F1F0-ATP Synthase Research Articles

CKB Promotes Mitochondrial ATP Production by Suppressing Permeability Transition Pore.

Creatine kinases are essential for maintaining cellular energy balance by facilitating the reversible transfer of a phosphoryl group from ATP to creatine, however, their role in mitochondrial ATP production remains unknown. This study shows creatine kinases, including CKMT1A, CKMT1B, and CKB, are highly expressed in cells relying on the mitochondrial F1F0 ATP synthase for survival. Interestingly, silencing CKB, but not CKMT1A or CKMT1B, leads to a loss of sensitivity to the inhibition of F1F0 ATP synthase in these cells. Mechanistically, CKB promotes mitochondrial ATP but reduces glycolytic ATP production by suppressing mitochondrial calcium (mCa2+) levels, thereby preventing the activation of mitochondrial permeability transition pore (mPTP) and ensuring efficient mitochondrial ATP generation. Further, CKB achieves this regulation by suppressing mCa2+ levels through the inhibition of AKT activity. Notably, the CKB-AKT signaling axis boosts mitochondrial ATP production in cancer cells growing in a mouse tumor model. Moreover, this study also uncovers a decline in CKB expression in peripheral blood mononuclear cells with aging, accompanied by an increase in AKT signaling in these cells. These findings thus shed light on a novel signaling pathway involving CKB that directly regulates mitochondrial ATP production, potentially playing a role in both pathological and physiological conditions.

Glucose Regulation of β-Cell KATP Channels: Is a New Model Needed?

The canonical model of glucose-induced increase in insulin secretion involves the metabolism of glucose via glycolysis and the citrate cycle, resulting in increased ATP synthesis by the respiratory chain and the closure of ATP-sensitive K+ (KATP) channels. The resulting plasma membrane depolarization, followed by Ca2+ influx through L-type Ca2+ channels, then induces insulin granule fusion. Merrins and colleagues have recently proposed an alternative model whereby KATP channels are controlled by pyruvate kinase, using glycolytic and mitochondrial phosphoenolpyruvate (PEP) to generate microdomains of high ATP/ADP immediately adjacent to KATP channels. This model presents several challenges. First, how mitochondrially generated PEP, but not ATP produced abundantly by the mitochondrial F1F0-ATP synthase, can gain access to the proposed microdomains is unclear. Second, ATP/ADP fluctuations imaged immediately beneath the plasma membrane closely resemble those in the bulk cytosol. Third, ADP privation of the respiratory chain at high glucose, suggested to drive alternating, phased-locked generation by mitochondria of ATP or PEP, has yet to be directly demonstrated. Finally, the approaches used to explore these questions may be complicated by off-target effects. We suggest instead that Ca2+ changes, well known to affect both ATP generation and consumption, likely drive cytosolic ATP/ADP oscillations that in turn regulate KATP channels and membrane potential. Thus, it remains to be demonstrated that a new model is required to replace the existing, mitochondrial bioenergetics-based model.

Isolation and Characterization of Heavy Metal-Tolerant Bacterial Isolates from Industrial Effluents in Uttar Pradesh, India

Industrial activities over the past century have significantly increased human exposure to pollutants such as heavy metals. Industrial emissions of heavy metals, which are carcinogenic, mutagenic and toxic, contaminate natural water supplies and the agricultural environment. Due to the high concentration of heavy metals in industrial effluents, the bacteria present there naturally develop resistance to heavy metals. The aim of this study is to isolate and characterize bacteria resistant to heavy metal, lead (Pb), copper (Cu), chromium (Cr), cadmium (Cd), and nickel (Ni) from sites contaminated by industrial effluents in Uttar Pradesh, India. A total of 58 bacterial isolates were isolated from 9 samples according to their various morphological parameters and 32 isolates were found positive for all heavy metals, which revealed that samples contained metal-resistant bacterial diversity. Two isolates were identified up to species level based on their physiological, biochemical and molecular characterization as Comamonas testosteroni (S4C1) and Bacillus cereus (S5C3). Both isolates are highly resistant to lead (Pb), copper (Cu), chromium (Cr), cadmium (Cd), and nickel (Ni) and they show different MICs against the above heavy metals at different levels. A growth experiment showed that the presence of heavy metals concentration had no discernible impact on the growth rate among the isolates. Gel analysis showed interesting patterns of protein expression were observed in the presence of various heavy metals. MALDI-TOF analysis found that specific proteins (S layer protein, F0F1ATP synthase subunit b, Flagellin, 50S ribosomal protein L4, Molecular chaperone) were overexpressed in the presence of heavy metals. As a result, identifying the heavy metal resistance bacteria and their proteins study could be useful as a preliminary investigation for the development of prospective bioremediation agents of potentially hazardous waste treatment technology.

