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While mechanisms controlling uncoupling protein-1 (UCP1) in thermogenic adipocytes play a pivotal role in non-shivering thermogenesis, it remains unclear whether F 1 Fo-ATP synthase function is also regulated in brown adipose tissue (BAT). Here, we show that inhibitory factor 1 (IF1, encoded by Atp5if1), an inhibitor of ATP synthase hydrolytic activity, is a critical negative regulator of brown adipocyte energy metabolism. In vivo, IF1 levels are diminished in BAT of cold-adapted mice compared to controls. Additionally, the capacity of ATP synthase to generate mitochondrial membrane potential (MMP) through ATP hydrolysis (the so-called "reverse mode" of ATP synthase) is increased in brown fat. In cultured brown adipocytes, IF1 overexpression results in an inability of mitochondria to sustain the MMP upon adrenergic stimulation, leading to a quiescent-like phenotype in brown adipocytes. In mice, adeno-associated virus-mediated IF1 overexpression in BAT suppresses adrenergic-stimulated thermogenesis and decreases mitochondrial respiration in BAT. Taken together, our work identifies downregulation of IF1 upon cold as a critical event for the facilitation of the reverse mode of ATP synthase as well as to enable energetic adaptation of BAT to effectively support non-shivering thermogenesis., (© 2024. The Author(s).)

Lysine acetylation is a conserved post-translational modification involved in energy metabolism in mitochondria and heart function. This study investigates the role of mitochondria-localized lysine acetyltransferase MOF (males absent on the first) in heart failure (HF). We find that MOF is upregulated in mitochondria during HF, and overexpression of mitochondria-targeted MOF (mtMOF) in mouse models results in mitochondria dysfunction, cardiac remodeling, and HF. Furthermore, sirtuin 3 (SIRT3) knockout aggravates mtMOF-induced damages, underscoring the role of MOF-catalyzed hyperacetylation in HF. Quantitative lysine acetylome analysis identifies ATP5B as a substrate of MOF. We demonstrate that the acetylation of ATP5B at K201, co-regulated by MOF and SIRT3, impairs mitochondrial respiration and energy metabolism both in vitro and in vivo. These findings suggest that the role of MOF in HF could be attributed to its regulation of ATP5B acetylation. Overall, our results highlight the disruptive impact of mitochondrial MOF on cardiac function and emphasize the significance of enzyme-catalyzed acetylation in mitochondria., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Triple-negative breast cancer (TNBC), a distinct subtype of breast cancer, is characterized by its high invasiveness, high metastatic potential, proneness to relapse, and poor prognosis. Effective treatment regimens for non-BRCA1/2 mutation TNBC are still lacking. As a result, there is a pressing clinical necessity to develop novel treatment approaches for non-BRCA1/2 mutation TNBC., Methods: For this research, the scRNA data was obtained from the GEO database, while the transcriptome data was obtained from the TCGA and METABRIC databases. Quality control procedures were conducted on single-cell sequencing data. and then annotation and the Copycat algorithm were applied for anlysis. Employing the high dimensional weighted gene coexpression network analysis (hdWGCNA) method, we analyzed the tumor epithelial cells from non-BRCA1/2 mutation TNBC to identify the functional module genes. PPI analysis and survival analysis were further emplyed to identify the key gene. siRNA-NC and siRNA-ATP5MF were transfected into two MDA-MB-231 and BT-549 TNBC cell lines. Cell growth was determined by CCK8 assay, colony formation and migration assay. Electron microscopy was used to examine the structure of mitochondria in cells. JC-1 staining was used to measure the potential of the mitochondrial membrane. A tumor xenograft animal model was established by injecting TNBC cells into nude mice. The animal model was usded to evaluated in vivo tumor response aftering ATP5MF silencing., Results: Using hdWGCNA, we have identified 136 genes in module 3. After PPI and survival analysis, we have identified ATP5MF as a potential therapeutic gene. High ATP5MF expression was associated with poor prognosis of non-BRCA1/2 mutation TNBC. The high expression of ATP5MF in TNBC tissues was evaluated using the TCGA database and IHC staining of clinical TNBC specimens. Silencing ATP5MF in two TNBC cell lines reduced the growth and colony formation of TNBC cells in vitro, and hindered the growth of TNBC xenografts in vivo. Additionally, ATP5MF knockdown impaired mitochondrial functions in TNBC cells., Conclusion: In summary, the metabolic protein ATP5MF plays a crucial role in the non-BRCA1/2 mutation TNBC cells, making it a potential novel diagnostic and therapeutic oncotarget for non-BRCA1/2 mutation TNBC., (© 2024. The Author(s).)

In developed countries, endometrial cancer (EC) is the most prevalent gynecological cancer. ATP5F1D is a subunit of ATP synthase, as well as an important component of the mitochondrial electron transport chain (ETC). ETC plays a compelling role in carcinogenesis. To date, little is known about the role of ATP5F1D in EC. We undertook data-independent acquisition mass spectrometry (DIA-MS) of 20 EC patients, comprising 10 high-grade and 10 low-grade cancer tissues. Biological functions of differentially expressed genes (DEGs) were analyzed by GO and KEGG. The expression level, clinicopathological features, diagnostic potency, prognostic value, RNA modifications, immune characteristics, and therapy response of ATP5F1D were investigated. In total, 77 DEGs were acquired by DIA analysis, which were closely related to regulating immune response and metabolic pathways. Among the five genes (NDUFB8, SLC26A2, RAF1, ATP5F1D, and GSTM5) involving in reactive oxygen species pathway, ATP5F1D showed the most significant differential expression (2.903-fold change). We found ATP5F1D had a high diagnostic value and was associated with a favorable prognosis in EC patients. After analyzing the RNA modifications of ATP5F1D, revealing a negative regulation between them. Additionally, ATP5F1D was closely related to tumor immune infiltration. Our results suggested T-cell dysfunction and TAM-M2 polarization might be the important mechanisms of ATP5F1D to facilitate tumor immune escape. Noticeably, EC patients with ATP5F1D-high expression had better immune treatment responses and were more sensitive to chemotherapy drugs. ATP5F1D can be used as a biomarker for diagnosis, prognosis, and immune infiltration of EC, and offers a crucial reference for personalized treatment of EC patients., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: In developed countries, endometrial cancer (EC) is the most prevalent gynecological cancer and its occurrence is associated with chronic inflammation. ATP5F1D is a subunit of ATP synthase (complex V), as well as the important component of mitochondrial electron transport chain (ETC). ETC play compelling roles in carcinogenesis. To date, little is known about the role of ATP5F1D in EC., Methods: ATP5F1D expression was identified in EC tissues and EC cell lines. We evaluated the influence of ATP5F1D on clinical features and prognosis based on TCGA database. The effects of ATP5F1D in EC malignant progression by applying loss-of-function assays in KLE and Ishikawa cell lines were detected by EdU, CCK-8, wound healing, Transwell, and flow cytometry assays. Additionally, electron microscope, LDH release, ELISA, mitochondrial ROS measurement, and Immunofluorescence were performed to demonstrate ATP5F1D can affect the pyroptosis of EC. To observe the anti-tumor effect on ATP5F1D silencing, we established an in vivo human endometrial tumor model using nude mice., Results: ATP5F1D expression was significantly upregulated in EC and was associated with favorable prognosis. ATP5F1D knockdown inhibited the proliferation, invasion, and migration of EC cells. Similarly, in nude mice, ATP5F1D knockdown suppressed the growth EC cells. Knocking down ATP5F1D lead to decrease the production of mitochondrial ROS (mtROS) and inhibited pyroptosis of EC cells., Conclusion: Downregulation of ATP5F1D as a new therapeutic strategy that could mediate pyroptosis via suppressing mtROS/NLRP3/caspase-1/GSDMD pathway to inhibit EC progression., 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 Elsevier B.V. All rights reserved.)

