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.

The PTEN-induced kinase 1 (PINK1)-Parkin pathway plays a vital role in maintaining a healthy pool of mitochondria in higher eukaryotic cells. While the downstream components of this pathway are well understood, the upstream triggers remain less explored. In this study, we conducted an extensive analysis of inhibitors targeting various mitochondrial electron transport chain (ETC) complexes to investigate their potential as activators of the PINK1-Parkin pathway. We identified cloflucarban, an antibacterial compound, as a novel pathway activator that simultaneously inhibits mitochondrial complexes III and V, and V. RNA interference (RNAi) confirmed that the dual inhibition of these complexes activates the PINK1-Parkin pathway. Intriguingly, we discovered that albumin, specifically bovine serum albumin (BSA) and human serum albumin (HSA) commonly present in culture media, can hinder carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-induced pathway activation. However, cloflucarban\'s efficacy remains unaffected by albumin, highlighting its reliability for studying the PINK1-Parkin pathway. This study provides insights into the activation of the upstream PINK1-Parkin pathway and underscores the influence of culture conditions on research outcomes. Cloflucarban emerges as a promising tool for investigating mitochondrial quality control and neurodegenerative diseases.

Heart failure (HF) is a disease related to bioenergetic mitochondrial abnormalities. However, the whole status of molecules involved in the oxidative phosphorylation system (OXPHOS) is unknown. Therefore, we analyzed the OXPHOS transcriptome of human cardiac tissue by RNA-seq analyses (mRNA n = 36; ncRNA n = 30) in HF patients (ischemic cardiomyopathy (ICM) and dilated cardiomyopathy (DCM)) and control subjects. We detected 28 altered genes in these patients, highlighting greater deregulation in ICM. Specifically, we found a general overexpression of complex V (ATP synthase) elements, among them, ATP5I (ICM, FC = 2.04; p < 0.01), ATP5MJ (ICM, FC = 1.33, p < 0.05), and ATP5IF1 (ICM, FC = 1.81; p < 0.001), which presented a significant correlation with established echocardiographic parameters of cardiac remodeling and ventricular function as follows: left ventricular end-systolic (p < 0.01) and end-diastolic (p < 0.01) diameters, and ejection fraction (p < 0.05). We also detected an increase in ATP5IF1 protein levels (ICM, FC = 1.75; p < 0.01) and alterations in the microRNA expression levels of miR-208b-3p (ICM, FC = -1.44, p < 0.001), miR-483-3p (ICM, FC = 1.37, p < 0.01), regulators of ATP5I. Therefore, we observed the deregulation of the OXPHOS transcriptome in ICM patients, highlighting the overexpression of complex V and its relationship with cardiac remodeling and function.

The homeostasis of the transmembrane potential of hydrogen ions in mitochondria is a prerequisite for the normal mitochondrial functioning. However, in different pathological conditions it is advisable to slightly reduce the membrane potential, while maintaining it at levels sufficient to produce ATP that will ensure the normal functioning of the cell. A number of chemical agents have been found to provide mild uncoupling; however, natural proteins residing in mitochondrial membrane can carry this mission, such as proteins from the UCP family, an adenine nucleotide translocator and a dicarboxylate carrier. In this study, we demonstrated that the butyl ester of rhodamine 19, C4R1, binds to the components of the mitochondrial ATP synthase complex due to electrostatic interaction and has a good uncoupling effect. The more hydrophobic derivative C12R1 binds poorly to mitochondria with less uncoupling activity. Mass spectrometry confirmed that C4R1 binds to the β-subunit of mitochondrial ATP synthase and based on molecular docking, a C4R1 binding model was constructed suggesting the binding site on the interface between the α- and β-subunits, close to the anionic amino acid residues of the β-subunit. The association of the uncoupling effect with binding suggests that the ATP synthase complex can provide induced uncoupling.

The maintenance of cellular function relies on the close regulation of adenosine triphosphate (ATP) synthesis and hydrolysis. ATP hydrolysis by mitochondrial ATP Synthase (CV) is induced by loss of proton motive force and inhibited by the mitochondrial protein ATPase inhibitor (ATPIF1). The extent of CV hydrolytic activity and its impact on cellular energetics remains unknown due to the lack of selective hydrolysis inhibitors of CV. We find that CV hydrolytic activity takes place in coupled intact mitochondria and is increased by respiratory chain defects. We identified (+)-Epicatechin as a selective inhibitor of ATP hydrolysis that binds CV while preventing the binding of ATPIF1. In cells with Complex-III deficiency, we show that inhibition of CV hydrolytic activity by (+)-Epichatechin is sufficient to restore ATP content without restoring respiratory function. Inhibition of CV-ATP hydrolysis in a mouse model of Duchenne Muscular Dystrophy is sufficient to improve muscle force without any increase in mitochondrial content. We conclude that the impact of compromised mitochondrial respiration can be lessened using hydrolysis-selective inhibitors of CV.

F1Fo 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. F1Fo 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. F1Fo 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 F1Fo 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 F1Fo 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.