The branched mitochondrial respiratory chain from the jellyfish Stomolophus sp2 as a probable adaptive response to environmental changes.

During their long evolutionary history, jellyfish have faced changes in multiple environmental factors, to which they may selectively fix adaptations, allowing some species to survive and inhabit diverse environments. Previous findings have confirmed the jellyfish's ability to synthesize large ATP amounts, mainly produced by mitochondria, in response to environmental challenges. This study characterized the respiratory chain from the mitochondria of the jellyfish Stomolophus sp2 (previously misidentified as Stomolophus meleagris). The in-gel activity from isolated jellyfish mitochondria confirmed that the mitochondrial respiratory chain contains the four canonical complexes I to IV and F0F1-ATP synthase. Specific additional activity bands, immunodetection, and mass spectrometry identification confirmed the occurrence of four alternative enzymes integrated into a branched mitochondrial respiratory chain of Stomolophus sp2: an alternative oxidase and three dehydrogenases (two NADH type II enzymes and a mitochondrial glycerol-3-phosphate dehydrogenase). The analysis of each transcript sequence, their phylogenetic relationships, and each protein's predicted models confirmed the mitochondrial alternative enzymes' identity and specific characteristics. Although no statistical differences were found among the mean values of transcript abundance of each enzyme in the transcriptomes of jellyfish exposed to three different temperatures, it was confirmed that each gene was expressed at all tested conditions. These first-time reported enzymes in cnidarians suggest the adaptative ability of jellyfish's mitochondria to display rapid metabolic responses, as previously described, to maintain energetic homeostasis and face temperature variations due to climate change.

Human Prune Regulates the Metabolism of Mammalian Inorganic Polyphosphate and Bioenergetics.

Inorganic polyphosphate (polyP) is an evolutionarily conserved and ubiquitous polymer that is present in all studied organisms. PolyP consists of orthophosphates (Pi) linked together by phosphoanhydride bonds. The metabolism of polyP still remains poorly understood in higher eukaryotes. Currently, only F0F1-ATP synthase, Nudt3, and Prune have been proposed to be involved in this metabolism, although their exact roles and regulation in the context of polyP biology have not been fully elucidated. In the case of Prune, in vitro studies have shown that it exhibits exopolyphosphatase activity on very short-chain polyP (up to four units of Pi), in addition to its known cAMP phosphodiesterase (PDE) activity. Here, we expand upon studies regarding the effects of human Prune (h-Prune) on polyP metabolism. Our data show that recombinant h-Prune is unable to hydrolyze short (13-33 Pi) and medium (45-160 Pi) chains of polyP, which are the most common chain lengths of the polymer in mammalian cells. Moreover, we found that the knockdown of h-Prune (h-Prune KD) results in significantly decreased levels of polyP in HEK293 cells. Likewise, a reduction in the levels of polyP is also observed in Drosophila melanogaster loss-of-function mutants of the h-Prune ortholog. Furthermore, while the activity of ATP synthase, and the levels of ATP, are decreased in h-Prune KD HEK293 cells, the expression of ATP5A, which is a main component of the catalytic subunit of ATP synthase, is upregulated in the same cells, likely as a compensatory mechanism. Our results also show that the effects of h-Prune on mitochondrial bioenergetics are not a result of a loss of mitochondrial membrane potential or of significant changes in mitochondrial biomass. Overall, our work corroborates the role of polyP in mitochondrial bioenergetics. It also demonstrates a conserved effect of h-Prune on the metabolism of short- and medium-chain polyP (which are the predominant chain lengths found in mammalian cells). The effects of Prune in polyP are most likely exerted via the regulation of the activity of ATP synthase. Our findings pave the way for modifying the levels of polyP in mammalian cells, which could have pharmacological implications in many diseases where dysregulated bioenergetics has been demonstrated.

