bims-cytox1 2025-09-21 papers

PLoS One. 2025 ;20(9): e0332065

Validation of blue- and clear-native polyacrylamide gel electrophoresis protocols to characterize mitochondrial oxidative phosphorylation complexes.

Jana Aref, Seungtae Lee, Supachaya Sriphoosanaphan, Micol Falabella, Shi-Yu Yang, Jan-Willem Taanman.

The mitochondrial oxidative phosphorylation (OXPHOS) system plays a pivotal role in the cell's energy conversion. The enzymes involved in OXPHOS are arranged in five protein-lipid complexes. The first four complexes (I-IV) form the mitochondrial respiratory chain, while Complex V is an F1Fo-ATP synthase. Mutations in genes involved in the biosynthesis of the OXPHOS complexes are an important cause of metabolic diseases. Blue-native polyacrylamide gel electrophoresis (BN-PAGE), originally developed by Hermann Schägger in the 1990s, has become instrumental in gaining insights into structure/function relationships of the OXPHOS system, including: (1) the assembly pathways of the complexes, (2) the composition of higher-order respiratory chain supercomplexes and (3) pathologic mechanisms in patients with a monogenetic OXPHOS disorder. We have used BN-PAGE for >20 years and validate here our recently published step-by-step laboratory protocol. This protocol describes the manual casting of native mini-gels and sample preparation for the resolution of individual OXPHOS complexes or respiratory chain supercomplexes. In addition to BN-PAGE, we explain the closely related clear-native (CN)-PAGE and two-dimensional BN/denaturing-PAGE techniques. Downstream applications include western blot analysis and in-gel enzyme activity staining for Complexes I, II, IV and V. Limitations of the technique are the comparative insensitivity of in-gel Complex IV activity staining and the lack of in-gel Complex III activity staining. Compared to other published BN-PAGE protocols, our protocol contains a shortened sample extraction procedure, advises when to use BN-PAGE and when to use CN-PAGE, and suggests a simple enhancement step for in-gel Complex V activity staining that markedly improves sensitivity. Our protocol is adaptable and yields robust, semi-quantitative and reproducible results.

DOI: https://doi.org/10.1371/journal.pone.0332065

J Oncol Res Ther. 2025 ;pii: 10299. [Epub ahead of print]10(3):

The Nuclear-Encoded Cytochrome c Oxidase Subunit COX4-1 Enhances Hypoxia Tolerance in Glioblastoma Cells.

Claudia R Oliva, Susanne Flor, Md Yousuf Ali, Corinne E Griguer.

Glioblastoma (GBM) is the most common and aggressive primary brain cancer in adults. While chemo- and radiotherapy are often effective in treating newly diagnosed GBM, increasing evidence suggests that treatment-induced metabolic alterations promote tumor recurrence and further resistance. In addition, GBM tumors are typically hypoxic, which further contributes to treatment resistance. Recent studies have shown that changes in glioma cell metabolism driven by a shift in the isoform expression of mitochondrial cytochrome c oxidase (CcO) subunit 4 (COX4), a key regulatory subunit of mammalian CcO, may underlie the treatment-induced metabolic alterations in GBM cells. However, the impact of hypoxia on GBM energetics is not fully understood. Using isogenic GBM cell lines expressing either COX4-1 or the alternative COX4 isoform, COX4-2, we found that COX4-1 expressing cells maintained a more oxidative metabolism under hypoxia, characterized by increased CcO activity and ATP production, enhanced assembly of CcO-containing mitochondrial supercomplexes, and reduced superoxide production. Furthermore, COX4-1 expression was sufficient to increase radioresistance under hypoxic conditions. Untargeted metabolomic analysis revealed that the most significantly upregulated pathways in COX4-1-expressing cells under hypoxia were purine and methionine metabolism. In contrast, COX4-2-expressing cells showed increased activation of glycolysis and the Warburg effect. Our study provides new insights into how CcO regulatory subunits influence cellular metabolic networks and radioresistance in GBM under hypoxia, identifying potential therapeutic targets for improved treatment strategies.

