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The mitochondrial permeability transition pore (mPTP) is a channel in the inner mitochondrial membrane whose sustained opening in response to elevated mitochondrial matrix Ca 2+ concentrations triggers necrotic cell death. The molecular identity of mPTP is unknown. One proposed candidate is the mitochondrial ATP synthase, whose canonical function is to generate most ATP in multicellular organisms. Here, we present mitochondrial, cellular, and in vivo evidence that, rather than serving as mPTP, the mitochondrial ATP synthase inhibits this pore. Our studies confirm previous work showing persistence of mPTP in HAP1 cell lines lacking an assembled mitochondrial ATP synthase. Unexpectedly, however, we observe that Ca 2+ -induced pore opening is markedly sensitized by loss of the mitochondrial ATP synthase. Further, mPTP opening in cells lacking the mitochondrial ATP synthase is desensitized by pharmacological inhibition and genetic depletion of the mitochondrial cis-trans prolyl isomerase cyclophilin D as in wild-type cells, indicating that cyclophilin D can modulate mPTP through substrates other than subunits in the assembled mitochondrial ATP synthase. Mitoplast patch clamping studies showed that mPTP channel conductance was unaffected by loss of the mitochondrial ATP synthase but still blocked by cyclophilin D inhibition. Cardiac mitochondria from mice whose heart muscle cells we engineered deficient in the mitochondrial ATP synthase also demonstrate sensitization of Ca 2+ -induced mPTP opening and desensitization by cyclophilin D inhibition. Further, these mice exhibit strikingly larger myocardial infarctions when challenged with ischemia/reperfusion in vivo. We conclude that the mitochondrial ATP synthase does not function as mPTP and instead negatively regulates this pore., Competing Interests: Competing interests statement:The authors declare no competing interest.

Apple necrotic mosaic virus (ApNMV) is associated with apple mosaic disease in China. However, the mechanisms of ApNMV infection, as well as host defence against the virus, are still poorly understood. Mitochondrial ATP synthase plays a fundamental role in the regulation of plant growth and development. However, mitochondrial ATP synthase function in response to virus infection remains to be defined. In the present study, a yeast two-hybrid (Y2H) screening revealed that the apple mitochondrial ATP synthase oligomycin sensitivity-conferring protein (OSCP) subunit (MdATPO) interacts with ApNMV coat protein (CP). It was further verified that overexpression of MdATPO in Nicotiana benthamiana inhibited viral accumulation. In contrast, silencing of NbATPO facilitated viral accumulation, indicating that ATPO plays a defensive role during ApNMV infection. Further investigation demonstrated that ApNMV infection accelerated abscisic acid (ABA) accumulation, and ABA negatively regulated ATPO transcription, which was related to the ability of ABA insensitive 5 (ABI5) to bind to the ABA-responsive elements (ABREs) of the ATPO promoter. Taken together, our results indicated that transcription factor ABI5 negatively regulated ATPO transcription by directly binding to its promoter, leading to the susceptibility of apple and N. benthamiana to ApNMV infection. The current study facilitates a comprehensive understanding of the intricate responses of the host to ApNMV infection., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Cancer poses a significant global health problem with profound personal and economic implications on National Health Care Systems. The reprograming of metabolism is a major trait of the cancer phenotype with a clear potential for developing effective therapeutic strategies to combat the disease. Herein, we summarize the relevant role that the mitochondrial ATP synthase and its physiological inhibitor, ATPase Inhibitory Factor 1 (IF1), play in metabolic reprogramming to an enhanced glycolytic phenotype. We stress that the interplay in the ATP synthase/IF1 axis has additional functional roles in signaling mitohormetic programs, pro-oncogenic or anti-metastatic phenotypes depending on the cell type. Moreover, the same axis also participates in cell death resistance of cancer cells by restrained mitochondrial permeability transition pore opening. We emphasize the relevance of the different post-transcriptional mechanisms that regulate the specific expression and activity of ATP synthase/IF1, to stimulate further investigations in the field because of their potential as future targets to treat cancer. In addition, we review recent findings stressing that mitochondria metabolism is the primary altered target in lung adenocarcinomas and that the ATP synthase/IF1 axis of OXPHOS is included in the most significant signature of metastatic disease. Finally, we stress that targeting mitochondrial OXPHOS in pre-clinical mouse models affords a most effective therapeutic strategy in cancer treatment.