Metabolic profile and physiological mechanisms underlying the promoting effects of TiO2NPs on the photosynthesis and growth of tomato

Titanium dioxide nanoparticles (TiO2NPs) are photocatalysts that can boost crop photosynthesis. However, the effects of TiO2NPs on photosynthesis performance in tomato plants as well as the underlying mechanisms are unknown. In this research, the effects of synthesized TiO2NPs on growth, photosynthetic performance and the mechanism underlying enhanced photosynthesis performance were assessed in tomato plants. The results showed that the positive effect of TiO2NPs on growth is concentration-dependent, with 0.5 g L−1 of TiO2NPs stimulated the maximum growth in tomato plants. However, TiO2NPs negatively influenced the growth of tomato plants when it was applied at ≥1 g L−1. At 0.5 g L−1 of TiO2NPs, agronomic traits such as plant height, stem diameter and root system were significantly promoted in the treated plants compared to the other concentrations. In addition, the photosynthetic performance of tomato plants was significantly promoted when they were treated with 0.5 g L−1 of TiO2NPs. Further investigation revealed that TiO2NPs significantly increased the net photosynthetic efficiency, the activity of Rubisco and chloroplast F1F0-ATP synthase, and the content of NADPH in the chloroplast. The results also showed that this photocatalyst positively improved stomatal conductance, transpiration rate, and water use efficiency in the treated plants when applied at 0.5 g L−1. In addition, TiO2NPs significantly increased the electron transport rate, actual quantum yield, and photochemical quenching coefficient, while it reduced the non-photochemical quenching parameter and non-photochemical quenching coefficient when applied at 0.5 g L−1. Taken together, the results of this study revealed that TiO2NPs can improve the growth of tomato plants by enhancing photosynthesis rate and improving photosynthetic parameters.

Epileptiform Discharges Reduce Neuronal ATP Production by Inhibiting F0F1-ATP Synthase Activity via A Zinc-α2-Glycoprotein-Dependent Mechanism.

Neuronal energy metabolism dysfunction, especially adenosine triphosphate (ATP) supply decrease, is observed in epilepsy and associated with epileptogenesis and prognosis. Zinc-α2-glycoprotein (ZAG) is known as an important modulator of energy metabolism and involved in neuronal glucose metabolism, fatty acid metabolism, and ketogenesis impairment in seizures, but its effect on neuronal ATP synthesis in seizures and the specific mechanism are unclear. In this study, we verified the localization of ZAG in primary cultured neuronal mitochondria by using double-labeling immunofluorescence, immune electron microscopy, and western blot. ZAG level in neuronal mitochondria was modulated by lentiviruses and detected by western blot. The F0F1-ATP synthase activity, ATP level, and acetyl-CoA level were measured. The binding between ZAG and F0F1-ATP synthase was determined by coimmunoprecipitation. We found that both ZAG and F0F1-ATP synthase existed in neuronal mitochondria, and there was mutual binding between them. Epileptiform discharge-induced decrease of mitochondrial ZAG level was reversed by ZAG overexpression. Epileptiform discharge or ZAG knockdown decreased F0F1-ATP synthase activity and ATP level in neurons, which were reversed by ZAG overexpression, while overexpression of ZAG along only increased F0F1-ATP synthase activity but not increased ATP level. Meanwhile, neither epileptiform discharges nor changes of ZAG level can alter the acetyl-CoA level. Moreover, epileptiform discharge did not alter F0F1-ATP synthase level. In conclusion, epileptiform discharge-induced ZAG decrease in neuronal mitochondria is correlated to F0F1-ATP synthase activity inhibition, which may possibly lead to ATP supply impairments. ZAG may be a potential therapeutic target for treating neuronal energy metabolism dysfunction in seizures with further researches.

Alterations of Mitochondrial Respiratory Function in Isolated Nephron Segments of Dahl Salt-Sensitive Rats in Salt-Induced Hypertension