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.

Coronavirus nonstructural protein 2 (Nsp2) is regarded as a virulence determinant and plays a critical role in virus replication, and innate immunity. Screening and identifying host cell proteins that interact with viral proteins is an effective way to reveal the functions of viral proteins. In this study, the host proteins that interacted with transmissible gastroenteritis virus (TGEV) Nsp2 were identified using immunoprecipitation combined with LC-MS/MS. 77 host cell proteins were identified as putative Nsp2 interaction host cell proteins and a protein-protein interaction (PPI) was constructed. The identified proteins were found to be associated with various subcellular locations and functional categories through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. It is hypothesized that the host cell proteins interacting with TGEV Nsp2 are mainly involved in the formation of the cytoplasmic translation initiation complex, mRNA binding, ribosomes, and proteasomes. Among these, the ATP5B, a core subunit of the mitochondrial ATP synthase was further studied. The Coimmunoprecipitation (Co-IP) and indirect immunofluorescence (IFA) results confirmed that TGEV Nsp2 interacted with ATP5B. Furthermore, the downregulation of ATP5B expression was found to promote TGEV replication, suggesting that ATP5B might function as a negative regulator of TGEV replication. Collectively, our results offer additional insights into the functions of Nsp2 and provide a novel antiviral target against TGEV.

Disorders of ATP synthase, the key enzyme in mitochondrial energy supply, belong to the most severe metabolic diseases, manifesting as early-onset mitochondrial encephalo-cardiomyopathies. Since ATP synthase subunits are encoded by both mitochondrial and nuclear DNA, pathogenic variants can be found in either genome. In addition, the biogenesis of ATP synthase requires several assembly factors, some of which are also hotspots for pathogenic variants. While variants of MT-ATP6 and TMEM70 represent the most common cases of mitochondrial and nuclear DNA mutations respectively, the advent of next-generation sequencing has revealed new pathogenic variants in a number of structural genes and TMEM70, sometimes with truly peculiar genetics. Here we present a systematic review of the reported cases and discuss biochemical mechanisms, through which they are affecting ATP synthase. We explore how the knowledge of pathophysiology can improve our understanding of enzyme biogenesis and function. Keywords: Mitochondrial diseases o ATP synthase o Nuclear DNA o Mitochondrial DNA o TMEM70.

Background: Male infertility due to spermatogenesis defects affects millions of men worldwide. However, the genetic etiology of the vast majority remains unclear. The present study was undertaken to assess the association of DNAH6 and ATPase6 genes in asthenozoospermia patients in the northern region of India., Methods: A total of 60 semen samples were collected for the study, of which 30 were from the case group and 30 were from the control group. The semen samples for the case group (asthenozoospermia) and control groups were collected from IVF and Reproductive Biology Centre, Maulana Azad Medical College, New Delhi. Sperm count and motility were classified as per World Health Organization (WHO 2021) protocol. A total genomic DNA was extracted as per the stranded TRIZOL method with little modification., Results: In-vitro molecular characterizations of DNAH6 and ATPase6 genes in both groups were checked by Polymerase Chain Reaction (PCR). The 675 bp and 375 bp amplicons were amplified using PCR for ATPase6 and DNAH6 genes. Our study results showed a significant (P ≤ 0.05) null deletion of DNAH6 and ATPase6 genes in asthenozoospermia patients as compared to the control. We found the significant null deletion of DNAH6 in case 45.0%, and the control group was 11.7%. However, in the case of APTase6, it was 26.7% and 10.0%, respectively., Conclusions: Our study concluded that the presence of DHAH6 and ATPase6 genes had a significant impact on male infertility., (© 2024. The Author(s).)

