bims-cytox1 2023-03-26 papers

EMBO Rep. 2023 Mar 20. e55760

A quantitative fluorescence-based approach to study mitochondrial protein import.

Naintara Jain, Ridhima Gomkale, Olaf Bernhard, Peter Rehling, Luis Daniel Cruz-Zaragoza.

Mitochondria play central roles in cellular energy production and metabolism. Most proteins required to carry out these functions are synthesized in the cytosol and imported into mitochondria. A growing number of metabolic disorders arising from mitochondrial dysfunction can be traced to errors in mitochondrial protein import. The mechanisms underlying the import of precursor proteins are commonly studied using radioactively labeled precursor proteins imported into purified mitochondria. Here, we establish a fluorescence-based import assay to analyze protein import into mitochondria. We show that fluorescently labeled precursors enable import analysis with similar sensitivity to those using radioactive precursors, yet they provide the advantage of quantifying import with picomole resolution. We adapted the import assay to a 96-well plate format allowing for fast analysis in a screening-compatible format. Moreover, we show that fluorescently labeled precursors can be used to monitor the assembly of the F1 F0 ATP synthase in purified mitochondria. Thus, we provide a sensitive fluorescence-based import assay that enables quantitative and fast import analysis.

Keywords: fluorescent precursor; in vitro import; mitochondria; presequence pathway; protein import

DOI: https://doi.org/10.15252/embr.202255760

FASEB J. 2023 Apr;37(4): e22891

Switching ubiquitous and muscle-specific isoforms of mitochondrial respiratory complex IV in skeletal muscle fine-tunes complex IV activity.

Xianglai Ye, Yaojun Xie, Yu Shi, Bo Wang, Xinyu Han, Xiaoxuan Zhou, Kexin Pan, Maofeng Wang, Hezhi Fang.

Respiratory complex IV (CIV, cytochrome c oxidase) is the terminal enzyme of the mitochondrial electron transport chain. Some CIV subunits have two or more isoforms, which are ubiquitously expressed or are expressed in specific tissues like the lung, muscle, and testis. Among the tissue-specific CIV isoforms, the muscle-specific isoforms are expressed in adult cardiac and skeletal muscles. To date, the physiological and biochemical association between the muscle-specific CIV isoforms and aerobic respiration in muscles remains unclear. In this study, we profiled the CIV organization and expression pattern of muscle-specific CIV isoforms in different mouse muscle tissues. We found extensive CIV-containing supramolecular organization in murine musculature at advanced developmental stages, while a switch in the expression from ubiquitous to muscle-specific isoforms of CIV was also detected. Such a switch was confirmed during the in vitro differentiation of mouse C2C12 myoblasts. Unexpectedly, a CIV expression decrease was observed during C2C12 differentiation, which was probably due to a small increase in the expression of muscle-specific isoforms coupled with a dramatic decrease in the ubiquitous isoforms. We also found that the enzymatic activity of CIV containing the muscle-specific isoform COX6A2 was higher than that with COX6A1 in engineered HEK293T cells. Overall, our results indicate that switching the expression from ubiquitous to muscle-specific CIV isoforms is indispensable for optimized oxidative phosphorylation in mature skeletal muscles. We also note that the in vitro C2C12 differentiation model is not suitable for the study of muscular aerobic respiration due to insufficient expression of muscle-specific CIV isoforms.

Keywords: electron transport complex IV; myoblasts; oxidative phosphorylation; protein isoforms; skeletal muscle

DOI: https://doi.org/10.1096/fj.202201223RR

J Cereb Blood Flow Metab. 2023 Mar 23. 271678X231165842

The relationship between cytochrome c oxidase, CBF and CMRO2 in mouse cortex: A NIRS-MRI study.

Mada Hashem, Ying Wu, Jeff F Dunn.

Quantifying relationships between cerebral blood flow (CBF), mitochondrial function (cytochrome c oxidase oxidation state), and metabolic rate of oxygen (CMRO2) could provide useful insight into normal neurovascular coupling, as well as regulation of oxidative metabolism in neurological disorders. This paper uses a multimodal NIRS-MRI method to quantify these parameters in rodent brain and, in so doing, provides novel information on the regulation of oxygen metabolism by stimulating with hypercapnia or variations in oxygenation. Under hypercapnia, although oxygenation, oxidation state, and CBF increased, there was no increase in CMRO2. Also, there was no correlation between CBF and CCO oxidation state. Conversely, changing oxygenation resulted in a strong correlation between the oxidation of CCO and CBF. This proves that the association between CBF and the redox state of CCO is not fixed and depends on the type of perturbation. Having a means to measure CBF and CCO oxidation state simultaneously will help understanding their contribution to intact neurovascular coupling and detecting abnormal cellular oxygen metabolism in many neurological disorders.