Rationale: Mammalian kidneys actively reabsorb nearly 99% of the glomerular filtrate to maintain body fluid and solute homeostasis which requires a great amount of ATP generation and O2 consumption to meet these demands. A high salt diet increases glomerular filtration of Na+ and places a high metabolic demand on the renal tubules to reabsorb more Na+in order for the organism to maintain Na+ balance. Dahl salt-sensitive (SS) rats which mimic the human condition become hypertensive when fed a high salt diet. They are also known to reabsorb greater amounts of Na+ in the proximal tubules (PT) and medullary thick ascending limbs (mTAL) thereby requiring greater ATP production and O2consumption. We hypothesized that the mitochondrial respiratory function of the PT and mTAL would be altered when SS rats are fed a high salt diet in ways that reduce the ability of the kidney to function efficiency thereby leading to salt-induced hypertension. Method: Agilent Seahorse XF96 Extracellular Flux Analyzer was used to assess mitochondrial respiratory function in freshly bulk isolated PT and mTAL from age-matched SS rats during the development of salt-induced hypertension fed with 0.4% NaCl diet (low salt; LS) or 4.0% NaCl diet (high salt; HS) for 7, 14, and 21 days. The Seahorse “Cell Mito Stress Test Protocol” was slightly modified with a substrate addition step to assess the segment-specific and substrate-dependent tubular O2 consumption rate (OCR) under different perturbations, which was used to determine alterations in different mitochondrial respiratory parameters, including H+ leak and ATP production dependent OCR, in the PT and mTAL during the development of salt-induced hypertension. Results: Renal tubular OCR data show that PT of SS rats preferred to use lactate, glutamine, and palmitate, while mTAL preferred to use glucose, pyruvate, and lactate for ATP production under LS diet condition. HS diet resulted in significantly reduced substrate-induced OCR in PT but increased OCR in mTAL. In addition, HS induced a higher level of OCR in the baseline as well as when oligomycin (an inhibitor of F1F0-ATP synthase) was added to both PT and mTAL suspensions indicating that an increased proton leakage/uncoupling of mitochondria occurs in SS rats during the development of salt-induced hypertension. Conclusion: Our emergent data demonstrated that the mitochondrial respiratory function is differentially altered in the PT and mTAL of SS rats in response to different circulatory substrates during the development of salt-induced hypertension. These changes in metabolism appear to be necessary to meet the increased tubular workloads and need for greater ATP production and O2 utilization in both the PT and mTAL of SS rats. NIH R01-HL151587. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Alpha Subunit of Mitochondrial Processing Peptidase Participated in Fertility Restoration in Honglian-CMS Rice

The cytoplasmic male sterility (CMS) and nuclear-controlled fertility restoration system is a favorable tool for the utilization of heterosis in plant hybrid breeding. Many restorer-of-fertility (Rf) genes have been characterized in various species over the decades, but more detailed work is needed to investigate the fertility restoration mechanism. Here, we identified an alpha subunit of mitochondrial processing peptidase (MPPA) that is involved in the fertility restoration process in Honglian-CMS rice. MPPA is a mitochondrial localized protein and interacted with the RF6 protein encoded by the Rf6. MPPA indirectly interacted with hexokinase 6, namely another partner of RF6, to form a protein complex with the same molecular weight as the mitochondrial F1F0-ATP synthase in processing the CMS transcript. Loss-of-function of MPPA resulted in a defect in pollen fertility, the mppa+/- heterozygotes showed semi-sterility phenotype and the accumulation of CMS-associated protein ORFH79, showing restrained processing of the CMS-associated atp6-OrfH79 in the mutant plant. Taken together, these results threw new light on the process of fertility restoration by investigating the RF6 fertility restoration complex. They also reveal the connections between signal peptide cleavage and the fertility restoration process in Honglian-CMS rice.

Prune regulates the metabolism of mammalian inorganic polyphosphates and bioenergetics.

Inorganic polyphosphate (polyP) is an evolutionarily conserved, ubiquitous polymer that is present in all studied organisms. It has shown a preferred localization within the mitochondria and it is composed of multiple orthophosphates (Pi) linked together by high energy phosphoanhydride bonds, identical to those found in ATP. The metabolism of polyP is well described in bacteria and yeast, but has yet to be elucidated in higher eukaryotes. However, recent evidence suggests that the mammalian F0F1 ATP synthase could be involved in both the synthesis and hydrolysis of polyP. The mitochondrial localized protein h-Prune, the human ortholog of Drosophila Prune, exhibits short chain exopolyphosphatase activity in vitro, in addition to its known cAMP phosphodiesterase activity. Here, we studied the role of prune orthologs in polyP metabolism in both mammalian HEK293 cells and Drosophila melanogaster. Our data shows that purified h-Prune is unable to hydrolyze polyP with chain lengths spanning from 13-33 Pi long. We then used interfering RNA to knock down the expression of prune orthologs and found that the polyP levels were significantly reduced in both HEK293 and Drosophila melanogaster. In HEK293 cells, ATP levels were also significantly decreased by h-Prune knockdown, although mitochondrial membrane potential was not affected. This suggests that h-Prune could either directly or indirectly influence the activity of the F0F1 ATP synthase. Concordantly, we found that h-Prune knockdown significantly decreased ATP synthase activity to levels comparable to inhibition by Oligomycin A. Our results collectively demonstrate the downstream ability of prune to regulate cellular polyP levels and bioenergetics, although further studies are required to elucidate the exact mechanism of this pathway.