Background: Sepsis represents a severe manifestation of infection often accompanied by metabolic disorders and mitochondrial dysfunction. Notably, mitochondrial DNA copy number (mtDNA-CN) and the expression of specific mitochondrial genes have emerged as sensitive indicators of mitochondrial function. To investigate the utility of mitochondrial gene expression in peripheral blood cells for distinguishing severe infections and predicting associated outcomes, we conducted a prospective cohort study., Methods: We established a prospective cohort comprising 74 patients with non-sepsis pneumonia and 67 cases of sepsis induced by respiratory infections, aging from 2 to 6 years old. We documented corresponding clinical data and laboratory information and collected blood samples upon initial hospital admission. Peripheral blood cells were promptly isolated, and both total DNA and RNA were extracted. We utilized absolute quantification PCR to assess mtDNA-CN, as well as the expression levels of mt-CO1, mt-ND1, and mt-ATP6. Subsequently, we extended these comparisons to include survivors and non-survivors among patients with sepsis using univariate and multivariate analyses. Receiver operating characteristic (ROC) curves were constructed to assess the diagnostic potential., Results: The mtDNA-CN in peripheral blood cells was significantly lower in the sepsis group. Univariate analysis revealed a significant reduction in the expression of mt-CO1, mt-ND1, and mt-ATP6 in patients with sepsis. However, multivariate analysis did not support the use of mitochondrial function in peripheral blood cells for sepsis diagnosis. In the comparison between pediatric sepsis survivors and non-survivors, univariate analysis indicated a substantial reduction in the expression of mt-CO1, mt-ND1, and mt-ATP6 among non-survivors. Notably, total bilirubin (TB), mt-CO1, mt-ND1, and mt-ATP6 levels were identified as independent risk factors for sepsis-induced mortality. ROC curves were then established for these independent risk factors, revealing areas under the curve (AUCs) of 0.753 for TB (95% CI 0.596-0.910), 0.870 for mt-CO1 (95% CI 0.775-0.965), 0.987 for mt-ND1 (95% CI 0.964-1.000), and 0.877 for mt-ATP6 (95% CI 0.793-0.962)., Conclusion: MtDNA-CN and mitochondrial gene expression are closely linked to the severity and clinical outcomes of infectious diseases. Severe infections lead to impaired mitochondrial function in peripheral blood cells. Notably, when compared to other laboratory parameters, the expression levels of mt-CO1, mt-ND1, and mt-ATP6 demonstrate promising potential for assessing the prognosis of pediatric sepsis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Jing, Zhang, Zhao, Li and Wen.)

Plastid retrograde signaling plays a key role in coordinating the expression of plastid genes and photosynthesis-associated nuclear genes (PhANGs). Although plastid retrograde signaling can be substantially compromised by mitochondrial dysfunction, it is not yet clear whether specific mitochondrial factors are required to regulate plastid retrograde signaling. Here, we show that mitochondrial ATP synthase beta -subunit mutants with decreased ATP synthase activity are impaired in plastid retrograde signaling in Arabidopsis thaliana . Transcriptome analysis revealed that the expression levels of PhANGs were significantly higher in the mutants affected in the AT5G08670 gene encoding the mitochondrial ATP synthase beta -subunit, compared to wild-type (WT) seedlings when treated with lincomycin (LIN) or norflurazon (NF). Further studies indicated that the expression of nuclear genes involved in chloroplast and mitochondrial retrograde signaling was affected in the AT5G08670 mutant seedlings treated with LIN. These changes might be linked to the modulation of some transcription factors (TFs), such as LHY ( Late Elongated Hypocotyl ), PIF ( Phytochrome-Interacting Factors ), MYB , WRKY , and AP2/ERF (Ethylene Responsive Factors). These findings suggest that the activity of mitochondrial ATP synthase significantly influences plastid retrograde signaling.

Background: The mechanism of cardiac reverse remodeling (CRR) mediated by the left ventricular assist device remains unclear. This study aims to identify the specific cell type responsible for CRR and develop the therapeutic target that promotes CRR., Methods: The nuclei were extracted from the left ventricular tissue of 4 normal controls, 4 CRR patients, and 4 no cardiac reverse remodeling patients and then subjected to single-nucleus RNA sequencing for identifying key cell types responsible for CRR. Gene overexpression in transverse aortic constriction and dilated cardiomyopathy heart failure mouse model (C57BL/6J background) and pathological staining were performed to validate the results of single-nucleus RNA sequencing., Results: Ten cell types were identified among 126 156 nuclei. Cardiomyocytes in CRR patients expressed higher levels of ATP5F1A than the other 2 groups. The macrophages in CRR patients expressed more anti-inflammatory genes and functioned in angiogenesis. Endothelial cells that elevated in no cardiac reverse remodeling patients were involved in the inflammatory response. Echocardiography showed that overexpressing ATP5F1A through cardiomyocyte-specific adeno-associated virus 9 demonstrated an ability to improve heart function and morphology. Pathological staining showed that overexpressing ATP5F1A could reduce fibrosis and cardiomyocyte size in the heart failure mouse model., Conclusions: The present results of single-nucleus RNA sequencing and heart failure mouse model indicated that ATP5F1A could mediate CRR and supported the development of therapeutics for overexpressing ATP5F1A in promoting CRR., Competing Interests: None.

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).)

A 23-month-old neutered male dog of unknown ancestry presented with a history of progressive neurological signs that included anxiety, cognitive impairment, tremors, seizure activity, ataxia, and pronounced visual impairment. The clinical signs were accompanied by global brain atrophy. Due to progression in the severity of disease signs, the dog was euthanized at 26 months of age. An examination of the tissues collected at necropsy revealed dramatic intracellular accumulations of autofluorescent inclusions in the brain, retina, and cardiac muscle. The inclusions were immunopositive for subunit c of mitochondrial ATP synthase, and their ultrastructural appearances were similar to those of lysosomal storage bodies that accumulate in some neuronal ceroid lipofuscinosis (NCL) diseases. The dog also exhibited widespread neuroinflammation. Based on these findings, the dog was deemed likely to have suffered from a form of NCL. A whole genome sequence analysis of the proband's DNA revealed a homozygous C to T substitution that altered the intron 3-exon 4 splice site of CLN6 . Other mutations in CLN6 cause NCL diseases in humans and animals, including dogs. The CLN6 protein was undetectable with immunolabeling in the tissues of the proband. Based on the clinical history, fluorescence and electron-microscopy, immunohistochemistry, and molecular genetic findings, the disorder in this dog was classified as an NCL resulting from the absence of the CLN6 protein. Screening the dog's genome for a panel of breed-specific polymorphisms indicated that its ancestry included numerous breeds, with no single breed predominating. This suggests that the CLN6 disease variant is likely to be present in other mixed-breed dogs and at least some ancestral breeds, although it is likely to be rare since other cases have not been reported to date.