ATP5F1B is a subunit of the mitochondrial ATP synthase or complex V of the mitochondrial respiratory chain. Pathogenic variants in nuclear genes encoding assembly factors or structural subunits are associated with complex V deficiency, typically characterized by autosomal recessive inheritance and multisystem phenotypes. Movement disorders have been described in a subset of cases carrying autosomal dominant variants in structural subunits genes ATP5F1A and ATP5MC3. Here, we report the identification of two different ATP5F1B missense variants (c.1000A>C; p.Thr334Pro and c.1445T>C; p.Val482Ala) segregating with early-onset isolated dystonia in two families, both with autosomal dominant mode of inheritance and incomplete penetrance. Functional studies in mutant fibroblasts revealed no decrease of ATP5F1B protein amount but severe reduction of complex V activity and impaired mitochondrial membrane potential, suggesting a dominant-negative effect. In conclusion, our study describes a new candidate gene associated with isolated dystonia and confirms that heterozygous variants in genes encoding subunits of the mitochondrial ATP synthase may cause autosomal dominant isolated dystonia with incomplete penetrance, likely through a dominant-negative mechanism., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain.)

The passage of protons across membranes through F1Fo-ATP synthases spins their rotors and drives the synthesis of ATP. While the principle of torque generation by proton transfer is known, the mechanisms and routes of proton access and release and their evolution are not fully understood. Here, we show that the entry site and path of protons in the lumenal half channel of mitochondrial ATP synthases are largely defined by a short N-terminal α-helix of subunit-a. In Trypanosoma brucei and other Euglenozoa, the α-helix is part of another polypeptide chain that is a product of subunit-a gene fragmentation. This α-helix and other elements forming the proton pathway are widely conserved across eukaryotes and in Alphaproteobacteria, the closest extant relatives of mitochondria, but not in other bacteria. The α-helix blocks one of two proton routes found in Escherichia coli, resulting in a single proton entry site in mitochondrial and alphaproteobacterial ATP synthases. Thus, the shape of the access half channel predates eukaryotes and originated in the lineage from which mitochondria evolved by endosymbiosis., Competing Interests: Conflict of interest statement. They authors declare they have no competing interests., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Mitochondria play essential and specific roles during erythroid differentiation. Recently, FAM210B , encoding a mitochondrial inner membrane protein, has been identified as a novel target of GATA-1, as well as an erythropoietin-inducible gene. While FAM210B protein is involved in regulate mitochondrial metabolism and heme biosynthesis, its detailed function remains unknown. Here, we generated both knockout and knockdown of endogenous FAM210B in human induced pluripotent stem-derived erythroid progenitor (HiDEP) cells using CRISPR/Cas9 methodology. Intriguingly, erythroid differentiation was more pronounced in the FAM210B-depleted cells, and this resulted in increased frequency of orthochromatic erythroblasts and decreased frequencies of basophilic/polychromatic erythroblasts. Comprehensive metabolite analysis and functional analysis indicated that oxygen consumption rates and the NAD (NAD + )/NADH ratio were significantly decreased, while lactate production was significantly increased in FAM210B deletion HiDEP cells, indicating involvement of FAM210B in mitochondrial energy metabolism in erythroblasts. Finally, we purified FAM210B-interacting protein from K562 cells that stably expressed His/biotin-tagged FAM210B. Mass spectrometry analysis of the His/biotin-purified material indicated interactions with multiple subunits of mitochondrial ATP synthases, such as subunit alpha (ATP5A) and beta (ATP5B). Our results suggested that FAM210B contributes prominently to erythroid differentiation by regulating mitochondrial energy metabolism. Our results provide insights into the pathophysiology of dysregulated hematopoiesis.

