived mammals exhibit delayed aging phenotypes and resistance to age-related pathologies including neurodegeneration. Multiple regulatory pathways have been proposed for the anti-aging mechanisms in NMR including enhanced mitochondrial function and suppressed oxidative stress. In this study, we investigated the assembly of the electron transfer chain (ETC) which constitutes the structural base for the regulation of both oxidative phosphorylation and the production of reactive oxygen species (ROS), in brains from young and old NMR and C57BL/6 mice. While ETC assembly declined with aging in C57BL/6 mice, we found that NMR display a robust respiratory chain assembly at older ages in both males and females. Among them, individual complex IV and supercomplexes containing complex I and III or complex III and IV showed the most pronounced differences between two species. Our results indicate that a preserved robust assembly of ETC during aging contributes to enhanced mitochondrial oxidative phosphorylation and suppressed oxidative stress, which may contribute to the longevity and resistance to age-related pathologies in NMR.

Keywords: Aging; Blue Native Gel; Mitochondrial Electron Transfer Chain; Naked Mole-Rat (NMR); Supercomplex

DOI: https://doi.org/10.61747/0ifp.202408002

Genetics. 2025 Apr 03. pii: iyaf037. [Epub ahead of print]

A new set of mutations in the second transmembrane helix of the Cox2p-W56R substantially improves its allotopic expression in Saccharomyces cerevisiae.

Kewin Gombeau, Stefan A Hoffmann, Yizhi Cai.

The dual genetic control of mitochondrial respiratory function, combined with the high mutation rate of the mitochondrial genome (mtDNA), makes mitochondrial diseases among the most frequent genetic diseases in humans (1 in 5,000 in adults). With no effective treatments available, gene therapy approaches have been proposed. Notably, several studies have demonstrated the potential for nuclear expression of a healthy copy of a dysfunctional mitochondrial gene, referred to as allotopic expression, to help recover respiratory function. However, allotopic expression conditions require significant optimization. We harnessed engineering biology tools to improve the allotopic expression of the COX2-W56R gene in the budding yeast Saccharomyces cerevisiae. Through conducting random mutagenesis and screening of the impact of vector copy number, promoter, and mitochondrial targeting sequence, we substantially increased the mitochondrial incorporation of the allotopic protein and significantly increased recovery of mitochondrial respiration. Moreover, CN-PAGE analyses revealed that our optimized allotopic protein does not impact cytochrome c oxidase assembly, or the biogenesis of respiratory chain supercomplexes. Importantly, the most beneficial amino acid substitutions found in the second transmembrane helix (L93S and I102K) are conserved residues in the corresponding positions of human MT-CO2 (L73 and L75), and we propose that mirroring these changes could potentially help improve allotopic Cox2p expression in human cells. To conclude, this study demonstrates the effectiveness of using engineering biology approaches to optimise allotopic expression of mitochondrial genes in the baker's yeast.

Keywords: allotopic expression; engineering biology; mitochondria; random mutagenesis; yeast

DOI: https://doi.org/10.1093/genetics/iyaf037