Molecular mechanism of reversed temperature-dependence of ATP synthesis in glacier ice worms.

The F0F1 ATP synthase enzyme is highly conserved across species. The F0F1 is a reversible motor, where the clockwise rotation of the rotor portion of F0 (known as the c-ring and present in the membrane embedded F0 domain) produces ATP and the counter-clockwise rotation induces ATP hydrolysis. In a surprising contrast with temperate organisms, glacier ice worms display elevated ATP levels as temperatures decline. The increased energy expenditure is used as a strategy for survival at cold temperatures, but the mechanism is unknown. To investigate the underlying mechanism of elevated ATP levels in glacier ice worms, we investigated the effects of temperature on the dynamics of the F0F1 ATP synthase of glacier ice worms compared to their temperate counterparts. More specifically, an ice worm-specific, 18 amino acid extension with regularly spaced histidine residues was previously found to be fused to the carboxy terminal of the ATP6 subunit generating a proton shuttling domain projecting away from the F0 exit pore. The role of this C-terminal extension in the temperature dependance ATP synthesis in ice worms is undetermined. We conducted all-atomistic MD simulations of the F0 domain subunits at different temperatures and used earthworms as a control to evaluate the effect of sequence on temperature dependence. We also measured the rotational diffusion of the c-ring as a function of temperature to evaluate the change in the rotation angles of the c-ring around the z axis in clock- and counter-clockwise directions. The ATP6 subunit is attached to the c-ring at the proton exit site. This suggests that the change in the c-ring rotational diffusion, as a function of temperature, can affect the ATP6 conformation, which likely affects the rate of ATP production.

Impact of engineering the ATP synthase rotor ring on photosynthesis in tobacco chloroplasts.

The chloroplast ATP synthase produces the ATP needed for photosynthesis and plant growth. The trans-membrane flow of protons through the ATP synthase rotates an oligomeric assembly of c subunits, the c-ring. The ion-to-ATP ratio in rotary F1F0-ATP synthases is defined by the number of c-subunits in the rotor c-ring. Engineering the c-ring stoichiometry is, therefore, a possible route to manipulate ATP synthesis by the ATP synthase and hence photosynthetic efficiency in plants. Here, we describe the construction of a tobacco (Nicotiana tabacum) chloroplast atpH (chloroplastic ATP synthase subunit c gene) mutant in which the c-ring stoichiometry was increased from 14 to 15 c-subunits. Although the abundance of the ATP synthase was decreased to 25% of wild-type (WT) levels, the mutant lines grew as well as WT plants and photosynthetic electron transport remained unaffected. To synthesize the necessary ATP for growth, we found that the contribution of the membrane potential to the proton motive force was enhanced to ensure a higher proton flux via the c15-ring without unwanted low pH-induced feedback inhibition of electron transport. Our work opens avenues to manipulate plant ion-to-ATP ratios with potentially beneficial consequences for photosynthesis.

OXPHOS deficiency induces mitochondrial DNA synthesis through non-canonical AMPK-dependent mRNA compartmentalization