Nuclear-encoded Atp23 was previously shown to have dual functions, including processing the yeast Atp6 precursor and assisting the assembly of yeast mitochondrial ATP synthase. However, it remains unknown whether there are genes functionally complementary to ATP23 to rescue atp23 null mutant. In the present paper, we screen and characterize three revertants of atp23 null mutant and reveal a T1121G point mutation in the mitochondrial gene COX1 coding sequence, which leads to Val374Gly mutation in Cox1, the suppressor in the revertants. This was verified further by the partial restoration of mitochondrial ATP synthase assembly in atp23 null mutant transformed with exogenous hybrid COX1   T1121G mutant plasmid. The predicted tertiary structure of the Cox1 p.Val374Gly mutation showed no obvious difference from wild-type Cox1. By further chase labeling with isotope [ 35 S]-methionine, we found that the stability of Atp6 of ATP synthase increased in the revertants compared with the atp23 null mutant. Taking all the data together, we revealed that the T1121G point mutation of mitochondrial gene COX1 could partially restore the unassembly of mitochondrial ATP synthase in atp23 null mutant by increasing the stability of Atp6. Therefore, this study uncovers a gene that is partially functionally complementary to ATP23 to rescue ATP23 deficiency, broadening our understanding of the relationship between yeast the cytochrome c oxidase complex and mitochondrial ATP synthase complex.

Reprogramming of cellular metabolism is a hallmark of cancer. Mitochondrial ATP synthase (MAS) produces most of the ATP that drives the cell. High expression of the MAS-composing proteins is found during cancer and is linked to a poor prognosis in glioblastoma, ovarian cancer, prostate cancer, breast cancer, and clear cell renal cell carcinoma. Cell surface-expressed ATP synthase, translocated from mitochondrion to cell membrane, involves the angiogenesis, tumorigenesis, and metastasis of cancer. ATP synthase has therefore been considered a therapeutic target. We review recent various ATP synthase inhibitors that suppress tumor growth and are being tested for the clinic., Competing Interests: The authors declare no competing interest., (© 2021 The Author(s).)

Two chaperones, Atp23p and Atp10p, were previously shown to regulate the assembly of yeast mitochondrial ATP synthase, and extra expression of ATP23 was found to partially rescue an atp10 deletion mutant, by an unknown mechanism. Here, we identified that the residues 112-115 (LRDK) of Atp23p were required for its function in assisting assembly of the synthase, and demonstrated both functions of Atp23p, processing subunit 6 precursor and assisting assembly of the synthase, were required for the partial rescue of atp10 deletion mutant. By chasing labeling with isotope 35 S-methionine, we found the stability of subunit 6 of the synthase increased in atp10 null strain upon overexpression of ATP23. Further co-immunoprecipitation (Co-IP) and blue native PAGE experiments showed that Atp23p and Atp10p were physically associated with each other in wild type. Moreover, we revealed the expression level of Atp23p increased in atp10 null mutant compared with the wild type. Furthermore, we found that, after 72 hours growth, atp10 null mutant showed leaky growth on respiratory substrates, presence of low level of subunit 6 and partial recovery of oligomycin sensitivity of mitochondrial ATPase activity. Further characterization revealed the expression of Atp23p increased after 24 hours growth in the mutant. These results indicated, in atp10 null mutant, ATP10 deficiency could be partially complemented with increased expression of Atp23p by stabilizing some subunit 6 of the synthase. Taken together, this study revealed the two chaperones Atp23p and Atp10p coordinated to regulate the assembly of mitochondrial ATP synthase, which advanced our understanding of mechanism of assembly of yeast mitochondrial ATP synthase., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Modulation of prostate stromal cells (PrSCs) within tumor tissues is gaining attention for the treatment of solid tumors. Using our original in vitro coculture system, we previously reported that leucinostatin (LCS)-A, a peptide mycotoxin, inhibited prostate cancer DU-145 cell growth through reduction of insulin-like growth factor 1 (IGF-I) expression in PrSCs. To further obtain additional bioactive compounds from LCS-A, we designed and synthesized a series of LCS-A derivatives as compounds that target PrSCs. Among the synthesized LCS-A derivatives, LCS-7 reduced IGF-I expression in PrSCs with lower toxicity to PrSCs and mice than LCS-A. As LCS-A has been suggested to interact with mitochondrial adenosine triphosphate (ATP) synthase, a docking study was performed to elucidate the mechanism of reduced IGF-I expression in the PrSCs. As expected, LCS-A and LCS-7 directly interacted with mitochondrial ATP synthase, and like LCS-A and LCS-7, other mitochondrial ATP synthase inhibitors also reduced the expression of IGF-I by PrSCs. Furthermore, LCS-A and LCS-7 significantly decreased the growth of mouse xenograft tumors. Based on these data, we propose that the mitochondrial ATP synthases-IGF-I axis of PrSCs plays a critical role on cancer cell growth and inhibition could be a potential anticancer target for prostate cancer., (© 2020 UICC.)