Arylamine N-acetyltransferase 1 (NAT1) is frequently upregulated in breast cancer. An unbiased analysis of proteomes of parental MDA-MB-231 breast cancer cells and two separate NAT1 knockout (KO) cell lines were performed. Among 4,890 proteins identified, 737 and 651 proteins were found significantly (p < 0.01) upregulated and downregulated, respectively, in NAT1 KO cells, compared to the parental cells. Each set of proteins was analyzed to identify Gene Ontology biological processes, molecular functions, and cellular components that were enriched in the set. Among the proteins upregulated in NAT1 KO cells, processes associated with MHC major histocompatibility complex I-mediated antigen presentation were significantly enriched. Multiple processes involved in mitochondrial functions were collectively downregulated in NAT1 KO cells, including multiple subunits of mitochondrial ATP synthase (Complex V of the electron transport chain). This was accompanied by a reduction in cell cycle-associated proteins and an increase in pro-apoptotic pathways in NAT1 KO cells. The current dataset contains additional representations of the biological processes and components that are differentially enriched in NAT1 KO MDA-MB-231 cells and will serve as a basis for generating novel hypotheses regarding the role of NAT1 in breast cancer. Data are available via ProteomeXchange with identifier PXD035953.

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.

Complex V or FoF1-ATPase is a multimeric protein found in bioenergetic membranes of cells and organelles like mitochondria/chloroplasts. The popular perception on Complex V deems it as a reversible molecular motor, working bi-directionally (breaking or making ATP) via a conformation-change based chemiosmotic rotary ATP synthesis (CRAS) mechanism, driven by proton-gradients or trans-membrane potential (TMP). In continuance of our pursuits against the CRAS model of cellular bioenergetics, herein we demonstrate the validity of the murburn model based in diffusible reactive (oxygen) species (DRS/DROS). Supported by new in silico derived data (that there are ∼12 adenosine nucleotide binding sites on the F1 bulb and not merely 3 sites, as perceived earlier), available structural information, known experimental observations, and thermodynamic/kinetic considerations (that de-solvation of protons from hydronium ions is facile), we deduce that Complex V serves as a physiological chemostat and a murzyme (enzyme working via murburn scheme, employing DRS). That is- Complex V uses ATP (via consumption at ε or proteins of F1 module) as a Michaelis-Menten substrate to serve as a pH-stat by inletting protons via the c-ring of Fo module. Physiologically, Complex V also functions as a murzyme by presenting ADP/Pi (or their reaction intermediates) on the αβ bulb, thereby enabling greater opportunities for DRS/proton-assisted ATP formation. Thus, the murburn paradigm succeeds the CRAS hypothesis for explaining the role of oxygen in mitochondrial physiologies of oxidative phosphorylation, thermogenesis, TMP and homeostasis.Communicated by Ramaswamy H. Sarma.

UNASSIGNED: 3,4-Dihydroxyl-phenyl lactic acid (DLA) and notoginsenoside R1 (R1) are known to protect ischemia and reperfusion (I/R) injury by targeting Sirtuin1/NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 10/the Mitochondrial Complex I (Sirt-1/NDUFA10/Complex I) and Rho-associated kinase/adenosine triphosphate (ROCK/ATP) ATP synthase δ subunit (ATP 5D), respectively. We hypothesized that a composite of the two may exhibit a more potent effect on I/R injury. The study was designed to test this hypothesis.
UNASSIGNED: Male Sprague-Dawley rats underwent left anterior descending artery occlusion and reperfusion, with or without DLA, R1, or a combination of 3,4-dihydroxyl-phenyl lactic acid and notoginsenoside R1 (DR) pretreatment. Heart function, myocardial morphology, myocardial infarct, myocardial blood flow (MBF), apoptosis, vascular diameter, and red blood cell (RBC) velocity in venules were evaluated. Myeloperoxidase (MPO), malondialdehyde (MDA), and 8-oxo-deoxyguanosine (8-OHdG) were assessed. The content of ATP, adenosine diphosphate (ADP), and adenosine monophosphate (AMP), the activity of mitochondrial respiratory chain Complex I and its subunit NDUFA10, the Mitochondrial Complex V (Complex V) and its subunit ATP 5D, Sirt-1, Ras homolog gene family, member A (RhoA), ROCK-1, and phosphorylated myosin light chain (P-MLC) were evaluated. R1 binding to Sirt-1 was determined by surface plasmon resonance.
UNASSIGNED: DLA inhibited the expression of Sirt-1, the reduction in Complex I activity and its subunit NDUFA10 expression, the increase in MPO, MDA, and 8-OhdG, and apoptosis. R1 inhibited the increase in the expression of RhoA/ROCK-1/P-MLC, the reduction of Complex V activity and its subunit ATP 5D expression, alleviated F-actin, and myocardial fiber rupture. Both DLA and R1 reduced the myocardial infarction size, increased the velocities of RBC in venules, and improved MBF and heart function impaired by I/R. DR exhibited effects similar to what was exerted, respectively, by DLA and R1 in terms of respiratory chain complexes and related signaling and outcomes, and an even more potent effect on myocardial infarct size, RBC velocity, heart function, and MBF than DLA and R1 alone.
UNASSIGNED: A combination of 3,4-dihydroxyl-phenyl lactic acid and notoginsenoside R1 revealed a more potent effect on I/R injury via the additive effect of DLA and R1, which inhibited not only apoptosis caused by low expression of Sirt-1/NDUFA10/Complex I but also myocardial fiber fracture caused by RhoA/ROCK-1 activation and decreased expression of ATP/ATP 5D/Complex V.