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Mitochondria serve a crucial role in cell growth and proliferation by supporting both ATP synthesis and the production of macromolecular precursors. Whereas oxidative phosphorylation (OXPHOS) depends mainly on the oxidation of intermediates from the tricarboxylic acid cycle, the mitochondrial production of proline and ornithine relies on reductive synthesis 1 . How these competing metabolic pathways take place in the same organelle is not clear. Here we show that when cellular dependence on OXPHOS increases, pyrroline-5-carboxylate synthase (P5CS)-the rate-limiting enzyme in the reductive synthesis of proline and ornithine-becomes sequestered in a subset of mitochondria that lack cristae and ATP synthase. This sequestration is driven by both the intrinsic ability of P5CS to form filaments and the mitochondrial fusion and fission cycle. Disruption of mitochondrial dynamics, by impeding mitofusin-mediated fusion or dynamin-like-protein-1-mediated fission, impairs the separation of P5CS-containing mitochondria from mitochondria that are enriched in cristae and ATP synthase. Failure to segregate these metabolic pathways through mitochondrial fusion and fission results in cells either sacrificing the capacity for OXPHOS while sustaining the reductive synthesis of proline, or foregoing proline synthesis while preserving adaptive OXPHOS. These findings provide evidence of the key role of mitochondrial fission and fusion in maintaining both oxidative and reductive biosyntheses in response to changing nutrient availability and bioenergetic demand., Competing Interests: Competing interests: C.M.R. has consulted regarding oncology drug development with Amgen, AstraZeneca, Chugai, D2G, Daiichi Sankyo, Hoffman-La Roche, Jazz and Legend, and serves on the scientific advisory boards of Auron, Bridge Medicines, DISCO, Earli and Harpoon Therapeutics. C.B.T. is a founder of Agios Pharmaceuticals, and is on the board of directors of Regeneron and Charles River Laboratories. The remaining authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Abiotic stress tends to induce oxidative damage to enzymes and organelles that in turns hampers the phosphorylation process and decreases the adenosine triphosphate (ATP) productivity. Artificial assemblies can alleviate abiotic stress and simultaneously provide nutrients to diminish the oxidative damage. Here, we have integrated natural acid phosphatase (ACP) and ATP synthase with plasmonic Au clusters in a biomimetic microreactor. ACP immobilized on the Au clusters is harnessed to generate proton influx to drive ATP synthase and concurrently supply phosphate to improve phosphorus availability to combat phosphorus-deficiency stress. In tandem with the reactive oxygen species (ROS) scavenging and the photothermal functionality of Au clusters, such an assembled microreactor exhibits an improved abiotic stress tolerance and achieves plasmon-accelerated ATP synthesis. This innovative approach offers an effective route to enhance the stress resistance of ATP synthase-based energy-generating systems, opening an exciting potential of these systems for biomimicking applications., (© 2024 Wiley-VCH GmbH.)

Cells depend on a continuous supply of adenosine triphosphate (ATP), the universal energy currency. In mitochondria, ATP is produced by a series of redox reactions, whereby an electrochemical gradient is established across the inner mitochondrial membrane. The ATP synthase harnesses the energy of the gradient to generate ATP from adenosine diphosphate (ADP) and inorganic phosphate. We determined the structure of ATP synthase within mitochondria of the unicellular flagellate Polytomella by electron cryo-tomography and subtomogram averaging at up to 4.2-angstrom resolution, revealing six rotary positions of the central stalk, subclassified into 21 substates of the F 1 head. The Polytomella ATP synthase forms helical arrays with multiple adjacent rows defining the cristae ridges. The structure of ATP synthase under native operating conditions in the presence of a membrane potential represents a pivotal step toward the analysis of membrane protein complexes in situ.

Hepatocellular carcinoma (HCC) is one of the most lethal malignant tumors worldwide. Brahma-related gene 1 (BRG1), as a catalytic ATPase, is a major regulator of gene expression and is known to mutate and overexpress in HCC. The purpose of this study was to investigate the mechanism of action of BRG1 in HCC cells. In our study, BRG1 was silenced or overexpressed in human HCC cell lines. Transwell and wound healing assays were used to analyze cell invasiveness and migration. Mitochondrial membrane potential (MMP) and mitochondrial permeability transition pore (mPTP) detection were used to evaluate mitochondrial function in HCC cells. Colony formation and cell apoptosis assays were used to evaluate the effect of BRG1/TOMM40/ATP5A1 on HCC cell proliferation and apoptosis/death. Immunocytochemistry (ICC), immunofluorescence (IF) staining and western blot analysis were used to determine the effect of BRG1 on TOMM40, ATP5A1 pathway in HCC cells. As a result, knockdown of BRG1 significantly inhibited cell proliferation and invasion, promoted apoptosis in HCC cells, whereas BRG1 overexpression reversed the above effects. Overexpression of BRG1 can up-regulate MMP level, inhibit mPTP opening and activate TOMM40, ATP5A1 expression. Our results suggest that BRG1, as an oncogene, promotes HCC progression by regulating TOMM40 affecting mitochondrial function and ATP5A1 synthesis. Targeting BRG1 may represent a new and effective way to prevent HCC development.

Maleic acid (MA) induces renal tubular cell dysfunction directed to acute kidney injury (AKI). AKI is an increasing global health burden due to its association with mortality and morbidity. However, targeted therapy for AKI is lacking. Previously, we determined mitochondrial-associated proteins are MA-induced AKI affinity proteins. We hypothesized that mitochondrial dysfunction in tubular epithelial cells plays a critical role in AKI. In vivo and in vitro systems have been used to test this hypothesis. For the in vivo model, C57BL/6 mice were intraperitoneally injected with 400 mg/kg body weight MA. For the in vitro model, HK-2 human proximal tubular epithelial cells were treated with 2 mM or 5 mM MA for 24 h. AKI can be induced by administration of MA. In the mice injected with MA, the levels of blood urea nitrogen (BUN) and creatinine in the sera were significantly increased (p < 0.005). From the pathological analysis, MA-induced AKI aggravated renal tubular injuries, increased kidney injury molecule-1 (KIM-1) expression and caused renal tubular cell apoptosis. At the cellular level, mitochondrial dysfunction was found with increasing mitochondrial reactive oxygen species (ROS) (p < 0.001), uncoupled mitochondrial respiration with decreasing electron transfer system activity (p < 0.001), and decreasing ATP production (p < 0.05). Under transmission electron microscope (TEM) examination, the cristae formation of mitochondria was defective in MA-induced AKI. To unveil the potential target in mitochondria, gene expression analysis revealed a significantly lower level of ATPase6 (p < 0.001). Renal mitochondrial protein levels of ATP subunits 5A1 and 5C1 (p < 0.05) were significantly decreased, as confirmed by protein analysis. Our study demonstrated that dysfunction of mitochondria resulting from altered expression of ATP synthase in renal tubular cells is associated with MA-induced AKI. This finding provides a potential novel target to develop new strategies for better prevention and treatment of MA-induced AKI., (© 2024. The Author(s).)

