Search

Many kinds of physical fields are able to affect biological activity, such as electromagnetic field, magnetic field, scalar wave, magnetic vector potential and so on. Some works investigated a high-penetrating emission (HPE) generated by a LED generator that can affect the conductivity of water and the growth of dry yeast. In this research, the biological effect of the HPE of LED generator on S. cerevisiae was studied. The results showed that this kind of HPE could promote the growth of S. cerevisiae. With the decrease of initial concentration and the extension of exposure time, the growth promoting effect of HPE increased significantly. When the initial cell concentration was 2 × 102 cell/ml and exposed for 24 h, the number of yeast cells increased by 19.5% compared with the control group. Further studies showed that adenosine triphosphate (ATP) content and the ratio of ATP and adenosine diphosphate (ADP, ATP/ADP ratio) of exposed cells were higher than those of unexposed cells, indicating that exposed cells had higher cell viability and energy status. In addition, the intracellular superoxide (O2−) content decreased significantly after the HPE exposed. In this study, the stainless steel box acts as a Faraday cage to shield the electromagnetic field, so the biological effect of the HPE is not produced by the electromagnetic field. Nevertheless, the mechanism of the HPE is still unknown and needs further research. This is the first report on the effect of HPE on mitochondrial parameters, contributing to further study on HPE and revealing potential for future medical applications.

Background ADP/ATP translocase 1 (ANT1) is involved in the exchange of cytosolic ADP and mitochondrial ATP, and its defection plays an important role in mitochondrial pathogenesis. To reveal an etiological implication of ANT1 for Parkinson’s disease (PD), a neurodegenerative disorder, a mouse model treated with 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine and neuroblastoma cell model induced by 1-methyl-4-pehny1-pyridine were utilized in this study. Results The tissue-specific abundance in ANT1 in mouse brains was accessed using the analysis of Western blot and immunohistochemistry. Down-regulated soluble ANT1 was found to be correlated with PD, and ANT1 was associated with PD pathogenesis via forming protein aggregates with α-synuclein. This finding was confirmed at cellular level using neuroblastoma cell models. ANT1 supplement in neuronal cells revealed the protective roles of ANT1 against cytotoxicity caused by MPP+. Protein interaction assay, coupled with the analysis of LC-MS/MS, silver-stained SDS-PAGE and Western blot against anti-ANT1 antibody respectively, illustrated the interaction of ANT1 with α-synuclein using the expressed α-synuclein as a bite. Additionally, a significant increasing ROSs was detected in the MPP+-treated cells. Conclusions This study indicated that ANT1 was a potentially causative factor of PD, and led to neuropathogenic injury via promoting the formation of protein aggregates with α-synuclein. This investigation potentially promotes an innovative understanding of ANT1 on the etiology of PD and provides valuable information on developing potential drug targets in PD treatment or reliable biomarkers in PD prognostication.

Mitochondrial ADP/ATP carriers import ADP into the mitochondrial matrix and export ATP to the cytosol to fuel cellular processes. Structures of the inhibited cytoplasmic- and matrix-open states have confirmed an alternating access transport mechanism, but the molecular details of substrate binding remain unresolved. Here, we evaluate the role of the solvent-exposed residues of the translocation pathway in the process of substrate binding. We identify the main binding site, comprising three positively charged and a set of aliphatic and aromatic residues, which bind ADP and ATP in both states. Additionally, there are two pairs of asparagine/arginine residues on opposite sides of this site that are involved in substrate binding in a state-dependent manner. Thus, the substrates are directed through a series of binding poses, inducing the conformational changes of the carrier that lead to their translocation. The properties of this site explain the electrogenic and reversible nature of adenine nucleotide transport.

The methanolic extract of the leaves of artichoke (Cynara scolymus L.) was found to inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Among the constituents of the extract, six sesquiterpene lactones (cynaropicrin, grosheimin, 11β,13-dihydrocynaropicrin, 3β-hydroxy-8α-[(S)-3-hydroxy-2-methylpropionyloxy]guaia-4(15),10(14),11(13)-trien-1α,5α,6βH-12,6-olide, 3β-hydroxy-8α-[2-methoxymethyl-2-propenoyloxy]guaia-4(15),10(14),11(13)-trien-1α,5α,6βH-12,6-olide, and deacylcynaropicrin) inhibited NO production and/or inducible nitric oxide synthase (iNOS) induction. The acyl group having an α,β-unsaturated carbonyl group at the 8-position and the α-methylene-γ-butyrolactone moiety were important for the strong inhibitory activity. Our results suggested that these sesquiterpene lactones inhibited the LPS-induced iNOS expression via the suppression of the JAK-STAT signaling pathway in addition to the κNF-κB signaling pathway. With regard to the target molecules of the sesquiterpene lactones, high-affinity proteins of cynaropicrin were purified from the cell extract. ATP/ADP translocase 2 and tubulin were identified and suggested to be involved in the cytotoxic effects of cynaropicrin, although the target molecules for the inhibition of iNOS expression were not clarified.