Background: MT-ATP6 is a mitochondrial gene which encodes for the intramembrane subunit 6 (or A) of the mitochondrial ATP synthase, also known asl complex V, which is involved in the last step of oxidative phosphorylation to produce cellular ATP through aerobic metabolism. Although classically associated with the NARP syndrome, recent evidence highlights an important role of MT-ATP6 pathogenic variants in complicated adult-onset ataxias., Methods: We describe two unrelated patients with adult-onset cerebellar ataxia associated with severe optic atrophy and mild cognitive impairment. Whole mitochondrial DNA sequencing was performed in both patients. We employed patients' primary fibroblasts and cytoplasmic hybrids (cybrids), generated from patients-derived cells, to assess the activity of respiratory chain complexes, oxygen consumption rate (OCR), ATP production and mitochondrial membrane potential., Results: In both patients, we identified the same novel m.8777 T > C variant in MT-ATP6 with variable heteroplasmy level in different tissues. We identifed an additional heteroplasmic novel variant in MT-ATP6, m.8879G > T, in the patients with the most severe phenotype. A significant reduction in complex V activity, OCR and ATP production was observed in cybrid clones homoplasmic for the m.8777 T > C variant, while no functional defect was detected in m.8879G > T homoplasmic clones. In addition, fibroblasts with high heteroplasmic levelsof m.8777 T > C variant showed hyperpolarization of mitochondrial membranes., Conclusions: We describe a novel pathogenic mtDNA variant in MT-ATP6 associated with adult-onset ataxia, reinforcing the value of mtDNA screening within the diagnostic workflow of selected patients with late onset ataxias., (© 2024. The Author(s).)

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.)

Mitochondrial ATP synthase (Complex V) catalyzes the last step of oxidative phosphorylation and provides most of the energy (ATP) required by human cells. The mitochondrial genes MT-ATP6 and MT-ATP8 encode two subunits of the multi-subunit Complex V. Since the discovery of the first MT-ATP6 variant in the year 1990 as the cause of Neuropathy, Ataxia, and Retinitis Pigmentosa (NARP) syndrome, a large and continuously increasing number of inborn variants in the MT-ATP6 and MT-ATP8 genes have been identified as pathogenic. Variants in these genes correlate with various clinical phenotypes, which include several neurodegenerative and multisystemic disorders. In the present review, we report the pathogenic variants in mitochondrial ATP synthase genes and highlight the molecular mechanisms underlying ATP synthase deficiency that promote biochemical dysfunctions. We discuss the possible structural changes induced by the most common variants found in patients by considering the recent cryo-electron microscopy structure of human ATP synthase. Finally, we provide the state-of-the-art of all therapeutic proposals reported in the literature, including drug interventions targeting mitochondrial dysfunctions, allotopic gene expression- and nuclease-based strategies, and discuss their potential translation into clinical trials.

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 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.

The coexistence of two pools of ATP synthase in mitochondria has been largely neglected despite in vitro indications for the existence of reversible active/inactive state transitions in the F1-domain of the enzyme. Herein, using cells and mitochondria from mouse tissues, we demonstrate the existence in vivo of two pools of ATP synthase: one active, the other IF1-bound inactive. IF1 is required for oligomerization and inactivation of ATP synthase and for proper cristae formation. Immunoelectron microscopy shows the co-distribution of IF1 and ATP synthase, placing the inactive "sluggish" ATP synthase preferentially at cristae tips. The intramitochondrial distribution of IF1 correlates with cristae microdomains of high membrane potential, partially explaining its heterogeneous distribution. These findings support that IF1 is the in vivo regulator of the active/inactive state transitions of the ATP synthase and suggest that local regulation of IF1-ATP synthase interactions is essential to activate the sluggish ATP synthase., (© 2023. Springer Nature Limited.)

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.)

Although decreased citrulline is used as a newborn screening (NBS) marker to identify proximal urea cycle disorders (UCDs), it is also a feature of some mitochondrial diseases, including MT-ATP6 mitochondrial disease. Here we describe biochemical and clinical features of 11 children born to eight mothers from seven separate families who were identified with low citrulline by NBS (range 3-5 μM; screening cutoff >5) and ultimately diagnosed with MT-ATP6 mitochondrial disease. Follow-up testing revealed a pattern of hypocitrullinemia together with elevated propionyl-(C3) and 3-hydroxyisovaleryl-(C5-OH) acylcarnitines, and a homoplasmic pathogenic variant in MT-ATP6 in all cases. Single and multivariate analysis of NBS data from the 11 cases using Collaborative Laboratory Integrated Reports (CLIR; https://clir.mayo.edu) demonstrated citrulline <1st percentile, C3 > 50th percentile, and C5-OH >90th percentile when compared with reference data, as well as unequivocal separation from proximal UCD cases and false-positive low citrulline cases using dual scatter plots. Five of the eight mothers were symptomatic at the time of their child(ren)'s diagnosis, and all mothers and maternal grandmothers evaluated molecularly and biochemically had a homoplasmic pathogenic variant in MT-ATP6, low citrulline, elevated C3, and/or elevated C5-OH. All molecularly confirmed individuals (n = 17) with either no symptoms (n = 12), migraines (n = 1), or a neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) phenotype (n = 3) were found to have an A or U mitochondrial haplogroup, while one child with infantile-lethal Leigh syndrome had a B haplogroup., (© 2023 Wiley Periodicals LLC.)

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.)

Mitochondrial ATP synthase is a multiprotein complex, which consists of a matrix-localized F1 domain (F1-ATPase) and an inner membrane-embedded Fo domain (Fo-ATPase). The assembly process of mitochondrial ATP synthase is complex and requires the function of many assembly factors. Although extensive studies on mitochondrial ATP synthase assembly have been conducted on yeast, much less study has been performed on plants. Here, we revealed the function of Arabidopsis prohibitin 3 (PHB3) in mitochondrial ATP synthase assembly by characterizing the phb3 mutant. The blue native PAGE (BN-PAGE) and in-gel activity staining assays showed that the activities of ATP synthase and F1-ATPase were significantly decreased in the phb3 mutant. The absence of PHB3 resulted in the accumulation of the Fo-ATPase and F1-ATPase intermediates, whereas the abundance of the Fo-ATPase subunit a was decreased in the ATP synthase monomer. Furthermore, we showed that PHB3 could interact with the F1-ATPase subunits β and δ in the yeast two-hybrid system (Y2H) and luciferase complementation imaging (LCI) assay and with Fo-ATPase subunit c in the LCI assay. These results indicate that PHB3 acts as an assembly factor required for the assembly and activity of mitochondrial ATP synthase.