Eukaryotic cells contain multiple copies of mitochondrial DNA (mtDNA) in discrete organelles or as tubular networks throughout the cytoplasm. The mtDNA copy number is dynamically regulated by mitochondrial biogenesis and mitophagy processes. However, the conditions regulating mtDNA replication, an essential component of biogenesis, are unknown. We observed that short-term (2 h) treatment of rat myoblasts with oligomycin, a specific inhibitor of the mitochondrial F1F0 ATP synthase, resulted in stimulation of mtDNA synthesis from the OH replication origin. This effect was abrogated by Compound C, an antagonist of the AMP-dependent protein kinase (AMPK), a universal intracellular energy sensor, and in AMPK-knockdown cells, indicating that mtDNA replication is regulated by AMPK under oxidative phosphorylation (OXPHOS)- deficient conditions. Using antibody decoration, enzymatically active AMPK, phosphorylated at T172 of the α1 subunit, was found to be located on the mitochondrial surface. Furthermore, oligomycin induced the compartmentalization of several mRNAs encoding OXPHOS components and mtDNA replication factors to mitochondria. Compartmentalization of mRNAs was inhibited by Compound C. We infer that AMPK is locally activated by inhibition of the F1F0 ATP synthase to stimulate association of mtDNA replication factor mRNAs, leading to stimulation of mtDNA synthesis. The findings have implications for the clonal expansion of OXPHOS-deficient mtDNA mutant mitochondria in human patients, with clinical consequences.

Type-B Energetic Processes and Their Associated Scientific Implications

Recently, our work has identified two thermodynamically distinct types (A and B) of energetic processes naturally occurring on Earth: Type-A energy processes such as the classical heat engines, ATP hydrolysis, and many of the known chemical, electrical, and mechanical processes apparently well follow the second law of thermodynamics; and Type-B energy processes, such as the newly discovered thermotrophic function that isothermally utilizes environmental heat energy to do useful work in driving ATP synthesis, which follows the first law of thermodynamics (conservation of mass and energy), but does not necessarily have to be constrained by the second law, owing to their special asymmetric functions. In mitochondria, their special asymmetric functions associated with Type-B processes comprise: 1) The transmembrane asymmetry of inner mitochondrial membrane structure with the protonic outlets of redox-driven proton-pumping protein complexes protruded away from the membrane surface by about 1-3 nm into the bulk liquid p-phase while the protonic inlet of the F0F1-ATP synthase located rightly at the transmembrane electrostatically localized proton (TELP) layer; and 2) The lateral asymmetry of mitochondrial cristae with an ellipsoidal shape that enhances the density of TELP at the cristae tips where the F0F1-ATP synthase enzymes are located in supporting the TELP-associated thermotrophic function. The identification of Type-B energy processes indicates that there is an entirely new world of physical and energy sciences yet to be fully uncovered. Innovative efforts on Type-B processes to enable isothermally utilizing endless environmental heat energy could help to liberate all peoples from their dependence of fossil fuel energy, thus helping to reduce greenhouse gas CO2 emissions and control climate change toward a sustainable future for the humanity on Earth.

BNIP3 regulates cardiac energy metabolism by interaction with F1FO ATP-synthase

Abstract Background The oxidative phosphorylation (OXPHOS) that takes place in the mitochondria produces chemical energy in form of ATP, the main energy source of the heart. Proper mitochondrial function determines the contractility of the heart. Changes in myocardial ATP levels are often associated with cardiovascular disease. However, mitochondrial dysfunction remains an unmet therapeutic challenge. The F1FO ATP-synthase protein complex catalyses the last step of OXPHOS, synthesising ATP and determining the respiratory function of cardiac mitochondria. An increased ATP pool during cardiovascular challenges, regulated by F1FO ATP-synthase, could therefore significantly improve the clinical outcome of affected patients. In neurons, Bcl2-familiy members like BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) have been suggested to be involved in the regulation of metabolic efficiency through interaction with the mitochondrial F1FO ATP synthase. A similar metabolic regulation in the cardiac context remains unclear. Furthermore, BNIP3 has a significant impact on the loss of cardiomyocytes under various pathological conditions and is thus a potential mediator of energy metabolism in cardiomyocytes. Methods and results With native gel-electrophoresis, cardiac BNIP3 of C57BL/6J mice was first identified in higher oligomeric complexes in similar molecular weight range as mitochondrial F1FO ATP-synthase. Using 60Å-precise proximity-ligation assays to determine possible binding partners, F1FO ATP-synthase was uncovered for the first time as an interacting protein of BNIP3. This interaction was confirmed by co-immunoprecipitation of BNIP3 and F1FO ATP-synthase and by spatial localization using electron microscopy. Peptide microarray studies elucidate the f subunit of F1FO ATP-synthase as the structural interaction site. Functional analysis using extracellular flux analyser technology revealed significantly elevated mitochondrial respiratory activity in Bnip3 knock out mice and human cardiomyocytes during BNIP3 inhibition. Increased ATP levels in C57BL/6J mice after acute inhibition of BNIP3 confirmed the observed regulatory effect of BNIP3 on F1FO ATP-synthase activity. Finally, depletion of BNIP3 in mice improves stress resistance with an augmented chronotropic capacity during dobutamine-induced cardiac stress. Conclusion Depletion of BNIP3 suggests a regulatory role of BNIP3 in cardiac mitochondrial energy homeostasis via interaction with F1F0 ATP-synthase in oligomeric complexes. Since acute inhibition of BNIP3 activity allows positive modulation of cardiac performance by elevating the available ATP pool, this may serve as a beneficial treatment for patients with cardiovascular disease in the future. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): German research foundation (DFG)