Keywords: Cerebral blood flow; cytochrome c oxidase; hypercapnia; neurovascular coupling; tissue oxygenation

DOI: https://doi.org/10.1177/0271678X231165842

Nature. 2023 Mar 22.

Structural basis of mitochondrial membrane bending by the I-II-III2-IV2 supercomplex.

Alexander Mühleip, Rasmus Kock Flygaard, Rozbeh Baradaran, Outi Haapanen, Thomas Gruhl, Victor Tobiasson, Amandine Maréchal, Vivek Sharma, Alexey Amunts.

Mitochondrial energy conversion requires an intricate architecture of the inner mitochondrial membrane1. Here we show that a supercomplex containing all four respiratory chain components contributes to membrane curvature induction in ciliates. We report cryo-electron microscopy and cryo-tomography structures of the supercomplex that comprises 150 different proteins and 311 bound lipids, forming a stable 5.8-MDa assembly. Owing to subunit acquisition and extension, complex I associates with a complex IV dimer, generating a wedge-shaped gap that serves as a binding site for complex II. Together with a tilted complex III dimer association, it results in a curved membrane region. Using molecular dynamics simulations, we demonstrate that the divergent supercomplex actively contributes to the membrane curvature induction and tubulation of cristae. Our findings highlight how the evolution of protein subunits of respiratory complexes has led to the I-II-III2-IV2 supercomplex that contributes to the shaping of the bioenergetic membrane, thereby enabling its functional specialization.

DOI: https://doi.org/10.1038/s41586-023-05817-y

bioRxiv. 2023 Mar 09. pii: 2023.03.09.531795. [Epub ahead of print]

Tetracycline-dependent inhibition of mitoribosome protein elongation in mitochondrial disease mutant cells suppresses IRE1α to promote cell survival.

Conor T Ronayne, Christopher F Bennett, Elizabeth A Perry, Noa Kantorovic, Pere Puigserver.

Mitochondrial diseases are a group of disorders defined by defects in oxidative phosphorylation caused by nuclear- or mitochondrial-encoded gene mutations. A main cellular phenotype of mitochondrial disease mutations are redox imbalances and inflammatory signaling underlying pathogenic signatures of these patients. Depending on the type of mitochondrial mutation, certain mechanisms can efficiently rescue cell death vulnerability. One method is the inhibition of mitochondrial translation elongation using tetracyclines, potent suppressors of cell death in mitochondrial disease mutant cells. However, the mechanisms whereby tetracyclines promote cell survival are unknown. Here, we show that in mitochondrial mutant disease cells, tetracycline-mediated inhibition of mitoribosome elongation promotes survival through suppression of the ER stress IRE1α protein. Tetracyclines increased levels of the splitting factor MALSU1 (Mitochondrial Assembly of Ribosomal Large Subunit 1) at the mitochondria with recruitment to the mitochondrial ribosome (mitoribosome) large subunit. MALSU1, but not other quality control factors, was required for tetracycline-induced cell survival in mitochondrial disease mutant cells during glucose starvation. In these cells, nutrient stress induced cell death through IRE1α activation associated with a strong protein loading in the ER lumen. Notably, tetracyclines rescued cell death through suppression of IRE1α oligomerization and activity. Consistent with MALSU1 requirement, MALSU1 deficient mitochondrial mutant cells were sensitive to glucose-deprivation and exhibited increased ER stress and activation of IRE1α that was not reversed by tetracyclines. These studies show that inhibition of mitoribosome elongation signals to the ER to promote survival, establishing a new interorganelle communication between the mitoribosome and ER with implications in basic mechanisms of cell survival and treatment of mitochondrial diseases.
Significance Statement: Mitochondrial diseases are a rare and heterogenous class of diseases that result from mutations in mitochondrial genes. Currently, there are no curative therapies due to a lack of mechanistic insights into pathological transformation and signaling. Our lab has discovered that the class of mitochondrial ribosome targeting antibiotics, tetracyclines, promote survival and fitness in models of mitochondrial disease, establishing a new paradigm of cell survival under nutrient stress conditions. In the current study, we present mechanistic insights into tetracyclines ability to rescue mitochondrial disease cells, detailing an interorganelle communication between mitochondrial protein translation and the unfolded protein response during endoplasmic reticulum stress.