Autism spectrum disorder (ASD) is a neurodevelopmental disability that causes social impairment, debilitated verbal or nonverbal conversation, and restricted/repeated behavior. Recent research reveals that mitochondrial dysfunction and oxidative stress might play a pivotal role in ASD condition. The goal of this case-control study was to investigate oxidative stress and related alterations in ASD patients. In addition, the impact of mitochondrial DNA (mtDNA) mutations, particularly MT-ATP6, and its link with oxidative stress in ASD was studied. We found that ASD patient's plasma had lower superoxide dismutase (SOD) and higher catalase (CAT) activity, resulting in lower SOD/CAT ratio. MT-ATP6 mutation analysis revealed that four variations, 8865 G>A, 8684 C>T, 8697 G>A, and 8836 A>G, have a frequency of more than 10% with missense and synonymous (silent) mutations. It was observed that abnormalities in mitochondrial complexes (I, III, V) are more common in ASD, and it may have resulted in MT-ATP6 changes or vice versa. In conclusion, our findings authenticate that oxidative stress and genetics both have an equal and potential role behind ASD and we recommend to conduct more such concurrent research to understand their unique mechanism for better diagnosis and therapeutic for ASD., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The mitochondrial DNA mutation m.9032T>C was previously identified in patients presenting with NARP (Neuropathy Ataxia Retinitis Pigmentosa). Their clinical features had a maternal transmission and patient's cells showed a reduced oxidative phosphorylation capacity, elevated reactive oxygen species (ROS) production and hyperpolarization of the mitochondrial inner membrane, providing evidence that m.9032T>C is truly pathogenic. This mutation leads to replacement of a highly conserved leucine residue with proline at position 169 of ATP synthase subunit a (L169P). This protein and a ring of identical c-subunits (c-ring) move protons through the mitochondrial inner membrane coupled to ATP synthesis. We herein investigated the consequences of m.9032T>C on ATP synthase in a strain of Saccharomyces cerevisiae with an equivalent mutation (L186P). The mutant enzyme assembled correctly but was mostly inactive as evidenced by a  > 95% drop in the rate of mitochondrial ATP synthesis and absence of significant ATP-driven proton pumping across the mitochondrial membrane. Intragenic suppressors selected from L186P yeast restoring ATP synthase function to varying degrees (30-70%) were identified at the original mutation site (L186S) or in another position of the subunit a (H114Q, I118T). In light of atomic structures of yeast ATP synthase recently described, we conclude from these results that m.9032T>C disrupts proton conduction between the external side of the membrane and the c-ring, and that H114Q and I118T enable protons to access the c-ring through a modified pathway., (© The Author(s) 2022. Published by Oxford University Press.)

F 1 F o adenosine triphosphate (ATP) synthase, also known as the complex V, is the central ATP-producing unit in the cells arranged in the mitochondrial and plasma membranes. F 1 F o ATP synthase also regulates the central metabolic processes in the human body driven by proton motive force (Δp). Numerous studies have immensely contributed toward highlighting its regulation in improving energy homeostasis and maintaining mitochondrial integrity, which otherwise gets compromised in illnesses. Yet, its role in the implication of non-communicable diseases remains unknown. F 1 F o ATP synthase dysregulation at gene level leads to reduced activity and delocalization in the cristae and plasma membranes, which is directly associated with non-communicable diseases: cardiovascular diseases, diabetes, neurodegenerative disorders, cancer, and renal diseases. Individual subunits of the F 1 F o ATP synthase target ligand-based competitive or non-competitive inhibition. After performing a systematic literature review to understand its specific functions and its novel drug targets, the present article focuses on the central role of F 1 F o ATP synthase in primary non-communicable diseases. Next, it discusses its involvement through various pathways and the effects of multiple inhibitors, activators, and modulators specific to non-communicable diseases with a futuristic outlook., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

The list of mitochondrial DNA (mtDNA) variants detected in individuals with neurodegenerative diseases is constantly growing. Evaluating their functional consequences and pathogenicity is not easy, especially when they are found in only a limited number of patients together with wild-type mtDNA (heteroplasmy). Owing to its amenability to mitochondrial genetic transformation and incapacity to stably maintain heteroplasmy, and the strong evolutionary conservation of the proteins encoded in mitochondria, Saccharomyces cerevisiae provides a convenient model to investigate the functional consequences of human mtDNA variants. We herein report the construction and energy-transducing properties of yeast models of eight MT-ATP6 gene variants identified in patients with various disorders: m.8843T>C, m.8950G>A, m.9016A>G, m.9025G>A, m.9029A>G, m.9058A>G, m.9139G>A and m.9160T>C. Significant defect in growth dependent on respiration and deficits in ATP production were observed in yeast models of m.8950G>A, m.9025G>A and m.9029A>G, providing evidence of pathogenicity for these variants. Yeast models of the five other variants showed very mild, if any, effect on mitochondrial function, suggesting that the variants do not have, at least alone, the potential to compromise human health., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

IF 1 is a natural inhibitor protein for mitochondrial F o F 1 ATP synthase that blocks catalysis and rotation of the F 1 by deeply inserting its N-terminal helices into F 1 . A unique feature of IF 1 is condition-dependent inhibition; although IF 1 inhibits ATP hydrolysis by F 1 , IF 1 inhibition is relieved under ATP synthesis conditions. To elucidate this condition-dependent inhibition mechanism, we have performed single-molecule manipulation experiments on IF 1 -inhibited bovine mitochondrial F 1 (bMF 1 ). The results show that IF 1 -inhibited F 1 is efficiently activated only when F 1 is rotated in the clockwise (ATP synthesis) direction, but not in the counterclockwise direction. The observed rotational-direction-dependent activation explains the condition-dependent mechanism of IF 1 inhibition. Investigation of mutant IF 1 with N-terminal truncations shows that the interaction with the γ subunit at the N-terminal regions is crucial for rotational-direction-dependent ejection, and the middle long helix is responsible for the inhibition of F 1 ., (© 2023. The Author(s).)