An In silico Study of Imidazo[1,2-a]pyridine Derivatives with Homology Modelled F1F0 ATP Synthase Against Mycobacterium Tuberculosis

Aim: To ascertain the binding manner and drug-likeliness profile of imidazo[1,2-a]pyridine derivatives as antitubercular agents on ATP synthase protein. Background: : Field-based 3D-QSAR, Homology modelling, Molecular Docking and ADME-T studies have been carried out to determine the binding mode and drug likeliness profile of imidazo[1,2-a]pyridine derivatives as anti-tubercular agents. Objective: To design new anti-tubercular agents using Field- based 3D-QSAR and molecular docking approach. Methods: A statistically significant 3D-QSAR model was generated with the dataset of 30 active agonists on ATP synthase whose pIC50 values range from 4.0 µM to 8.30 µM. The same dataset was analysed for ADME-T properties and docked to the homology modeled ATP synthase protein. Moreover, information from 3D-QSAR contour maps was used in designing of new molecules. Results: The constructed 3D-QSAR model had a high correlation coefficient (R2=0.9688) and cross-validation coefficient (Q2=0.9045) and F value (176) at 3 component PLS factor. The homology modeled protein ‘ac9’ was validated with various parameters like Ramachandran plot (92.5 %), ERRAT plot (98.43 %), and ProSA (-1.78 chain ‘C’; -2.74 chain ‘A’). The protein was also examined for physico-chemical properties which showed the acidic and hydrophobic nature of protein. The docking score of dataset compound no. PF19 (-9.97 Kcal/mol) was found to be almost similar with that of Bedaquiline (-10.08 Kcal/mol). Based on previous results from 3D-QSAR and Docking, four new molecules was designed. The newly designed molecules (M1-M4) were docked amongst them M3 (-9.82 Kcal/mol) scored the highest. They were further analysed for drug-likeliness, ADME-T and synthetic assesbility. The findings suggested that these compounds had a strong possibility to become ATP-synthase inhibitors. Conclusion: The various Insilico approaches used in the present study offer new avenues for designing novel molecules against ATP synthase from M. tuberculosis and can be employed for the drug discovery programme.

Complete mitochondrial genome sequencing and identification of candidate genes responsible for C5-type cytoplasmic male sterility in cabbage (B. oleracea var. capitata).

Cytoplasmic male sterility (CMS) is widely used in cruciferous vegetables hybrid breeding. The C5-type CMS cabbage line exhibits stable male sterility and offers great value for cabbage breeding. However, the underlying CMS mechanism remains unclear. Here, the complete mitochondrial genome was sequenced and assembled for this line. The genome size was 221,862 bp. Mitochondrial genome comparison showed that the mitochondrial genome was likely generated by recombination with a nap-type CMS B. napus strain. Sixty-seven unknown-function open reading frames (ORFs) were identified. Seven orfs, orf114a, orf123a, orf188a, orf222a, orf261a, orf286a, and orf322a, were specifically identified in this genome. The presence of these candidate CMS genes decreased ATPase activity and ATP content by affecting the transcript levels of energy metabolism-related genes and F1F0-ATP synthase assembly. Among them, orf188a, orf222a, orf261a, orf286a, and orf322a possessed a transmembrane structure, and orf188a was cotranscribed with rps7 and trnfM. orf222a was partially homologous to atp8 and coexpressed with nad5. orf261a and orf322a were cotranscribed with cox1 and atp9, respectively. Additionally, orf114a was cotranscribed with atp8. Yeast two-hybrid assays showed that the ORF222a protein interacts with a B. oleracea ATP17 homolog (Bo7g114140) during F0-type ATP synthase assembly, reducing the quantity and activity of assembled F1F0-ATP synthase. Cytological sections showed that premature separation of the tapetum from the connective tissue and delayed tapetal programmed cell death (PCD) might be the immediate causes of CMS in C5-type CMS cabbage lines. Our results may help uncover the molecular mechanism of C5-type CMS in B. oleracea from the perspectives of the whole mitochondrial genome and cytology of anther development.