Background: Neonatal Encephalopathy (NE) is a mitochondrial ATP synthase (mATPase) disease, which results in the death of infants. The case presented here is reportedly caused by complex V deficiency as a result of mutation of Arginine to Cysteine at residue 329 in the mATPase. A recent breakthrough was the discovery of J147, which targets mATPase in the treatment of Alzheimer's disease. Based on the concepts of computational target-based drug design, this study investigated the possibility of employing J147 as a viable candidate in the treatment of NE., Objective/methods: The structural dynamic implications of this drug on the mutated enzyme are yet to be elucidated. Hence, integrative molecular dynamics simulations and thermodynamic calculations were employed to investigate the activity of J147 on the mutated enzyme in comparison to its already established inhibitory activity on the wild-type enzyme., Results: A correlated structural trend occurred between the wild-type and mutant systems whereby all the systems exhibited an overall conformational transition. Equal observations in favorable free binding energies further substantiated uniformity in the mobility, and residual fluctuation of the wild-type and mutant systems. The similarity in the binding landscape suggests that J147 could as well modulate mutant mATPase activity in addition to causing structural modifications in the wild-type enzyme., Conclusion: Findings suggest that J147 can stabilize the mutant protein and restore it to a similar structural state as the wild-type which depicts functionality. These details could be employed in drug design for potential drug resistance cases due to mATPase mutations that may present in the future., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Mitochondria play a central role in the energy metabolism of plants. At the same time, they provide energy for plant stress responses. We here report a first view on the mitochondrial Oxidative Phosphorylation (OXPHOS) system of the halophile (salt tolerant) plant Cakile maritima . Mitochondria were purified from suspension cultures of C. maritima and for comparison of Arabidopsis thaliana , a closely related glycophyte (salt sensitive) plant. Mitochondria were treated with digitonin and solubilized protein complexes were analyzed by 2D Blue native/SDS polyacrylamide gel electrophoresis. The OXPHOS systems of the two compared plants exhibit some distinct differences. C. maritima mitochondria include a very abundant respiratory supercomplex composed of monomeric complex I and dimeric complex III. At the same time the complexes II and IV are of reduced abundance. The stability of the OXPHOS complexes was investigated by combined salt and temperature treatments of isolated mitochondria. ATP synthase (complex V) is of increased stability in C. maritima . Also, the I + III 2 supercomplex is present in high abundance during stress treatments. These results give insights into the mitochondrial contribution to the plant salt stress response.

The discovery of J147 represented a significant milestone in the treatment of age-related disorders, which was further augmented by the recent identification of mitochondrial ATP synthase as the therapeutic target. However, the underlying molecular events associated with the modulatory activity of J147 have remained unresolved till date. Herein, we present, for the first time, a dynamical approach to investigate the allosteric regulation of mATP synthase by J147, using a reliable human αγβ protein model. The highlight of our findings is the existence of the J147-bound protein in distinct structural associations at different MD simulation periods coupled with concurrent open↔close transitions of the β catalytic and α allosteric (ATP5A) sites as defined by Cα distances (d), TriCα (Θ) and dihedral (φ) angular parameters. Firstly, there was an initial pairing of the αγ subunits away from the β subunit followed by the formation of the 'non-catalytic' αβ pair at a distance from the γ subunit. Interestingly, J147-induced structural arrangements were accompanied by the systematic transition of the β catalytic site from a closed to an open state, while there was a concurrent transition of the allosteric site from an open α E conformation to a closed state. Consequentially, J147 reduced the structural activity of the whole αγβ complex, while the unbound system exhibited high atomistic deviations and structural flexibility. Furthermore, J147 exhibited favorable binding at the allosteric site of mATP synthase with considerable electrostatic energy contributions from Gln215, Gly217, Thr219, Asp312, Asp313, Glu371 and Arg406. These findings provide details on the possible effects of J147 on mitochondrial bioenergetics, which could facilitate the structure-based design of novel small-molecule modulators of mATP synthase in the management of Alzheimer's disease and other neurodegenerative disorders., (© 2019 Wiley-VHCA AG, Zurich, Switzerland.)