Perfluorooctane sulfonate (PFOS), an officially listed persistent organic pollutant, is a widely distributed perfluoroalkyl substance. Epidemiological studies have shown that PFOS is intimately linked to the occurrence of insulin resistance (IR). However, the detailed mechanism remains obscure. In previous studies, we found that mitochondrial calcium overload was concerned with hepatic IR induced by PFOS. In this study, we found that PFOS exposure noticeably raised lysosomal calcium in L-02 hepatocytes from 0.5 h. In the PFOS-cultured L-02 cells, inhibiting autophagy alleviated lysosomal calcium overload. Inhibition of mitochondrial calcium uptake aggravated the accumulation of lysosomal calcium, while inhibition of lysosomal calcium outflowing reversed PFOS-induced mitochondrial calcium overload and IR. Transient receptor potential mucolipin 1 (TRPML1), the calcium output channel of lysosomes, interacted with voltage-dependent anion channel 1 (VDAC1), the calcium intake channel of mitochondria, in the PFOS-cultured cells. Moreover, we found that ATP synthase F 1 subunit beta (ATP5B) interacted with TRPML1 and VDAC1 in the L-02 cells and the liver of mice under PFOS exposure. Inhibiting ATP5B expression or restraining the ATP5B on the plasma membrane reduced the interplay between TRPML1 and VDAC1, reversed the mitochondrial calcium overload and deteriorated the lysosomal calcium accumulation in the PFOS-cultured cells. Our research unveils the molecular regulation of the calcium crosstalk between lysosomes and mitochondria, and explains PFOS-induced IR in the context of activated autophagy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The mitochondrial protein IF1 is upregulated in many tumors and acts as a pro-oncogenic protein through its interaction with the ATP synthase and the inhibition of apoptosis. We have recently characterized the molecular nature of the IF1-Oligomycin Sensitivity Conferring Protein (OSCP) subunit interaction; however, it remains to be determined whether this interaction could be targeted for novel anti-cancer therapeutic intervention. We generated mitochondria-targeting peptides to displace IF1 from the OSCP interaction. The use of one selective peptide led to displacement of the inhibitor IF1 from ATP synthase, as shown by immunoprecipitation. NMR spectroscopy analysis, aimed at clarifying whether these peptides were able to directly bind to the OSCP protein, identified a second peptide which showed affinity for the N-terminal region of this subunit overlapping the IF1 binding region. In situ treatment with the membrane-permeable derivatives of these peptides in HeLa cells, that are silenced for the IF1 inhibitor protein, showed significant inhibition in mitochondrial permeability transition and no effects on mitochondrial respiration. These peptides mimic the effects of the IF1 inhibitor protein in cancer HeLa cells and confirm that the IF1-OSCP interaction inhibits apoptosis. A third peptide was identified which counteracts the anti-apoptotic role of IF1, showing that OSCP is a promising target for anti-cancer therapies.

Control of protein stoichiometry is essential for cell function. Mitochondrial oxidative phosphorylation (OXPHOS) presents a complex stoichiometric challenge as the ratio of the electron transport chain (ETC) and ATP synthase must be tightly controlled, and assembly requires coordinated integration of proteins encoded in the nuclear and mitochondrial genome. How correct OXPHOS stoichiometry is achieved is unknown. We identify the Mitochondrial Regulatory hub for respiratory Assembly (MiRA) platform, which synchronizes ETC and ATP synthase biogenesis in yeast. Molecularly, this is achieved by a stop-and-go mechanism: the uncharacterized protein Mra1 stalls complex IV assembly. Two "Go" signals are required for assembly progression: binding of the complex IV assembly factor Rcf2 and Mra1 interaction with an Atp9-translating mitoribosome induce Mra1 degradation, allowing synchronized maturation of complex IV and the ATP synthase. Failure of the stop-and-go mechanism results in cell death. MiRA controls OXPHOS assembly, ensuring correct stoichiometry of protein machineries encoded by two different genomes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The hydrolytic activity of the ATP synthase in bovine mitochondria is inhibited by a protein called IF 1 , but bovine IF 1 has no effect on the synthetic activity of the bovine enzyme in mitochondrial vesicles in the presence of a proton motive force. In contrast, it has been suggested based on indirect observations that human IF I inhibits both the hydrolytic and synthetic activities of the human ATP synthase and that the activity of human IF 1 is regulated by the phosphorylation of Ser-14 of mature IF 1 . Here, we have made both human and bovine IF 1 which are 81 and 84 amino acids long, respectively, and identical in 71.4% of their amino acids and have investigated their inhibitory effects on the hydrolytic and synthetic activities of ATP synthase in bovine submitochondrial particles. Over a wide range of conditions, including physiological conditions, both human and bovine IF 1 are potent inhibitors of ATP hydrolysis, with no effect on ATP synthesis. Also, substitution of Ser-14 with phosphomimetic aspartic and glutamic acids had no effect on inhibitory properties, and Ser-14 is not conserved throughout mammals. Therefore, it is unlikely that the inhibitory activity of mammalian IF 1 is regulated by phosphorylation of this residue., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Mutations in the small genome present in mitochondria often result in severe pathologies. Different genetic strategies have been explored, aiming to rescue such mutations. A number of these strategies were based on the capacity of human mitochondria to import RNAs from the cytosol and designed to repress the replication of the mutated genomes or to provide the organelles with wild-type versions of mutant transcripts. However, the mutant RNAs present in mitochondria turned out to be an obstacle to therapy and little attention has been devoted so far to their elimination. Here, we present the development of a strategy to knockdown mitochondrial RNAs in human cells using the transfer RNA-like structure of Brome mosaic virus or Tobacco mosaic virus as a shuttle to drive trans-cleaving ribozymes into the organelles in human cell lines. We obtained a specific knockdown of the targeted mitochondrial ATP6 mRNA, followed by a deep drop in ATP6 protein and a functional impairment of the oxidative phosphorylation chain. Our strategy provides a powerful approach to eliminate mutant organellar transcripts and to analyse the control and communication of the human organellar genetic system., (© The Author(s) (2023). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, CEMCS, CAS.)

The tumor suppressor Liver Kinase B1 (LKB1) is a multifunctional serine/threonine protein kinase that regulates cell metabolism, polarity, and growth and is associated with Peutz-Jeghers Syndrome and cancer predisposition. The LKB1 gene comprises 10 exons and 9 introns. Three spliced LKB1 variants have been documented, and they reside mainly in the cytoplasm, although two possess a nuclear-localization sequence (NLS) and are able to shuttle into the nucleus. Here, we report the identification of a fourth and novel LKB1 isoform that is, interestingly, targeted to the mitochondria. We show that this mitochondria-localized LKB1 (mLKB1) is generated from alternative splicing in the 5' region of the transcript and translated from an alternative initiation codon encoded by a previously unknown exon 1b (131 bp) hidden within the long intron 1 of LKB1 gene. We found by replacing the N-terminal NLS of the canonical LKB1 isoform, the N-terminus of the alternatively spliced mLKB1 variant encodes a mitochondrial transit peptide that allows it to localize to the mitochondria. We further demonstrate that mLKB1 colocalizes histologically with mitochondria-resident ATP Synthase and NAD-dependent deacetylase sirtuin-3, mitochondrial (SIRT3) and that its expression is rapidly and transiently upregulated by oxidative stress. We conclude that this novel LKB1 isoform, mLKB1, plays a critical role in regulating mitochondrial metabolic activity and oxidative stress response., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The globin protein superfamily has diverse functions. Haemoglobin has been found in non-erythroid locations, including within the mitochondria. Using co-immunoprecipitation and in silico methods, we investigated the interaction of mitochondrial haemoglobin with ATP synthase and its associated proteins, including inhibitory factor 1 (IF1). We measured the expression of mitochondrial haemoglobin in response to hypoxia. In vitro and in silico evidence of interactions between mitochondrial haemoglobin and ATP synthase were found, and we report upregulated mitochondrial haemoglobin expression in response to hypoxia within skeletal muscle tissue. Our observations indicate that mitochondrial pH and ATP synthase activity are implicated in the mitochondrial haemoglobin response to hypoxia.

