bims-cytox1 2025-05-25 papers

Science. 2025 May 22. eadr3498

Silencing mitochondrial gene expression in living cells.

Mitochondria fulfill central functions in metabolism and energy supply. They express their own genome, which encodes key subunits of the oxidative phosphorylation system. However, central mechanisms underlying mitochondrial gene expression remain enigmatic. A lack of suitable technologies to target mitochondrial protein synthesis in cells has limited experimental access. Here, we silenced the translation of specific mitochondrial mRNAs in living human cells by delivering synthetic peptide-morpholino chimeras. This approach allowed us to perform a comprehensive temporal monitoring of cellular responses. Our study provides insights into mitochondrial translation, its integration into cellular physiology, and provides a strategy to address mitochondrial gene expression in living cells. The approach can potentially be used to analyze mechanisms and pathophysiology of mitochondrial gene expression in a range of cellular model systems.

DOI: https://doi.org/10.1126/science.adr3498

Cell Rep. 2025 May 20. pii: S2211-1247(25)00494-2. [Epub ahead of print]44(6): 115723

SMIM20 promotes complex IV biogenesis and Ca2+ signaling in mice heart.

Angela Boshnakovska, Julius Ryan Pronto, Tanja Gall, Abhishek Aich, Jan Prochazka, Zuzana Nichtova, Radislav Sedlacek, Izzatullo Sobitov, Sofia Ainatzi, Christof Lenz, Dörthe M Katschinski, Henning Urlaub, Niels Voigt, Peter Rehling, Laura S Kremer.

Mitochondria are key to cellular energetics, metabolism, and signaling. Their dysfunction is linked to devastating diseases, including mitochondrial disorders, diabetes, neurodegenerative diseases, cardiac disorders, and cancer. Here, we present a knockout mouse model lacking the complex IV assembly factor SMIM20/MITRAC7. SMIM20-/- mice display cardiac pathology with reduced heart weight and cardiac output. Heart mitochondria present with reduced levels of complex IV associated with increased complex I activity, have altered fatty acid oxidation, and display elevated levels of ROS production. Interestingly, mutant mouse ventricular myocytes show unphysiological Ca2+ handling, which can be attributed to the increase in mitochondrial ROS production. Our study presents an example of a tissue-specific phenotype in the context of OXPHOS dysfunction. Moreover, our data suggest a link between complex IV dysfunction and Ca2+ handling at the endoplasmic reticulum through ROS signaling.

Keywords: CP: Cell biology; CP: Molecular biology; OXPHOS; assembly factor; cytochrome c oxidase; mitochondria; mitochondrial disease

DOI: https://doi.org/10.1016/j.celrep.2025.115723

J Vis Exp. 2025 May 02.

Analysis of the Expression and Complexes Assembly of the Mitochondrial Respiratory Chain Proteins in the Fission Yeast Schizosaccharomyces pombe.

Guangchun Lu, Jinjie Shang, Gang Feng.

The mitochondrial respiratory chain is crucial for cellular energy metabolism, serving as the core of oxidative phosphorylation. The mitochondrial respiratory chain comprises five enzyme complexes and their interacting supercomplexes. Analysis of the expression and complexes assembly of these proteins is vital to understanding mitochondrial function. This can be studied by combining biochemical and genetic methods in an excellent model organism fission yeast Schizosaccharomyces pombe (S. pombe), which provides a compensatory system to budding yeast for studies of mitochondrial biology. Here, we present a detailed protocol for the isolation of S. pombe mitochondria and analysis of expression levels and complexes assembly of the mitochondrial respiratory proteins by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and blue native-PAGE (BN-PAGE). Briefly, mitochondria from the wild-type and gene mutants are purified, and then their complexes are solubilized and subjected to SDS-PAGE/BN-PAGE and immunoblotting. This method enables the characterization of a gene's novel function in the mitochondrial respiratory chain.

DOI: https://doi.org/10.3791/68336

mBio. 2025 May 23. e0126925

GSNOR plays roles in growth, pathogenicity, and stress resistance by modulating mitochondrial protein COX6B S-nitrosylation in Colletotrichum gloeosporioides.

Xing Yang, Yongbao Yang, Aoxue Wang, Zhihao Zhao, Hongli Luo, Bang An, Qiannan Wang.

