bims-resufa 2025-05-25 papers

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

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

Am J Physiol Regul Integr Comp Physiol. 2025 May 23.

Corticosterone Induces Fatty Liver Syndrome in Chickens via Glucocorticoid Receptor and Inhibition of Mitochondrial Supercomplex Formation.

Huimin Chen, Ke Zhang, Hui Yu, Ziting Guan, Ruqian Zhao, Lei Wu.

Stress is a primary contributor to fatty liver syndrome (FLS) in chickens. Mitochondrial functionality is pivotal in FLS progression, with diminished supercomplex (SC) formation disrupting electron transport and escalating reactive oxygen species (ROS) production. However, the impact of stress on mitochondrial SC in chicken FLS remains elusive. This study employed corticosterone (CORT) to model chronic stress and examined its consequences on mitochondrial performance and SC configuration in both vivo and vitro FLS models. Notably, the CORT-treated hepatocytes exhibited elevated triglyceride content (P < 0.05), accompanied by increased mitochondrial ROS (P < 0.01). Moreover, CORT-exposed broilers displayed reduced body weight (P < 0.05) alongside heightened liver-to-body weight ratio (P < 0.01), indicative of liver steatosis with increased triglyceride levels in both liver and plasma (P < 0.01). Mitochondrial alterations in reduced ATP content (P < 0.05). Gene expression analysis revealed enrichment in the mitochondrial respiratory chain pathway, with down-regulated mRNA expression of Complex I-associated SC assembly factors NDUFAF5, NDUFAF7, and TIMMDC1 (P < 0.05). Meanwhile, the glucocorticoid receptor (GR) protein level and its specific binding to the NDUFAF5 gene promoter were reduced in the CORT group (P < 0.01 and P < 0.05, respectively), accompanied by a decrease in NDUFAF5 protein expression in liver, primary hepatocytes, and AML12 cells (P < 0.05). GR knockdown in AML12 cells reduced NDUFAF5 protein expression (P < 0.05). Thus, these findings imply that GR-mediated transcriptional regulation of Complex I assembly factor NDUFAF5 may influence SC assembly, shedding light on stress-induced FLS mechanisms in broilers.

Keywords: broiler; chronic stress; glucocorticoid; liver; mitochondrial supercomplex

DOI: https://doi.org/10.1152/ajpregu.00313.2024