As the last step of the OXPHOS system, mitochondrial ATP synthase (or complex V) is responsible for ATP production by using the generated proton gradient, but also has an impact on other important functions linked to this system. Mutations either in complex V structural subunits, especially in mtDNA-encoded ATP6 gene, or in its assembly factors, are the molecular cause of a wide variety of human diseases, most of them classified as neurodegenerative disorders. The role of ATP synthase alterations in cancer development or metastasis has also been postulated. In this work, we reported the generation and characterization of the first mt-Atp6 pathological mutation in mouse cells, an m.8414A>G transition that promotes an amino acid change from Asn to Ser at a highly conserved residue of the protein (p.N163S), located near the path followed by protons from the intermembrane space to the mitochondrial matrix. The phenotypic consequences of the p.N163S change reproduce the effects of MT-ATP6 mutations in human diseases, such as dependence on glycolysis, defective OXPHOS activity, ATP synthesis impairment, increased ROS generation or mitochondrial membrane potential alteration. These observations demonstrate that this mutant cell line could be of great interest for the generation of mouse models with the aim of studying human diseases caused by alterations in ATP synthase. On the other hand, mutant cells showed lower migration capacity, higher expression of MHC-I and slightly lower levels of HIF-1α, indicating a possible reduction of their tumorigenic potential. These results could suggest a protective role of ATP synthase inhibition against tumor transformation that could open the door to new therapeutic strategies in those cancer types relying on OXPHOS metabolism.

The mitochondrial F 1 F O -ATP synthase produces the bulk of cellular ATP. The soluble F 1 domain contains the catalytic head that is linked via the central stalk and the peripheral stalk to the membrane embedded rotor of the F O domain. The assembly of the F 1 domain and its linkage to the peripheral stalk is poorly understood. Here we show a dual function of the mitochondrial Hsp70 (mtHsp70) in the formation of the ATP synthase. First, it cooperates with the assembly factors Atp11 and Atp12 to form the F 1 domain of the ATP synthase. Second, the chaperone transfers Atp5 into the assembly line to link the catalytic head with the peripheral stalk. Inactivation of mtHsp70 leads to integration of assembly-defective Atp5 variants into the mature complex, reflecting a quality control function of the chaperone. Thus, mtHsp70 acts as an assembly and quality control factor in the biogenesis of the F 1 F O -ATP synthase., (© 2023. The Author(s).)

Mitochondrial ATP synthase is a multifunctional enzyme complex involved in ATP production. We previously reported that the ATP synthase catalytic beta subunit (Atp2p in yeast) is regulated by the 2A-like protein phosphatase Sit4p, which targets Atp2p at T124/T317 impacting on ATP synthase levels and mitochondrial respiration. Here we report that Atp2-T124/T317 is also potentially regulated by Cdc5p, a polo-like mitotic kinase. Since both Cdc5p and Sit4p have established roles in cell cycle regulation, we investigated whether Atp2-T124/T317 phosphorylation was cell cycle-related. We present evidence that Atp2p levels and phosphorylation vary during cell cycle progression, with an increase at G2/M phase. Atp2-T124/T317 phosphorylation stimulates mitochondrial membrane potential, respiration and ATP levels at G2/M phase, indicating that dynamic Atp2p phosphorylation contributes to mitochondrial activity at this specific cell cycle phase. Preventing Atp2p phosphorylation delays G2/M to G1 transition, suggesting that enhanced bioenergetics at G2/M may help meet the energetic demands of cell cycle progression. However, mimicking constitutive T124/T317 phosphorylation or overexpressing Atp2p leads to mitochondrial DNA instability, indicating that reversible Atp2p phosphorylation is critical for homeostasis. These results indicate that transient phosphorylation of Atp2p, a protein at the core of the ATP production machinery, impacts on mitochondrial bioenergetics and supports cell cycle progression at G2/M., Competing Interests: Declarations of competing interest None., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Two multisubunit protein complexes for membrane protein insertion were recently identified in the endoplasmic reticulum (ER): the guided entry of tail anchor proteins (GET) complex and ER membrane complex (EMC). The structures of both of their hydrophobic core subunits, which are required for the insertion reaction, revealed an overall similarity to the YidC/Oxa1/Alb3 family members found in bacteria, mitochondria, and chloroplasts. This suggests that these membrane insertion machineries all share a common ancestry. To test whether these ER proteins can functionally replace Oxa1 in yeast mitochondria, we generated strains that express mitochondria-targeted Get2-Get1 and Emc6-Emc3 fusion proteins in Oxa1 deletion mutants. Interestingly, the Emc6-Emc3 fusion was able to complement an Δoxa1 mutant and restored its respiratory competence. The Emc6-Emc3 fusion promoted the insertion of the mitochondrially encoded protein Cox2, as well as of nuclear encoded inner membrane proteins, although was not able to facilitate the assembly of the Atp9 ring. Our observations indicate that protein insertion into the ER is functionally conserved to the insertion mechanism in bacteria and mitochondria and adheres to similar topological principles., Competing Interests: The authors have declared that no competing interests exist.

Alzheimer's Disease (AD) is the most common neurodegenerative disorder in our society, as the population ages, its incidence is expected to increase in the coming decades. The etiopathology of this disease still remains largely unclear, probably because of the highly complex and multifactorial nature of AD. However, the presence of mitochondrial dysfunction has been broadly described in AD neurons and other cellular populations within the brain, in a wide variety of models and organisms, including post-mortem humans. Mitochondria are complex organelles that play a crucial role in a wide range of cellular processes, including bioenergetics. In fact, in mammals, including humans, the main source of cellular ATP is the oxidative phosphorylation (OXPHOS), a process that occurs in the mitochondrial electron transfer chain (ETC). The last enzyme of the ETC, and therefore the ulterior generator of ATP, is the ATP synthase. Interestingly, in mammalian cells, the ATP synthase can also degrade ATP under certain conditions (ATPase), which further illustrates the crucial role of this enzyme in the regulation of cellular bioenergetics and metabolism. In this collaborative review, we aim to summarize the knowledge of the presence of dysregulated ATP synthase, and of other components of mammalian mitochondrial bioenergetics, as an early event in AD. This dysregulation can act as a trigger of the dysfunction of the organelle, which is a clear component in the etiopathology of AD. Consequently, the pharmacological modulation of the ATP synthase could be a potential strategy to prevent mitochondrial dysfunction in AD.