Keywords: COX4–1; Cytochrome c oxidase; Glioma; Hypoxia; Mitochondrial supercomplexes; Radioresistance

DOI: https://doi.org/10.29011/2574-710x.10299

Biol Chem. 2025 Sep 15.

Manipulating mitochondrial gene expression.

Drishan Dahal, Luis D Cruz-Zargoza, Peter Rehling.

Mitochondria are essential for cellular metabolism, serving as the primary source of adenosine triphosphate (ATP). This energy is generated by the oxidative phosphorylation (OXPHOS) system located in the inner mitochondrial membrane. Impairments in this machinery are linked to serious human diseases, especially in tissues with high energy demands. Assembly of the OXPHOS system requires the coordinated expression of genes encoded by both the nuclear and mitochondrial genomes. The mitochondrial DNA encodes for 13 protein components, which are synthesized by mitochondrial ribosomes and inserted into the inner membrane during translation. Despite progress, key aspects of how mitochondrial gene expression is regulated remain elusive, largely due to the organelle's limited genetic accessibility. However, emerging technologies now offer new tools to manipulate various stages of this process. In this review, we explore recent strategies that expand our ability to target mitochondria genetically.

Keywords: RNA; gene expression; genetic tools; mitochondria

DOI: https://doi.org/10.1515/hsz-2025-0170

Life Sci. 2025 Sep 11. pii: S0024-3205(25)00596-X. [Epub ahead of print] 123961

Cisplatin transported by COX17 induces cochlear damage by binding Myosin IIA to regulate cell pyroptosis induced by mitochondrial dysfunction.

Jiahui Peng, Guofang Tao, Xubo Chen, Yufeng Ai, Ruosha Lai, Wei Liu, Bing Hu, Yingying Xu, Bingbin Xie, Lihua Li.

AIMS: Cytochrome c oxidase copper chaperone 17 (COX17) could transport cisplatin to cancer cell mitochondria. Whether COX17 transports cisplatin to cochlear mitochondria and induces pyroptosis to participate in cisplatin-induced ototoxicity remains unknown.
MATERIALS AND METHODS: A cisplatin-induced hearing loss model was constructed in rats, and the role of COX17 and Myosin IIA in cisplatin-induced hearing loss and cochlear injury was evaluated by auditory brainstem response test and H&E staining. Cisplatin binding to Myosin IIA was verified through cisplatin agar-pull-down assays and drug affinity responsiveness target stability. The interaction between Myosin IIA and F-actin was verified using co-immunoprecipitation. Various techniques were used to study how cisplatin causes damage to cochlear hair cells and induces pyroptosis, including immunofluorescence staining, and flow cytometry.
KEY FINDINGS: Our findings indicated that downregulating COX17 inhibits cisplatin accumulation in mitochondria, leading to improved cell survival, and inhibits pyrodeath. Upon entering the mitochondria, cisplatin transported by COX17 binds to Myosin IIA, enhancing its expression and subsequently promoting the expression of F-actin and p-DRP1 while inhibiting the expression of Mfn1, Mfn2, and OPA1. Furthermore, the interaction between Myosin IIA and F-actin was determined. Downregulation of Myosin IIA or treatment with mitochondrial fission inhibitors resulted in reduced mitochondrial ROS release and increased pyroptosis. In vivo studies demonstrated that downregulating COX17 or Myosin IIA improved cisplatin-induced hearing loss and cochlear damage in rats.
SIGNIFICANCE: These findings offer new insights and potential targets for the preventing and treatment of cisplatin-induced ototoxicity.

Keywords: COX17; Cisplatin ototoxicity; Myosin IIA; mitochondrial damage; pyroptosis

DOI: https://doi.org/10.1016/j.lfs.2025.123961