We generated two pairs of mother-child iPSCs lines for Maternally Inherited Leigh Syndrome (MILS) carrying the m.8993 T > G and m.9176 T > G mutations in the MT-ATP6 gene. We delivered reprogramming factors OCT4, SOX2, KLF4, and c-MYC via Sendai virus. All iPSCs lines had a normal karyotype, expressed pluripotency markers, and differentiated into the three germ layers. Both patient-iPSCs retained the same degrees of heteroplasmy as their source fibroblasts (>97.0 %). In maternal iPSCs, the heteroplasmy remained 0.0 % in the case of the m.8993 T > G mutation and dropped from 55.0 % to 1.0 % in the case of m.9176 T > G mutation., 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 © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Schizophrenia is a chronic and debilitating mental disorder, with unknown pathophysiology. Converging lines of evidence suggest that mitochondrial functioning may be compromised in schizophrenia. Postmortem brain samples of individuals with schizophrenia showed dysregulated expression levels of genes encoding enzyme complexes comprising the mitochondrial electron transport chain (ETC), including ATP synthase, the fifth ETC complex. However, there are inconsistencies regarding the direction of change, i.e., up- or down-regulation, and differences between female and male patients were hardly examined. We have performed a systematic meta-analysis of the expression of 16 ATP synthase encoding genes in postmortem brain samples of individuals with schizophrenia vs. healthy controls of three regions: Brodmann Area 10 (BA10), BA22/Superior Temporal Gyrus (STG) and the cerebellum. Eight independent datasets were integrated (overall 294brain samples, 145 of individuals with schizophrenia and 149 controls). The meta-analysis was applied to all individuals with schizophrenia vs. the controls, and also to female and male patients vs. age-matched controls, separately. A significant down-regulation of two ATP synthase encoding genes was detected in schizophrenia, ATP5A1 and ATP5H, and a trend towards down-regulation of five further ATP synthase genes. The down-regulation tendency was shown for both females and males with schizophrenia. Our findings support the hypothesis that schizophrenia is associated with reduced ATP synthesis via the oxidative phosphorylation system, which is caused by reduced cellular demand of ATP. Abnormal cellular energy metabolism can lead to alterations in neural function and brain circuitry, and thereby to the cognitive and behavioral aberrations characteristic of schizophrenia., Competing Interests: Declaration of competing interest All authors declare no conflict of interests in this study., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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).)

The presence of circulating cancer cells in the bloodstream is positively correlated with metastasis. We hypothesize that fluid shear stress (FSS) occurring during circulation alters mitochondrial function, enhancing metastatic behaviors of cancer cells. MCF7 and MDA-MB-231 human breast cancer cells subjected to FSS exponentially increased proliferation. Notably, FSS-treated cells consumed more oxygen but were resistant to uncoupler-mediated ATP loss. We found that exposure to FSS downregulated the F 1 F O ATP synthase c-subunit and overexpression of the c-subunit arrested cancer cell migration. Approaches that regulate c-subunit abundance may reduce the likelihood of breast cancer metastasis., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Mytilidae, also known as marine mussels, are widely distributed in the oceans worldwide. Members of Mytilidae show a tremendous range of ecological adaptions, from the species distributed in freshwater to those that inhabit in deep-sea. Mitochondria play an important role in energy metabolism, which might contribute to the adaptation of Mytilidae to different environments. In addition, some bivalve species are thought to lack the mitochondrial protein-coding gene ATP synthase F0 subunit 8. Increasing studies indicated that the absence of atp8 may be caused by annotation difficulties for atp8 gene is characterized by highly divergent, variable length., Results: In this study, the complete mitochondrial genomes of three marine mussels (Xenostrobus securis, Bathymodiolus puteoserpentis, Gigantidas vrijenhoeki) were newly assembled, with the lengths of 14,972 bp, 20,482, and 17,786 bp, respectively. We annotated atp8 in the sequences that we assembled and the sequences lacking atp8. The newly annotated atp8 sequences all have one predicted transmembrane domain, a similar hydropathy profile, as well as the C-terminal region with positively charged amino acids. Furthermore, we reconstructed the phylogenetic trees and performed positive selection analysis. The results showed that the deep-sea bathymodiolines experienced more relaxed evolutionary constraints. And signatures of positive selection were detected in nad4 of Limnoperna fortunei, which may contribute to the survival and/or thriving of this species in freshwater., Conclusions: Our analysis supported that atp8 may not be missing in the Mytilidae. And our results provided evidence that the mitochondrial genes may contribute to the adaptation of Mytilidae to different environments., (© 2022. The Author(s).)

Episodic weakness is typically associated with a group of disorders so called periodic paralyses. Their major causes are mutation of ion channels, and have rarely been linked to mitochondrial disorders. We report a 20-year-old man with episodic weakness and axonal sensorimotor neuropathy since the age of 10 years. Analysis of the next generation sequencing data of the entire mitochondrial genome extracted from the blood revealed a homoplasmic m.9185T > C variant in MT-ATP6. Acetazolamide may be responsive for episodic weakness, and supplements with l-carnitine with coenzyme-Q10 seem to be beneficial as well. To the best of our knowledge, this is the first report in Taiwan which reveals episodic weakness and sensorimotor polyneuropathy as a unique phenotype of MT-ATP6 mutations., Competing Interests: Declaration of competing interest The authors have no conflicts of interest relevant to this article., (Copyright © 2021. Published by Elsevier B.V.)