F-ATP synthase is a leading candidate as the mitochondrial permeability transition pore (PTP) but the mechanism(s) leading to channel formation remain undefined. Here, to shed light on the structural requirements for PTP formation, we test cells ablated for g, OSCP and b subunits, and ρ 0 cells lacking subunits a and A6L. Δg cells (that also lack subunit e) do not show PTP channel opening in intact cells or patch-clamped mitoplasts unless atractylate is added. Δb and ΔOSCP cells display currents insensitive to cyclosporin A but inhibited by bongkrekate, suggesting that the adenine nucleotide translocator (ANT) can contribute to channel formation in the absence of an assembled F-ATP synthase. Mitoplasts from ρ 0 mitochondria display PTP currents indistinguishable from their wild-type counterparts. In this work, we show that peripheral stalk subunits are essential to turn the F-ATP synthase into the PTP and that the ANT provides mitochondria with a distinct permeability pathway., (© 2021. The Author(s).)

The mitochondrial permeability transition pore (PTP), which drives regulated cell death when Ca 2+ concentration suddenly increases in mitochondria, was related to changes in the Ca 2+ -activated F 1 F O -ATPase. The effects of the gadolinium cation (Gd 3+ ), widely used for diagnosis and therapy, and reported as PTP blocker, were evaluated on the F 1 F O -ATPase activated by Mg 2+ or Ca 2+ and on the PTP. Gd 3+ more effectively inhibits the Ca 2+ -activated F 1 F O -ATPase than the Mg 2+ -activated F 1 F O -ATPase by a mixed-type inhibition on the former and by uncompetitive mechanism on the latter. Most likely Gd 3+ binding to F 1 , is favoured by Ca 2+ insertion. The maximal inactivation rates (k inact ) of pseudo-first order inactivation are similar either when the F 1 F O -ATPase is activated by Ca 2+ or by Mg 2+ . The half-maximal inactivator concentrations (K I ) are 2.35 ± 0.35 mM and 0.72 ± 0.11 mM, respectively. The potency of a mechanism-based inhibitor (k inact /K I ) also highlights a higher inhibition efficiency of Gd 3+ on the Ca 2+ -activated F 1 F O -ATPase (0.59 ± 0.09 mM -1 ∙s -1 ) than on the Mg 2+ -activated F 1 F O -ATPase (0.13 ± 0.02 mM -1 ∙s -1 ). Consistently, the PTP is desensitized in presence of Gd 3+ . The Gd 3+ inhibition on both the mitochondrial Ca 2+ -activated F 1 F O -ATPase and the PTP strengthens the link between the PTP and the F 1 F O -ATPase when activated by Ca 2+ and provides insights on the biological effects of Gd 3+ ., (Copyright © 2021. Published by Elsevier B.V.)

Neutrophils play a key role in the innate immunity with their ability to generate and release inflammatory mediators that promote the inflammatory response and consequently restore the hemostasis. As active participants in several steps of the normal inflammatory response, neutrophils are also involved in chronic inflammatory diseases such as asthma, atherosclerosis, and arthritis. Given their dual role in the modulation of inflammation, regulating the inflammatory response of neutrophils has been suggested as an important therapeutic approach by numerous researchers. The neutrophils have a relatively short lifespan, which can be problematic for some in vitro experiments. To address this issue, researchers have used the human monomyelocyte cell line PLB-985 as an in vitro model for exploratory experiments addressing neutrophil-related physiological functions. PLB-985 cells can be differentiated into a neutrophil-like phenotype upon exposure to several agonists, including dimethyl sulfoxide (DMSO). Whether this differentiation of PLB-985 affects important features related to the neutrophil's normal functions (i.e., mitochondrial activity, eicosanoid production) remains elusive, and characterizing these changes will be the focal point of this study. Our results indicate that the differentiation affected the proliferation of PLB-985 cells, without inducing apoptosis. A significant decrease in mitochondrial respiration was observed in differentiated PLB-985 cells. However, the overall mitochondria content was not affected. Immunoblotting with mitochondrial antibodies revealed a strong modulation of the succinate dehydrogenase A, superoxide dismutase 2, ubiquinol-cytochrome c reductase core protein 2 and ATP synthase subunit α in differentiated PLB-985 cells. Finally, eicosanoids (leukotriene B 4 , 12-hydroxyheptadecatrienoic and 15-hydroxyeicosatetraenoic acids) production was significantly increased in differentiated cells. In summary, our data demonstrate that the differentiation process of PLB-985 cells does not impact their viability despite a reduced respiratory state of the cells. This process is also accompanied by modulation of the inflammatory state of the cell. Of importance, our data suggest that PLB-985 cells could be suitable in vitro candidates to study mitochondrial-related dysfunctions in inflammatory diseases., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Transduction of electrochemical proton gradient into ATP synthesis is performed by F 1 F O -ATP synthase. The reverse reaction is prevented by the regulatory subunit Inh1. Knockout of the inh1 gene in the basidiomycete Ustilago maydis was generated in order to study the function of this protein in the mitochondrial metabolism and cristae architecture. Deletion of inh1 gen did not affect cell growth, glucose consumption, and biomass production. Ultrastructure and fluorescence analyzes showed that size, cristae shape, network, and distribution of mitochondria was similar to wild strain. Membrane potential, ATP synthesis, and oxygen consumption in wild type and mutant strains had similar values. Kinetic analysis of ATPase activity of complex V in permeabilized mitochondria showed similar values of V max and K M for both strains, and no effect of pH was observed. Interestingly, the dimeric state of complex V occurs in the mutant strain, indicating that this subunit is not essential for dimerization. ATPase activity of the isolated monomeric and dimeric forms of complex V indicated V max values 4-times higher for the mutant strain than for the WT strain, suggesting that the absence of Inh1 subunit increased ATPase activity, and supporting a regulatory role for this protein; however, no effect of pH was observed. ATPase activity of WT oligomers was stimulated several times by dodecyl-maltoside (DDM), probably by removal of ADP from F 1 sector, while DDM induced an inactive form of the mutant oligomers., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Our laboratory has demonstrated that functional N-methyl-d-aspartate-like receptors are present on neuronal mitochondria (NMDA m ). This novel site gates the influx of Ca 2+ and causes a several-fold increase in ATP levels. Although elevations in ATP in other cell types have been linked to increases in mitochondrial Ca 2+ , it has not been established whether the same holds true for calcium uptake via NMDA m . In this study, we have investigated the effect of NMDA m activation on a variety of bioenergetic parameters. Our findings suggest that mitochondrial bioenergetics are not only modulated by NMDA m activation in a Ca 2+ -dependent but also in a Ca 2+ -independent manner., (Copyright © 2021 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