Computational Model of the Effect of Mitochondrial Dysfunction on Excitation–Contraction Coupling in Skeletal Muscle

It has become well established that mitochondria not only regulate myoplasmic calcium in skeletal muscle, but also use that calcium to stimulate oxidative phosphorylation (OXPHOS). While experimental approaches have allowed for imaging of mitochondrial calcium and membrane potentials in isolated fibers, capturing the role of mitochondria and the impact of mitochondrial impairments on excitation–contraction coupling (ECC) remains difficult to explore in intact muscle. Computational models have been widely used to examine the structure and function of skeletal muscle contraction; however, models of ECC to date lack communication between the myoplasm and mitochondria for regulating calcium and ATP during sustained contractions. To address this, a mathematical model of mitochondrial calcium handling and OXPHOS was integrated into a physiological model of ECC incorporating action potential propagation, calcium handling between the sarcoplasmic reticulum (SR) and the myoplasm, and crossbridge cycling. The model was used to examine the protective role of mitochondria during repeated stimulation and the impact of mitochondrial dysfunction on ECC resulting from progressive OXPHOS inhibition. Pathological myoplasmic calcium accumulation occurred through distinct mechanisms in the model in the case of either electron transport chain, F1F0 ATP synthase, or adenine nucleotide transporter impairments. To investigate the effect of each impairment on force, a model of calcium-stimulated apoptosis was utilized to capture dysfunction-induced reductions in muscle mass, driving whole muscle force loss. The model presented in this study can be used to examine the role of mitochondria in the regulation of calcium, ATP, and force generation during voluntary contraction.

Mitochondria – More than just batteries for cellular function

Mitochondria are complex small organelles of eukaryotic cells and build the cellular source of energy. Several morphological features of mitochondria such as the double membrane and the circular DNA structure support the thesis that they originated from a prokaryotic eubacterial ancestor that has been taken up by the eukaryotic cell very early during the eukaryotic evolution. Since this "uptake-event" mitochondria were integrated into cellular processes and regulation which was realized by the transfer of mitochondrial genes into the host cell genome. 1 The mitochondrial genome reduced to for instance 13 encoded protein subunits of the oxidative phosphorylation system in human cells. Mitochondria offer energy for the cell by producing about 95% of cellular ATP.2 Nutrients, mainly pyruvate from the glycolysis enter the tricarboxylic acid cycle and undergo iterative oxidations whereas electrons are transferred to the reduction equivalents NADH and FADH2 . These redox equivalents transport electrons to the electron transport chain located on the inner mitochondrial membrane and protons are pumped into the perimembranal room. The F1 F0 -ATP synthase generates ATP driven by protons flowing down an electrochemical gradient during a process named oxidative phosphorylation. As a byproduct reactive oxygen species are generated. Mitochondria are more than simple batteries for the cell, they are furthermore involved in numerous vital cellular processes, among them are calcium homeostasis, cell death, fatty acid oxidation, reactive oxygen species (ROS) signaling, cholesterol synthesis and nucleotide synthesis, topics that are frequently published in Acta Physiologica.

Inorganic Polyphosphate and F0F1-ATP Synthase of Mammalian Mitochondria.

Inorganic polyphosphate is a polymer which plays multiple important roles in yeast and bacteria. In higher organisms the role of polyP has been intensively studied in last decades and involvements of this polymer in signal transduction, cell death mechanisms, energy production, and many other processes were demonstrated. In contrast to yeast and bacteria, where enzymes responsible for synthesis and hydrolysis of polyP were identified, in mammalian cells polyP clearly plays important role in physiology and pathology but enzymes responsible for synthesis of polyP or consumption of this polymer are still not identified. Here, we discuss the role of mitochondrial F0F1-ATP synthase in polyP synthesis with results, which confirm this proposal. We also discuss the role of other enzymes which may play important roles in polyP metabolism.