Major progress has been made in defining the basis of the mitochondrial permeability transition, a Ca 2+ -dependent permeability increase of the inner membrane that has puzzled mitochondrial research for almost 70 years. Initially considered an artefact of limited biological interest by most, over the years the permeability transition has raised to the status of regulator of mitochondrial ion homeostasis and of druggable effector mechanism of cell death. The permeability transition is mediated by opening of channel(s) modulated by matrix cyclophilin D, the permeability transition pore(s) (PTP). The field has received new impulse (a) from the hypothesis that the PTP may originate from a Ca 2+ -dependent conformational change of F-ATP synthase and (b) from the reevaluation of the long-standing hypothesis that it originates from the adenine nucleotide translocator (ANT). Here, we provide a synthetic account of the structure of ANT and F-ATP synthase to discuss potential and controversial mechanisms through which they may form high-conductance channels; and review some intriguing findings from the wealth of early studies of PTP modulation that still await an explanation. We hope that this review will stimulate new experiments addressing the many outstanding problems, and thus contribute to the eventual solution of the puzzle of the permeability transition., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: An inability of a man to conceive a potentially fertile woman after a year of unprotected intercourse is defined as male infertility. It is reported that 30-40% of males in their reproductive years have abnormalities in sperm production, either qualitatively or quantitatively, or both. However, genetic factors result in up to 15% of male infertility cases. The present study aimed to analyze the possible correlations between sub-fertility and polymorphisms in sperm mitochondrial CO3, ATP6 and ATP8 genes in sub-fertile men., Methods and Results: For 67 sub-fertile and 44 fertile male samples, Sanger sequencing of selected mitochondrial DNA genes was done. A total of twelve SNPs in the MT-CO3 gene: rs2248727, rs7520428, rs3134801, rs9743, rs28358272, rs2853824, rs2856985, rs2854139, rs41347846, rs28380140, rs3902407, and 28,411,821, fourteen SNPs in the MT-ATP6: rs2001031, rs2000975, rs2298011, rs7520428, rs9645429, rs112660509, rs6650105, rs6594033, rs6594034, rs6594035, rs3020563, rs28358887, rs2096044, and rs9283154, and ten SNPs in the MT-ATP8: rs9285835, rs9285836, rs9283154, rs8179289, rs121434446, rs1116906, rs2153588, rs1116905, rs1116907, and rs3020563 were detected in the case and control groups at different nucleotide positions. Only the rs7520428 in the MT-CO3 and MT-ATP6 showed a statistically significant difference between sub-fertile and fertile groups in the genotype's and allele's frequency test (P < 0.0001 for both)., Conclusion: The results of our study suggest that male sub-fertility is linked with rs7520428 SNP in MT-CO3 and MT-ATP6. The studied polymorphic variations in the MT-ATP8 gene, on the contrary, did not reveal any significant association with male sub-fertility., (© 2022. The Author(s).)

We describe the case of identical twin boys who presented with low body weight despite excessive caloric intake. An evaluation of their fibroblasts showed elevated oxygen consumption and decreased mitochondrial membrane potential. Exome analysis revealed a de novo heterozygous variant in ATP5F1B , which encodes the β subunit of mitochondrial ATP synthase (also called complex V). In yeast, mutations affecting the same region loosen coupling between the proton motive force and ATP synthesis, resulting in high rates of mitochondrial respiration. Expression of the mutant allele in human cell lines recapitulates this phenotype. These data support an autosomal dominant mitochondrial uncoupling syndrome with hypermetabolism. (Funded by the National Institutes of Health.)., (Copyright © 2022 Massachusetts Medical Society.)

We used a non-integrative self-replicating RNA vector to establish four iPSC lines: two iPSC lines from a young male carrying the mutation m.9185 T>C in the mitochondrial gene MT-ATP6 (present at virtual homoplasmic level), and two iPSC lines from his healthy mother (carrying the mutation in only about 4 % of mtDNA copies). All iPSC lines exhibited pluripotency characteristics, were capable to give rise to cells of the three germ layers in vitro, and presented a normal karyotype. The derived iPSC lines retained the MT-ATP6 mutation at levels similar to those observed in the parental fibroblasts., 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 © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Mitochondrial complex V plays an important role in oxidative phosphorylation by catalyzing the generation of ATP. Most complex V subunits are nuclear encoded and not yet associated with recognized Mendelian disorders. Using exome sequencing, we identified a rare homozygous splice variant (c.87+3A>G) in ATP5PO, the complex V subunit which encodes the oligomycin sensitivity conferring protein, in three individuals from two unrelated families, with clinical suspicion of a mitochondrial disorder. These individuals had a similar, severe infantile and often lethal multi-systemic disorder that included hypotonia, developmental delay, hypertrophic cardiomyopathy, progressive epileptic encephalopathy, progressive cerebral atrophy, and white matter abnormalities on brain MRI consistent with Leigh syndrome. cDNA studies showed a predominant shortened transcript with skipping of exon 2 and low levels of the normal full-length transcript. Fibroblasts from the affected individuals demonstrated decreased ATP5PO protein, defective assembly of complex V with markedly reduced amounts of peripheral stalk proteins, and complex V hydrolytic activity. Further, expression of human ATP5PO cDNA without exon 2 (hATP5PO-∆ex2) in yeast cells deleted for yATP5 (ATP5PO homolog) was unable to rescue growth on media which requires oxidative phosphorylation when compared to the wild type construct (hATP5PO-WT), indicating that exon 2 deletion leads to a non-functional protein. Collectively, our findings support the pathogenicity of the ATP5PO c.87+3A>G variant, which significantly reduces but does not eliminate complex V activity. These data along with the recent report of an affected individual with ATP5PO variants, add to the evidence that rare biallelic variants in ATP5PO result in defective complex V assembly, function and are associated with Leigh syndrome., (© 2022 SSIEM.)

Mitochondrial ATP synthase is vital not only for cellular energy production but also for energy dissipation and cell death. ATP synthase c-ring was suggested to house the leak channel of mitochondrial permeability transition (mPT), which activates during excitotoxic ischemic insult. In this present study, we purified human c-ring from both eukaryotic and prokaryotic hosts to biophysically characterize its channel activity. We show that purified c-ring forms a large multi-conductance, voltage-gated ion channel that is inhibited by the addition of ATP synthase F 1 subcomplex. In contrast, dissociation of F 1 from F O occurs during excitotoxic neuronal death suggesting that the F 1 constitutes the gate of the channel. mPT is known to dissipate the osmotic gradient across the inner membrane during cell death. We show that ATP synthase c-subunit knock down (KD) prevents the osmotic change in response to high calcium and eliminates large conductance, Ca 2+ and CsA sensitive channel activity of mPT. These findings elucidate the gating mechanism of the ATP synthase c-subunit leak channel (ACLC) and suggest how ACLC opening is regulated by cell stress in a CypD-dependent manner., (© 2022. 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.)

Intramuscular fat (IMF) content is a key determinant of beef quality, making it a key topic of research interest. ATP5B serves as the catalytic component of the mitochondrial ATP synthase complex and plays essential roles in controlling fat contents and oxidative metabolism in bovine skeletal muscle. In this study, we determined that bovine ATP5B was highly expressed in longissimus thoracis. To elucidate the molecular mechanisms involved in bovine ATP5B regulation, we cloned and characterized the promoter region of ATP5B. Applying 5'-rapid amplification of cDNA end analysis (RACE), we identified two transcriptional start sites (TSSs) in its promoter region. Using a series of 5'-deletion promoter plasmids in luciferase reporter assay, we found that the proximal minimal promoter of ATP5B was located within the region -539/220 relative to the TSS. Site-directed mutation in combination with chromatin immunoprecipitation (ChIP) assays demonstrated that MyoD and GATA1 binding to the promoter region drives bovine ATP5B transcription. Taken together, these results provide new insight into the regulatory mechanisms of ATP5B transcription in mediating the IMF content of beef.