The mitochondrial ATP synthase is producing most of the energy required to support eucaryotic life. It is located in the mitochondrial inner-membrane and couples the dissipation of the proton gradient produced by the electron transfer chain with ATP production. It is composed of two domains, the F 1 domain located in the matrix and the F O domain embedded in the inner membrane. The mitochondrial ATP synthase belongs to the F-type ATP synthase family together with bacterial and chloroplastic enzymes. The composition of the mitochondrial ATP synthase is well conserved across species, except in plants where several subunits found in opisthokonts were not identified and additional, plant-specific, subunits were found. The assembly of the F-type ATP synthase has been extensively studied in bacteria, yeast and mammals. The overall assembly pattern is conserved but species-specific steps have been identified. In plant, little is known about the assembly of the mitochondrial ATP synthase. We have mined our previously published complexome profiling dataset in order to identity assembly steps of the ATP synthase in the reference plant Arabidopsis thaliana. Several assembly intermediates were identified and we propose a model for the assembly pathway of the ATP synthase of plant mitochondria. In addition, combining complexome profiling with homology searches, we found that the previously described plant-specific subunits are actually present in other organisms. Overall, our work show that the subunit composition and the assembly pathway of the plant mitochondria ATP synthase are mostly conserved with other mitochondrial enzymes., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The mitochondrial ATP synthase emerges as key hub of cellular functions controlling the production of ATP, cellular signaling, and fate. It is regulated by the ATPase inhibitory factor 1 (IF1), which is highly abundant in neurons. Herein, we ablated or overexpressed IF1 in mouse neurons to show that IF1 dose defines the fraction of active/inactive enzyme in vivo, thereby controlling mitochondrial function and the production of mitochondrial reactive oxygen species (mtROS). Transcriptomic, proteomic, and metabolomic analyses indicate that IF1 dose regulates mitochondrial metabolism, synaptic function, and cognition. Ablation of IF1 impairs memory, whereas synaptic transmission and learning are enhanced by IF1 overexpression. Mechanistically, quenching the IF1-mediated increase in mtROS production in mice overexpressing IF1 reduces the increased synaptic transmission and obliterates the learning advantage afforded by the higher IF1 content. Overall, IF1 plays a key role in neuronal function by regulating the fraction of ATP synthase responsible for mitohormetic mtROS signaling., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: M.P.M. consults for Antipodean Pharmaceutical Inc., which is commercializing MitoQ. The rest of authors declare that they have no conflict of interest.

The f subunit is localized at the base of the ATP synthase peripheral stalk. Its function in the human enzyme is poorly characterized. Because full disruption of its ATP5J2 gene with the CRISPR-Cas9 strategy in the HAP1 human model has been shown to cause alterations in the amounts of other ATP synthase subunits, here we investigated the role of the f subunit in HeLa cells by regulating its levels through RNA interference. We confirm the role of the f subunit in ATP synthase dimer stability and observe that its downregulation per se does not alter the amounts of the other enzyme subunits or ATP synthase synthetic/hydrolytic activity. We show that downregulation of the f subunit causes abnormal crista organization and decreases permeability transition pore (PTP) size, whereas its re-expression in f subunit knockdown cells rescues mitochondrial morphology and PTP-dependent swelling., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Excessive mitochondrial fission has been implicated in the etiology of neuronal cell death in traumatic brain injury (TBI). In the present study, we examined the efficacy of melatonin (Mel) as a neuroprotective agent against TBI-induced oxidative damage and mitochondrial dysfunction. We assessed the impact of Mel post-treatment (10 mg/kg b.wt., i.p.) at different time intervals in TBI-subjected Wistar rats. We found that the Mel treatment significantly attenuated brain edema, oxidative damage, mitochondrial fission, and promoted mitochondrial fusion. Additionally, Mel-treated rats showed restoration of mitochondrial membrane potential and oxidative phosphorylation with a concomitant reduction in cytochrome-c release. Further, Mel treatment significantly inhibited the translocation of Bax and Drp1 proteins to mitochondria in TBI-subjected rats. The restorative role of Mel treatment in TBI rats was supported by the mitochondrial ultra-structural analysis, which showed activation of mitochondrial fusion mechanism. Mel enhanced mitochondrial biogenesis by upregulation of PGC-1α protein. Our results demonstrated the remedial role of Mel in ameliorating mitochondrial dysfunctions that are modulated in TBI-subjected rats and provided support for mitochondrial-mediated neuroprotection as a putative therapeutic agent in the brain trauma.

The mechanisms controlling the post-natal maturation of astrocytes play a crucial role in ensuring correct synaptogenesis. We show that mitochondrial biogenesis in developing astrocytes is necessary for coordinating post-natal astrocyte maturation and synaptogenesis. The astrocytic mitochondrial biogenesis depends on the transient upregulation of metabolic regulator peroxisome proliferator-activated receptor gamma (PPARγ) co-activator 1α (PGC-1α), which is controlled by metabotropic glutamate receptor 5 (mGluR5). At tissue level, the loss or downregulation of astrocytic PGC-1α sustains astrocyte proliferation, dampens astrocyte morphogenesis, and impairs the formation and function of neighboring synapses, whereas its genetic re-expression is sufficient to restore the mitochondria compartment and correct astroglial and synaptic defects. Our findings show that the developmental enhancement of mitochondrial biogenesis in astrocytes is a critical mechanism controlling astrocyte maturation and supporting synaptogenesis, thus suggesting that astrocytic mitochondria may be a therapeutic target in the case of neurodevelopmental and psychiatric disorders characterized by impaired synaptogenesis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Sodium deoxycholate (NaDOC) inhibits the intestinal Ca 2+ absorption and ursodeoxycholic acid (UDCA) stimulates it. The aim of this study was to determine whether NaDOC and UDCA produce differential effects on the redox state of duodenal mitochondria altering the Krebs cycle and the electron transport chain (ETC) functioning, which could lead to perturbations in the mitochondrial dynamics and biogenesis. Rat intestinal mitochondria were isolated from untreated and treated animals with either NaDOC, UDCA, or both. Krebs cycle enzymes, ETC components, ATP synthase, and mitochondrial dynamics and biogenesis markers were determined. NaDOC decreased isocitrate dehydrogenase (ICDH) and malate dehydrogenase activities affecting the ETC and ATP synthesis. NaDOC also induced oxidative stress and increased the superoxide dismutase activity and impaired the mitochondrial biogenesis and functionality. UDCA increased the activities of ICDH and complex II of ETC. The combination of both bile acids conserved the functional activities of Krebs cycle enzymes, ETC components, oxidative phosphorylation, and mitochondrial biogenesis. In conclusion, the inhibitory effect of NaDOC on intestinal Ca 2+ absorption is mediated by mitochondrial dysfunction, which is avoided by UDCA. The stimulatory effect of UDCA alone is associated with amelioration of mitochondrial functioning. This knowledge could improve treatment of diseases that affect the intestinal Ca 2+ absorption.

Mitochondrial Permeability Transition (PT) is a phenomenon of increased permeability of the inner mitochondrial membrane in response to high levels of Ca 2+ and/or reactive oxygen species (ROS) in the matrix. PT occurs upon the opening of a pore, namely the permeability transition pore (PTP), which dissipates the membrane potential uncoupling the respiratory chain. mPT activation and PTP formation can occur through multiple molecular pathways. The specific focus of this review is to discuss the possible molecular mechanisms of PTP that involve the participation of mitochondrially targeted amyloid peptides Aβ, α-synuclein and c subunit of the ATP synthase (ATPase). As activators of PTP, amyloid peptides are uniquely different from other activators because they are capable of forming channels in lipid bilayers. This property rises the possibility that in this permeabilization pathway the formation of the channel involves the direct participation of peptides, making it uniquely different from other PTP induction mechanisms. In this pathway, a critical step of PTP activation involves the import of amyloidogenic peptides from the cytosol into the matrix. In the matrix these peptides, which would fold into α-helical structure in native conditions, interact with cyclophilin D (CypD) and upon stimulation by elevated ROS and/or the Ca 2+ spontaneously misfold into β-sheet ion conducting pores, causing PTP opening., (Copyright © 2020 Elsevier B.V. All rights reserved.)