DOI: https://doi.org/10.1101/2023.03.09.531795

Hum Reprod. 2023 Mar 23. pii: dead052. [Epub ahead of print]

A shared pattern of altered gene expression in human embryos affected by mitochondrial diseases.

Kalliopi Chatzovoulou, Anne Mayeur, Nicolas Cagnard, Mohammed Zarhrate, Christine Bole, Patrick Nitschke, Fabienne Jabot-Hanin, Agnès Rötig, Sophie Monnot, Arnold Munnich, Nelly Frydman, Julie Steffann.

STUDY QUESTION: Does mitochondrial deficiency affect human embryonic preimplantation development?
SUMMARY ANSWER: The presence of a pathogenic mitochondrial variant triggers changes in the gene expression of preimplantation human embryos, compromising their development, cell differentiation, and survival.
WHAT IS KNOWN ALREADY: Quantitative and qualitative anomalies of mitochondrial DNA (mtDNA) are reportedly associated with impaired human embryonic development, but the underlying mechanisms remain unexplained.
STUDY DESIGN, SIZE, DURATION: Taking advantage of the preimplantation genetic testing for mitochondrial disorders in at-risk couples, we have compared gene expression of 9 human embryos carrying pathogenic variants in either mtDNA genes or nuclear genes encoding mitochondrial protein to 33 age-matched control embryos.
PARTICIPANTS/MATERIALS, SETTING, METHODS: Single-embryo transcriptomic analysis was performed on whole human blastocyst embryos donated to research.
MAIN RESULTS AND THE ROLE OF CHANCE: Specific pathogenic mitochondrial variants downregulate gene expression in preimplantation human embryos [566 genes in oxidative phosphorylation (OXPHOS)-deficient embryos], impacting transcriptional regulators, differentiation factors, and nuclear genes encoding mitochondrial proteins. These changes in gene expression primarily alter OXPHOS and cell survival pathways.
LIMITATIONS, REASONS FOR CAUTION: The number of OXPHOS-deficient embryos available for the study was limited owing to the rarity of this material. However, the molecular signature shared by all these embryos supports the relevance of the findings.
WIDER IMPLICATIONS OF THE FINDINGS: While identification of reliable markers of normal embryonic development is urgently needed in ART, our study prompts us to consider under-expression of the targeted genes reported here, as predictive biomarkers of mitochondrial dysfunction during preimplantation development.
STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the 'Association Française contre les Myopathies (AFM-Téléthon)' and the 'La Fondation Maladies Rares'. No competing interests to declare.
TRIAL REGISTRATION NUMBER: N/A.

Keywords: RNA sequencing; differential gene expression; human embryo development; mitochondrial metabolism; mitochondrial mutation; transcriptome

DOI: https://doi.org/10.1093/humrep/dead052

bioRxiv. 2023 Mar 09. pii: 2023.03.09.531918. [Epub ahead of print]

Ophiobolin A Covalently Targets Complex IV Leading to Mitochondrial Metabolic Collapse in Cancer Cells.

Flor A Gowans, Danny Q Thach, Yangzhi Wang, Belen E Altamirano Poblano, Dustin Dovala, John A Tallarico, Jeffrey M McKenna, Markus Schirle, Thomas J Maimone, Daniel K Nomura.

Ophiobolin A (OPA) is a sesterterpenoid fungal natural product with broad anti-cancer activity. While OPA possesses multiple electrophilic moieties that can covalently react with nucleophilic amino acids on proteins, the proteome-wide targets and mechanism of OPA remain poorly understood in many contexts. In this study, we used covalent chemoproteomic platforms to map the proteome-wide reactivity of OPA in a highly sensitive lung cancer cell line. Among several proteins that OPA engaged, we focused on two targets-cysteine C53 of HIG2DA and lysine K72 of COX5A-that are part of complex IV of the electron transport chain and contributed significantly to the anti-proliferative activity. OPA activated mitochondrial respiration in a HIG2DA and COX5A-dependent manner, led to an initial spike in mitochondrial ATP, but then compromised mitochondrial membrane potential leading to ATP depletion. We have used chemoproteomic strategies to discover a unique anti-cancer mechanism of OPA through activation of complex IV leading to compromised mitochondrial energetics and rapid cell death.

DOI: https://doi.org/10.1101/2023.03.09.531918