Cytoplasmic male sterility (CMS) is a powerful tool for the exploitation of hybrid heterosis and the study of signaling and interactions between the nucleus and the cytoplasm. C-type CMS (CMS-C) in maize has long been used in hybrid seed production, but the underlying sterility factor and its mechanism of action remain unclear. In this study, we demonstrate that the mitochondrial gene atp6c confers male sterility in CMS-C maize. The ATP6C protein shows stronger interactions with ATP8 and ATP9 than ATP6 during the assembly of F 1 F o -ATP synthase (F-type ATP synthase, ATPase), thereby reducing the quantity and activity of assembled F 1 F o -ATP synthase. By contrast, the quantity and activity of the F 1 ' component are increased in CMS-C lines. Reduced F 1 F o -ATP synthase activity causes accumulation of excess protons in the inner membrane space of the mitochondria, triggering a burst of reactive oxygen species (ROS), premature programmed cell death of the tapetal cells, and pollen abortion. Collectively, our study identifies a chimeric mitochondrial gene (ATP6C) that causes CMS in maize and documents the contribution of ATP6C to F 1 F o -ATP synthase assembly, thereby providing novel insights into the molecular mechanisms of male sterility in plants., (Copyright © 2022 The Author. Published by Elsevier Inc. All rights reserved.)

We report the generation of four human iPSC lines (8993-A12, 8993-B12, 8993-C11, and 8993-D7) from fibroblasts of four patients affected by maternally inherited Leigh syndrome (MILS) carrying homoplasmic mutations m.8993T > G or m.8993T > C in the mitochondrial gene MT-ATP6. We used Sendai viruses to deliver reprogramming factors OCT4, SOX2, KLF4, and c-MYC. The established iPSC lines expressed pluripotency markers, exhibited a normal karyotype, were capable to form cells of the three germ layers in vitro, and retained the MT-ATP6 mutations at the same homoplasmic level of the parental fibroblasts., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Pathogenic variants that disrupt human mitochondrial protein synthesis are associated with a clinically heterogeneous group of diseases. Despite an impairment in oxidative phosphorylation being a common phenotype, the underlying molecular pathogenesis is more complex than simply a bioenergetic deficiency. Currently, we have limited mechanistic understanding on the scope by which a primary defect in mitochondrial protein synthesis contributes to organelle dysfunction. Since the proteins encoded in the mitochondrial genome are hydrophobic and need co-translational insertion into a lipid bilayer, responsive quality control mechanisms are required to resolve aberrations that arise with the synthesis of truncated and misfolded proteins. Here, we show that defects in the OXA1L-mediated insertion of MT-ATP6 nascent chains into the mitochondrial inner membrane are rapidly resolved by the AFG3L2 protease complex. Using pathogenic MT-ATP6 variants, we then reveal discrete steps in this quality control mechanism and the differential functional consequences to mitochondrial gene expression. The inherent ability of a given cell type to recognize and resolve impairments in mitochondrial protein synthesis may in part contribute at the molecular level to the wide clinical spectrum of these disorders., (© The Author(s) 2021. Published by Oxford University Press.)

Objectives: Mitochondrial DNA (mtDNA)-associated Leigh syndrome (LS) is characterized by maternal inheritance, and the heteroplasmic mutant load of mtDNA pathogenic variants is known to affect clinical phenotypes. Among mtDNA pathogenic variants, variants of the MT-ATP6 gene account for most of reported cases. In this report, we aimed to describe the clinical and genetic findings of MT-ATP6-associated LS patients diagnosed at a single tertiary institution in Korea., Methods: Thirteen patients with genetically confirmed MT-ATP6-associated LS were selected. We reviewed each patient's clinical findings, including general characteristics, biochemical parameters, brain MR images, muscle biopsy results, and heteroplasmic mutant load over a long-term follow-up period., Results: MT-ATP6-associated LS was of predominantly early onset (age <2 years), although we identified 2 late-onset (>60 months) LS patients. The heteroplasmic mutant load estimated by next-generation sequencing was 96%-100% in all nucleotide change groups. Compared with other forms of MT-ATP6-associated LS, the m.8993T>G point mutation elicited a significantly higher rate of symptom onset before 2 years of age. Brain MRI showed bilateral basal ganglia involvement in all patients, followed by cerebral atrophy, brainstem and thalamus involvement, and cerebellar atrophy. After follow-up (median 7.2 years, range 1.4 to 11.5 years), LS with m.8993T>G point mutations had a slightly more severe clinical progression compared with other forms of MT-ATP6-associated LS., Conclusions: MT-ATP6-associated LS patients presented with a broad spectrum of clinical diagnoses and had a very high heteroplasmic mutant load. This study provides valuable data on MT-ATP6-associated LS that will inform subsequent studies on LS., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The mitochondrial F 1 F o ATP synthase of the parasite Trypanosoma brucei has been previously studied in detail. This unusual enzyme switches direction in functionality during the life cycle of the parasite, acting as an ATP synthase in the insect stages, and as an ATPase to generate mitochondrial membrane potential in the mammalian bloodstream stages. Whereas the trypanosome F 1 moiety is relatively highly conserved in structure and composition, the F o subcomplex and the peripheral stalk have been shown to be more variable. Interestingly, a core subunit of the latter, the normally conserved subunit b, has been resistant to identification by sequence alignment or biochemical methods. Here, we identified a 17 kDa mitochondrial protein of the inner membrane, Tb927.8.3070, that is essential for normal growth, efficient oxidative phosphorylation, and membrane potential maintenance. Pull-down experiments and native PAGE analysis indicated that the protein is both associated with the F 1 F o ATP synthase and integral to its assembly. In addition, its knockdown reduced the levels of F o subunits, but not those of F 1 , and disturbed the cell cycle. Finally, analysis of structural homology using the HHpred algorithm showed that this protein has structural similarities to F o subunit b of other species, indicating that this subunit may be a highly diverged form of the elusive subunit b., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)