C-Src kinase is localized in several subcellular compartments, including mitochondria where it is involved in the regulation of organelle functions and overall metabolism. Surprisingly, the characterization of the intramitochondrial Src interactome has never been fully determined. Using in vitro proximity-dependent biotin identification (BioID) coupled to mass spectrometry, we identified 51 candidate proteins that may interact directly or indirectly with c-Src within the mitochondrial matrix. Pathway analysis suggests that these proteins are involved in a large array of mitochondrial functions such as protein folding and import, mitochondrial organization and transport, oxidative phosphorylation, tricarboxylic acid cycle and metabolism of amino and fatty acids. Among these proteins, we identified 24 tyrosine phosphorylation sites in 17 mitochondrial proteins (AKAP1, VDAC1, VDAC2, VDAC3, LonP1, Hsp90, SLP2, PHB2, MIC60, UBA1, EF-Tu, LRPPRC, ACO2, OAT, ACAT1, ETFβ and ATP5β) as potential substrates for intramitochondrial Src using in silico prediction of tyrosine phospho-sites. Interaction of c-Src with SLP2 and ATP5β was confirmed using coimmunoprecipitation. This study suggests that the intramitochondrial Src could target several proteins and regulate different mitochondrial functions., (Copyright © 2019 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

The ATP synthase complexes in mitochondria make the ATP required to sustain life by a rotary mechanism. Their membrane domains are embedded in the inner membranes of the organelle, and they dimerize via interactions between their membrane domains. The dimers form extensive chains along the tips of the cristae with the two rows of monomeric catalytic domains extending into the mitochondrial matrix at an angle to each other. Disruption of the interface between dimers by mutation affects the morphology of the cristae severely. By analysis of particles of purified dimeric bovine ATP synthase by cryo-electron microscopy, we have shown that the angle between the central rotatory axes of the monomeric complexes varies between ca. 76 and 95°. These particles represent active dimeric ATP synthase. Some angular variations arise directly from the catalytic mechanism of the enzyme, and others are independent of catalysis. The monomer-monomer interaction is mediated mainly by j subunits attached to the surface of wedge-shaped protein-lipid structures in the membrane domain of the complex, and the angular variation arises from rotational and translational changes in this interaction, and combinations of both. The structures also suggest how the dimeric ATP synthases might be interacting with each other to form the characteristic rows along the tips of the cristae via other interwedge contacts, molding themselves to the range of oligomeric arrangements observed by tomography of mitochondrial membranes, and at the same time allowing the ATP synthase to operate under the range of physiological conditions that influence the structure of the cristae., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Synaptic failure underlies cognitive impairment in Alzheimer's disease (AD). Cumulative evidence suggests a strong link between mitochondrial dysfunction and synaptic deficits in AD. We previously found that oligomycin-sensitivity-conferring protein (OSCP) dysfunction produces pronounced neuronal mitochondrial defects in AD brains and a mouse model of AD pathology (5xFAD mice). Here, we prevented OSCP dysfunction by overexpressing OSCP in 5xFAD mouse neurons in vivo (Thy-1 OSCP/5xFAD mice). This approach protected OSCP expression and reduced interaction of amyloid-beta (Aβ) with membrane-bound OSCP. OSCP overexpression also alleviated F1Fo ATP synthase deregulation and preserved mitochondrial function. Moreover, OSCP modulation conferred resistance to Aβ-mediated defects in axonal mitochondrial dynamics and motility. Consistent with preserved neuronal mitochondrial function, OSCP overexpression ameliorated synaptic injury in 5xFAD mice as demonstrated by preserved synaptic density, reduced complement-dependent synapse elimination, and improved synaptic transmission, leading to preserved spatial learning and memory. Taken together, our findings show the consequences of OSCP dysfunction in the development of synaptic stress in AD-related conditions and implicate OSCP modulation as a potential therapeutic strategy., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Neuroprotective and other functional proteins of mitochondria were quantified in extracts of plasma neural-derived exosomes from ten first-episode psychosis (FP) patients and ten matched psychiatrically normal controls (ctls). Astrocyte-derived extracellular vesicles (ADEVs) and neuron-derived extracellular vesicles (NDEVs) were immunoabsorbed separately from physically precipitated plasma total EVs. Extracted mitochondrial ATP synthase was specifically immunofixed to plastic wells for quantification of catalytic activity based on conversion of NADH to NAD + . Other extracted mitochondrial functional proteins were quantified by ELISAs. All protein levels were normalized with EV content of the CD81 exosome marker. FP patient ADEV level but not NDEV level of mitochondrial ATP synthase activity was significantly lower than that of ctls. FP patient ADEV and NDEV levels of the functionally critical mitochondrial proteins mitofusin 2 and cyclophilin D, but not of transcription factor A of mitochondria, and of the mitochondrial short open-reading frame neuroprotective and metabolic regulatory peptides humanin and MOTS-c were significantly lower than those of ctls. In contrast, FP patient NDEV, but not ADEV, level of the mitochondrial-tethering protein syntaphilin, but not of myosin VI, was significantly higher than that of ctls. The distinctively different neural levels of some mitochondrial proteins in FP patients than ctls now should be correlated with diverse clinical characteristics. Drugs that increase depressed levels of proteins and mimetics of deficient short open-reading frame peptides may be of therapeutic value in early phases of schizophrenia., (© 2021 Federation of American Societies for Experimental Biology.)

Although the mitochondrial permeability transition pore (PTP) is presumably formed by either ATP synthase or the ATP/ADP carrier (AAC), little is known about their differential roles in PTP activation. We explored the role of AAC and ATP synthase in PTP formation in Saccharomyces cerevisiae using bisindolylpyrrole (BP), an activator of the mammalian PTP. The yeast mitochondrial membrane potential, as indicated by tetramethylrhodamine methyl ester signals, dissipated over 2-4 h after treatment of cells with 5 μM BP, which was sensitive to cyclosporin A (CsA) and Cpr3 deficiency and blocked by porin1/2 deficiency. The BP-induced depolarization was inhibited by a specific AAC inhibitor, bongkrekate, and consistently blocked in a yeast strain lacking all three AACs, while it was not affected in the strain with defective ATP synthase dimerization, suggesting the involvement of an AAC-associated pore. Upon BP treatment, isolated yeast mitochondria underwent CsA- and bongkrekate-sensitive depolarization without affecting the mitochondrial calcein signals, indicating the induction of a low conductance channel. These data suggest that, upon BP treatment, yeast can form a porin1/2- and Cpr3-regulated PTP, which is mediated by AACs but not by ATP synthase dimers. This implies that yeast may be an excellent tool for the screening of PTP modulators.