Epilepsy is a common disease presenting with recurrent seizures. Hippocampal sclerosis (HS) is the commonest histopathological alteration in patients with temporal lobe epilepsy (TLE) undergoing surgery. HS physiopathogenesis is debatable. We have recently studied, by using mass spectrometry-based proteomics, an experimental model of TLE induced by electrical stimulation. Specifically, protein expressions of both the beta subunit of mitochondrial ATP synthase (ATP5B) and of membrane ATPases were found to be reduced. Here, we investigated tissue distribution of ATP5B and sodium/potassium-transporting ATPase subunit alpha-3 (NKAα3), a protein associated with neuromuscular excitability disorders, in human hippocampi resected "en bloc" for HS treatment (n = 15). We used immunohistochemistry and the stained area was digitally evaluated (increase in binary contrast of microscopic fields) in the hippocampal sectors (CA1-CA4) and dentate gyrus. All HS samples were classified as Type 1, according to the International League Against Epilepsy (ILAE) 2013 Classification (predominant cell loss in CA1 and CA4). ATP5B was significantly decreased in all sectors and dentate gyrus of HS patients compared with individuals submitted to necropsy and without history of neurological alterations (n = 10). NKAα3 expression showed no difference. Moreover, we identified a negative correlation between frequency of pre-operative seizures and number of neurons in CA1. In conclusion, our data showed similarity between changes in protein expression in a model of TLE and individuals with HS. ATP5B reduction would be at least in part due to neuronal loss. Future investigations on ATP5B activity could provide insights into the process of such cell loss.

We used electron cryotomography and subtomogram averaging to determine the in situ structures of mitochondrial ATP synthase dimers from two organisms belonging to the phylum euglenozoa: Trypanosoma brucei, a lethal human parasite, and Euglena gracilis, a photosynthetic protist. At a resolution of 32.5 Å and 27.5 Å, respectively, the two structures clearly exhibit a noncanonical F 1 head, in which the catalytic (αβ) 3 assembly forms a triangular pyramid rather than the pseudo-sixfold ring arrangement typical of all other ATP synthases investigated so far. Fitting of known X-ray structures reveals that this unusual geometry results from a phylum-specific cleavage of the α subunit, in which the C-terminal α C fragments are displaced by ∼20 Å and rotated by ∼30° from their expected positions. In this location, the α C fragment is unable to form the conserved catalytic interface that was thought to be essential for ATP synthesis, and cannot convert γ-subunit rotation into the conformational changes implicit in rotary catalysis. The new arrangement of catalytic subunits suggests that the mechanism of ATP generation by rotary ATPases is less strictly conserved than has been generally assumed. The ATP synthases of these organisms present a unique model system for discerning the individual contributions of the α and β subunits to the fundamental process of ATP synthesis., Competing Interests: The authors declare no conflict of interest.

Background: Aberrant DNA methylation status of some genes has been shown to be involved in chemoresistance of acute myeloid leukemia (AML). We have recently found that down-regulation of the β subunit of mitochondrial ATP synthase (ATPsyn-β) leads to adriamycin resistance in acute and chronic myeloid leukemia cells, and hypermethylation of the ATPsyn-β gene promoter is associated with chemoresistance in chronic myeloid leukemia., Objective: To further investigate the relationship between methylation of ATPsyn-β gene, mRNA expression as well as chemoresistance in AML., Methods: Quantitative RT-PCR and methylation specific PCR were performed to assess mRNA expression and methylation status of ATPsyn-β gene on primary bone marrow nuclear cells (BMMCs), and cell proliferation assay was used to determine the sensitivity of BMMCs to adriamycin., Results: Hypermethylation status of ATPsyn-β gene promoter existed in those relapsed/refractory AML patients, and this hypermethylation of the gene was associated with a suppressed mRNA expression levels. Four patients at diagnosis and relapse underwent gene methylation status shift from hypermethylation to hypomethylation, which was accompanied by reduced mRNA expression of the gene. 5-azacitidine(5-Aza)- a demethylating agent, could restore ATPsyn-β mRNA expression and increase the adriamycin sensitivity of primary leukemic cells from seven relapsed/ refractory AML patients., Conclusions: Hypermethylation of ATPsyn-β gene promoter is associated with a down-regulated mRNA expression and chemoresistance in AML patients.