Nitric oxide (NO) serves as a versatile signaling molecule governing diverse biological processes, primarily through post-translational modifications such as S-nitrosylation. The enzyme glutathione S-nitrosoglutathione reductase (GSNOR) plays a central role in NO homeostasis by modulating cellular levels of S-nitrosoglutathione (GSNO), thereby controlling protein S-nitrosylation dynamics. However, the functional significance of GSNOR in fungal pathogenicity remains insufficiently characterized. In this study, we investigated the function of CgGSNOR in the phytopathogenic fungus Colletotrichum gloeosporioides. Deletion of CgGSNOR disrupted nitrosative homeostasis, leading to elevated NO accumulation, increased protein S-nitrosylation levels, and mitochondrial dysfunction as evidenced by reduced ATP production and altered ROS levels. Proteomic and structural analyses identified cytochrome c oxidase subunit 6B (CgCOX6B) as a key target of S-nitrosylation. Functional characterization revealed that CgCOX6B is essential for appressorial turgor maintenance and fungal pathogenicity. Site-directed mutagenesis demonstrated that three conserved cysteine residues (Cys42, Cys62, and Cys73) are critical for CgCOX6B function and are susceptible to S-nitrosylation-induced disruption. Notably, the CgCOX6B knockout strain exhibited increased sensitivity to Iprodione, a widely used fungicide, and this sensitivity was further amplified by NO donor treatment. Together, our findings uncover a GSNOR-dependent redox regulatory axis that links NO signaling, mitochondrial function, and fungal pathogenicity, offering potential targets for antifungal strategies via manipulation of NO signaling networks.IMPORTANCEColletotrichum gloeosporioides is a globally significant fungal pathogen responsible for anthracnose diseases, causing losses across a wide range of crops. Although nitric oxide (NO) signaling and its post-translational regulatory mechanism, S-nitrosylation, are known to play pivotal roles in fungal biology, their specific contributions to pathogenicity remain poorly characterized. This study identifies glutathione S-nitrosoglutathione reductase (GSNOR) as a critical regulator of NO homeostasis in C. gloeosporioides and demonstrates its critical role in regulating fungal growth, conidiation, and pathogenicity. We uncover cytochrome c oxidase subunit 6B (COX6B) as a key target of S-nitrosylation, required for fungal energy metabolism, host infection, and resistance to fungicides. Furthermore, we reveal that exogenous NO supplementation using sodium nitroprusside synergistically enhances the antifungal activity of Iprodione. These findings advance our understanding of redox regulation in fungal pathogenesis and highlight GSNOR and COX6B as promising molecular targets for developing antifungal approaches to reduce crop losses.

Keywords: Colletotrichum gloeosporioides; S-nitrosylation; cytochrome c oxidase subunit 6B (COX6B); fungal pathogenicity; glutathione S-nitrosoglutathione reductase (GSNOR)

DOI: https://doi.org/10.1128/mbio.01269-25

Nat Struct Mol Biol. 2025 May 19.

Structure, assembly and inhibition of the Toxoplasma gondii respiratory chain supercomplex.

Andrew E MacLean, Shikha Shikha, Mariana Ferreira Silva, Max J Gramelspacher, Aaron Nilsen, Katherine M Liebman, Sovitj Pou, Rolf W Winter, Amit Meir, Michael K Riscoe, J Stone Doggett, Lilach Sheiner, Alexander Mühleip.

The apicomplexan mitochondrial electron transport chain is essential for parasite survival and displays a divergent subunit composition. Here we report cryo-electron microscopy structures of an apicomplexan III2-IV supercomplex and of the drug target complex III2. The supercomplex structure reveals how clade-specific subunits form an apicomplexan-conserved III2-IV interface with a unique, kinked architecture, suggesting that supercomplexes evolved independently in different eukaryotic lineages. A knockout resulting in supercomplex disassembly challenges the proposed role of III2-IV in electron transfer efficiency as suggested for mammals. Nevertheless, knockout analysis indicates that III2-IV is critical for parasite fitness. The complexes from the model parasite Toxoplasma gondii were inhibited with the antimalarial atovaquone, revealing interactions underpinning species specificity. They were also inhibited with endochin-like quinolone (ELQ)-300, an inhibitor in late-stage preclinical development. Notably, in the apicomplexan binding site, ELQ-300 is flipped compared with related compounds in the mammalian enzyme. On the basis of the binding modes and parasite-specific interactions discovered, we designed more potent ELQs with subnanomolar activity against T. gondii. Our findings reveal critical evolutionary differences in the role of supercomplexes in mitochondrial biology and provide insight into cytochrome b inhibition, informing future drug discovery.

DOI: https://doi.org/10.1038/s41594-025-01531-7