Mitochondrial ATP synthase plays a key role in inducing membrane curvature to establish cristae. In Apicomplexa causing diseases such as malaria and toxoplasmosis, an unusual cristae morphology has been observed, but its structural basis is unknown. Here, we report that the apicomplexan ATP synthase assembles into cyclic hexamers, essential to shape their distinct cristae. Cryo-EM was used to determine the structure of the hexamer, which is held together by interactions between parasite-specific subunits in the lumenal region. Overall, we identified 17 apicomplexan-specific subunits, and a minimal and nuclear-encoded subunit-a. The hexamer consists of three dimers with an extensive dimer interface that includes bound cardiolipins and the inhibitor IF 1 . Cryo-ET and subtomogram averaging revealed that hexamers arrange into ~20-megadalton pentagonal pyramids in the curved apical membrane regions. Knockout of the linker protein ATPTG11 resulted in the loss of pentagonal pyramids with concomitant aberrantly shaped cristae. Together, this demonstrates that the unique macromolecular arrangement is critical for the maintenance of cristae morphology in Apicomplexa.

In mammalian mitochondria, messenger RNA is processed and matured from large primary transcripts in structures known as RNA granules. The identity of the factors and process transferring the matured mRNA to the mitoribosome for translation is unclear. Nascent mature transcripts are believed to associate initially with the small mitoribosomal subunit prior to recruitment of the large subunit to form the translationally active monosome. When the small subunit fails to assemble, however, the stability of mt-mRNA is only marginally affected, and under these conditions, the LRPPRC/SLIRP RNA-binding complex has been implicated in maintaining mt-mRNA stability. Here, we exploit the activity of a bacterial ribotoxin, VapC20, to show that in the absence of the large mitoribosomal subunit, mt-mRNA species are selectively lost. Further, if the small subunit is also depleted, the mt-mRNA levels are recovered. As a consequence of these data, we suggest a natural pathway for loading processed mt-mRNA onto the mitoribosome., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Progress in animal modeling of polymorphisms and mutations in mitochondrial DNA (mtDNA) is not as developed as nuclear transgenesis due to a host of cellular and physiological distinctions. mtDNA mutation modeling is of critical importance as mutations in the mitochondrial genome give rise to a variety of pathological conditions and play a contributing role in many others. Nuclear localization and transcription of mtDNA genes followed by cytoplasmic translation and transport into mitochondria (allotopic expression, AE) provide an opportunity to create in vivo modeling of a targeted mutation in mitochondrial genes. Accordingly, such technology has been suggested as a strategy for gene replacement therapy in patients harboring mitochondrial DNA mutations. Here, we use our AE approach to transgenic mouse modeling of the pathogenic human T8993G mutation in mtATP6 as a case study for designing AE animal models.

Several methods are available to measure ATP production by isolated mitochondria or permeabilized cells but have several limitations, depending upon the particular assay employed. These limitations may include poor sensitivity or specificity, complexity of the method, poor throughput, changes in mitochondrial inner membrane potential as ATP is consumed, and/or inability to simultaneously assess other mitochondrial functional parameters. Here we describe a novel nuclear magnetic resonance (NMR)-based assay that can be carried out with high efficiency in a manner that alleviates the above problems.

Melanoma is an aggressive form of skin cancer characterized by rapid invasion and metastasis. CD147 is known to be functioning in cell invasion. In this study, we showed that CD147 was translocated from the cell membrane to the mitochondria in advanced melanoma. Melanoma patients with CD147 localized to the mitochondria confer a worse prognosis. The mitochondrial CD147 levels are correlated with the invasion potential of various melanoma cell lines as well as mitochondrial energy metabolism. Depletion of CD147 decreased the activity of mitochondrial complex V. STRING analysis for protein-protein interaction networks (PPIN) in CD147-depleted melanoma cells showed that mitochondrial proteins HSP60 and ATP5B, a subunit of mitochondrial complex V, were node proteins. HSP60 upregulation was correlated with a worse prognosis of melanoma patients. Co-immunoprecipitation (Co-IP) assay indicates that CD147 interacts with HSP60. These data suggested that mitochondrial CD147 may prompt HSP60 to activate ATP5B, thereby promoting the mitochondrial aerobic oxidation and the invasive abilities of melanoma cells. Correlation analysis of the data acquired from patients was helpful to draw a 5-year survival curve for patients who screened positive and negative for mitochondrial CD147. This study unravels the function of CD147 in tumor invasion and highlights it as a potential tumor therapeutic target., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Early detection and treatment of endometrial hyperplasia (EH) is mandatory for endometrial cancer prevention. Several bioactive agents of plant origin have been shown to elicit their chemotherapeutic effect against tumors and cancer via induction of mitochondrial permeability transition(mPT) pore opening. This research was therefore aimed at evaluating the potential chemopreventive effect of methyl palmitate (MP), on estradiol benzoate(EB)-induced EH, looking at the mitochondrial-mediated pathway and other possible mechanisms of action. Mitochondria were isolated using differential centrifugation. The mPT pore, mitochondrial ATPase (mATPase) activity, lipid peroxidation and cytochrome c release were determined by standard methods using spectrophotometer. Uterine interleukin 1b, MDA levels and SOD, GSH activities, were determined using commercially available kits. The uterine histological and immunohistochemical assessment of estrogen receptor (ERα), IL-1b and caspas-3 were carried out. The fibroblast cell count density was determined using histomorphometry. At all the concentrations of MP used, there was no significant induction of mPT pore opening, neither any enhancement of mATPase activity nor release of cytochrome c when compared to the control. Similar pattern of results were recorded for the in vivo study. However, there was marked increase in the uterine MDA and interleukin 1b levels, with concurrent decrease in SOD and GSH activities, in the EB-treated group, which was significantly reversed by MP co-administration. Endometrial Hyperplasia observed in the EB-treated group was ameliorated by MP co-administration. The immunoexpression of ERα and IL-1b in the EB-treated group was reversed by MP co-administration. This study suggests anti-inflammatory, antioxidant and anti-proliferative potential of MP against EB-induced EH.

The effects of insulin on the bioenergetic and thermogenic capacity of brown adipocyte mitochondria were investigated by focusing on key mitochondrial proteins. Two-month-old male Wistar rats were treated acutely or chronically with a low or high dose of insulin. Acute low insulin dose increased expression of all electron transport chain complexes and complex IV activity, whereas high dose increased complex II expression. Chronic low insulin dose decreased complex I and cyt c expression while increasing complex II and IV expression and complex IV activity. Chronic high insulin dose decreased complex II, III, cyt c , and increased complex IV expression. Uncoupling protein (UCP) 1 expression was decreased after acute high insulin but increased following chronic insulin treatment. ATP synthase expression was increased after acute and decreased after chronic insulin treatment. Only a high dose of insulin increased ATP synthase activity in acute and decreased it in chronic treatment. ATPase inhibitory factor protein expression was increased in all treated groups. Confocal microscopy showed that key mitochondrial proteins colocalize differently in different mitochondria within a single brown adipocyte, indicating mitochondrial mosaicism. These results suggest that insulin modulates the bioenergetic and thermogenic capacity of rat brown adipocytes in vivo by modulating mitochondrial mosaicism.

The adenosine triphosphate (ATP) synthase in human mitochondria is a membrane bound assembly of 29 proteins of 18 kinds organized into F 1 -catalytic, peripheral stalk (PS), and c 8 -rotor ring modules. All but two membrane components are encoded in nuclear genes, synthesized on cytoplasmic ribosomes, imported into the mitochondrial matrix, and assembled into the complex with the mitochondrial gene products ATP6 and ATP8. Intermediate vestigial ATPase complexes formed by disruption of nuclear genes for individual subunits provide a description of how the various domains are introduced into the enzyme. From this approach, it is evident that three alternative pathways operate to introduce the PS module (including associated membrane subunits e, f, and g). In one pathway, the PS is built up by addition to the core subunit b of membrane subunits e and g together, followed by membrane subunit f. Then this b-e-g-f complex is bound to the preformed F 1 -c 8 module by subunits OSCP and F 6 The final component of the PS, subunit d, is added subsequently to form a key intermediate that accepts the two mitochondrially encoded subunits. In another route to this key intermediate, first e and g together and then f are added to a preformed F 1 -c 8 -OSCP-F 6 -b-d complex. A third route involves the addition of the c 8 -ring module to the complete F 1 -PS complex. The key intermediate then accepts the two mitochondrially encoded subunits, stabilized by the addition of subunit j, leading to an ATP synthase complex that is coupled to the proton motive force and capable of making ATP., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

The physical and functional organisation of the OXPHOS system in mitochondria in vivo remains elusive. At present, different models of OXPHOS arrangement, representing either highly ordered respiratory strings or, vice versa, a set of randomly dispersed supercomplexes and respiratory complexes, have been suggested. In the present study, we examined a supramolecular arrangement of the OXPHOS system in pea shoot mitochondria using digitonin solubilisation of its constituents, which were further analysed by classical BN-related techniques and a multidimensional gel electrophoresis system when required. As a result, in addition to supercomplexes I 1 III 2 , I 1 III 2 IV n and III 2 IV 1 - , and individual complexes I-V previously detected in plant mitochondria, new OXPHOS structures were also revealed. Of them, (1) a megacomplex (II 2 , dimer V 2 , and individual complexes I-V previously detected in plant mitochondria, new OXPHOS structures were also revealed. Of them, (1) a megacomplex (II x III y IV z )n including complex II, (2) respirasomes I 2 III 4 IV n with two copies of complex I and dimeric complex III 2 , (3) a minor new supercomplex IV 1 Va 2 comigrating with I 1 III 2 , and (4) a second minor form of ATP synthase, Va, were found. The activity of singular complexes I, IV, and V was higher than the activity of the associated forms. The detection of new supercomplex IV 1 Va 2 , along with assemblies I 1 III 2 and I 1 - 2 III 2 - 4 IV n , prompted us to suggest the occurrence of in vivo oxphosomes comprising complexes I, III 2 , IV, and V. The putative oxphosome's stoichiometry, historical background, assumed functional significance, and subcompartmental location are discussed herein., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Cardiac hypertrophy is a major risk factor for congestive heart failure, a leading cause of morbidity and mortality. Abrogating hypertrophic progression is a well-recognized therapeutic goal. Mitochondrial dysfunction is a hallmark of numerous human diseases, including cardiac hypertrophy and heart failure. F1Fo-ATP synthase catalyzes the final step of oxidative energy production in mitochondria. Oligomycin sensitivity conferring protein (OSCP), a key component of the F1Fo-ATP synthase, plays an essential role in mitochondrial energy metabolism. However, the effects of OSCP-targeted therapy on cardiac hypertrophy remain unknown. In the present study, we found that impaired cardiac expression of OSCP is concomitant with mitochondrial dysfunction in the hypertrophied heart. We used cardiac-specific, adeno-associated virus-mediated gene therapy of OSCP to treat mice subjected to pressure overload induced by transverse aortic constriction (TAC). OSCP gene therapy protected the TAC-mice from cardiac dysfunction, cardiomyocyte hypertrophy, and fibrosis. OSCP gene therapy also enhanced mitochondrial respiration capacities in TAC-mice. Consistently, OSCP gene therapy attenuated reactive oxygen species and opening of mitochondrial permeability transition pore in the hypertrophied heart. Together, adeno-associated virus type 9-mediated, cardiac-specific OSCP overexpression can protect the heart via improving mitochondrial function. This result may provide insights into a novel therapy for cardiac hypertrophy and heart failure.

The mitochondrial ATP synthase is a multi-subunit enzyme complex located in the inner mitochondrial membrane which is essential for oxidative phosphorylation under physiological conditions. In this review, we analyse the enzyme functions involved in cancer progression by dissecting specific conditions in which ATP synthase contributes to cancer development or metastasis. Moreover, we propose the role of ATP synthase in the formation of the permeability transition pore (PTP) as an additional mechanism which controls tumour cell death. We further describe transcriptional and translational modifications of the enzyme subunits and of the inhibitor protein IF1 that may promote adaptations leading to cancer metabolism. Finally, we outline ATP synthase gene mutations and epigenetic modifications associated with cancer development or drug resistance, with the aim of highlighting this enzyme complex as a potential novel target for future anti-cancer therapy.

Dendritic spine injury underlies synaptic failure in many neurological disorders. Mounting evidence suggests a mitochondrial pathway of local nonapoptotic caspase signaling in mediating spine pruning. However, it remains unclear whether this caspase signaling plays a key role in spine loss when severe mitochondrial functional defects are present. The answer to this question is critical especially for some pathological states, in which mitochondrial deficits are prominent and difficult to fix. F1Fo ATP synthase is a pivotal mitochondrial enzyme and the dysfunction of this enzyme involves in diseases with spinopathy. Here, we inhibited F1Fo ATP synthase function in primary cultured hippocampal neurons by using non-lethal oligomycin A treatment. Oligomycin A induced mitochondrial defects including collapsed mitochondrial membrane potential, dissipated ATP production, and elevated reactive oxygen species (ROS) production. In addition, dendritic mitochondria underwent increased fragmentation and reduced positioning to dendritic spines along with increased caspase 3 cleavage in dendritic shaft and spines in response to oligomycin A. Concurring with these dendritic mitochondrial changes, oligomycin A-insulted neurons displayed spine loss and altered spine architecture. Such oligomycin A-mediated changes in dendritic spines were substantially prevented by the inhibition of caspase activation by using a pan-caspase inhibitor, quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methyl ketone (Q-VD-OPh). Of note, the administration of Q-VD-OPh showed no protective effect on oligomycin A-induced mitochondrial dysfunction. Our findings suggest a pivotal role of caspase 3 signaling in mediating spine injury and the modulation of caspase 3 activation may benefit neurons from spine loss in diseases, at least, in those with F1Fo ATP synthase defects.

The current state of research on the mitochondrial permeability transition pore (PTP) can be described in terms of three major problems: molecular identity, atomic structure and gating mechanism. In this review these three problems are discussed in the light of recent findings with special emphasis on the discovery that the PTP is mitochondrial F-ATP synthase (mtF o F 1 ). Novel features of the mitochondrial F-ATP synthase emerging from the success of single particle cryo electron microscopy (cryo-EM) to determine F-ATP synthase structures are surveyed along with their possible involvement in pore formation. Also, current findings from the gap junction field concerning the involvement of lipids in channel closure are examined. Finally, an earlier proposal denoted as the 'Death Finger' is discussed as a working model for PTP gating., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)