For more than 40 years, the oligomeric state of members of the mitochondrial carrier family (SLC25) has been the subject of debate. Initially, the consensus was that they were dimeric, based on the application of a large number of different techniques. However, the structures of the mitochondrial ADP/ATP carrier, a member of the family, clearly demonstrated that its structural fold is monomeric, lacking a conserved dimerisation interface. A re-evaluation of previously published data, with the advantage of hindsight, concluded that technical errors were at the basis of the earlier dimer claims. Here, we revisit this topic, as new claims for the existence of dimers of the bovine ADP/ATP carrier have emerged using native mass spectrometry of mitochondrial membrane vesicles. However, the measured mass does not agree with previously published values, and a large number of post-translational modifications are proposed to account for the difference. Contrarily, these modifications are not observed in electron density maps of the bovine carrier. If they were present, they would interfere with the structure and function of the carrier, including inhibitor and substrate binding. Furthermore, the reported mass does not account for three tightly bound cardiolipin molecules, which are consistently observed in other studies and are important stabilising factors for the transport mechanism. The monomeric carrier has all of the required properties for a functional transporter and undergoes large conformational changes that are incompatible with a stable dimerisation interface. Thus, our view that the native mitochondrial ADP/ATP carrier exists and functions as a monomer remains unaltered.

Effective functioning of the mitochondrial complexes of the oxidative phosphorylation (OXPHOS) system is necessary for ATP synthesis. The OXPHOS complexes exist both as individual forms and supercomplexes, whose formation and stability are supported by specific protein and lipid factors. In this paper, we report on the types and activities of OXPHOS complexes and supercomplexes from wheat (Triticum aestivum L.) root mitochondria analyzed by blue native polyacrylamide gel electrophoresis (BN-PAGE). The activity of OXPHOS complexes decreased when a mixture of rotenone, an inhibitor of complex I, and antimycin A, an inhibitor of complex III (R + AA) was applied to the BN-PAGE gels. By contrast, the types and activities of the OXPHOS complexes and supercomplexes did not change when they were isolated from the R + AA treated roots. However, the amount of the mitochondrial membrane-bound low molecular mass proteins in these roots markedly increased. The proteins were identified as ANT1 and ANT2 (ADP/ATP translocators) and ABA 8′-hydroxylase. We suggest that these low molecular mass proteins contribute to fine control mechanisms that stabilize mitochondrial supercomplexes and help to overcome an inhibitor-induced energy deficit by enhancing ADP/ATP transfer and ultimately improving the supply of ATP.

Biological oxidations do not take place by direct transfer of electrons (e − ) from substrate to oxygen. They are carried out in successive stages by different e − acceptors with increasing reduction potential. This allows for a stepwise release of energy and its best utilization by the cell. The respiratory chain , located in the mitochondrial inner membrane, comprises a series of H or electrons (e − ) acceptors arranged according to increasing reduction potential, associated with enzymes that catalyze e − transfer. It is composed of complex I or NADH–ubiquinone reductase, complex II or succinate–ubiquinone reductase, ubiquinone or coenzyme Q, complex III or ubiquinone–cytochrome c reductase, cytochrome c , located on the outer face of inner membrane, complex IV or cytochrome oxidase. Finally 4 e − are transferred to 2 O atoms, which with 4 H + form 2 H 2 O. The energy produced by the flow of e − is coupled to phosphoryl transfer, synthesizing adenosine triphosphate (ATP) from ADP in a process known as oxidative phosphorylation . Each e − pair from substrates of NAD-linked dehydrogenases generates three molecules of ATP, while substrates oxidized by FAD-dependent enzymes produce two ATP. The chemio-osmotic hypothesis explains the mechanism underlying oxidative phosphorylation. The energy generated by the flow of reducing equivalents is used to pump protons from the mitochondrial matrix outward into the inner membrane, at the site of complexes I, III, and IV. The proton gradient created across the mitochondrial inner membrane drives proton flux through the F 1 F 0 or ATP synthase complex , which couples proton transport to phosphate addition to ADP. Compounds that reduce or eliminate the proton gradient inhibit phosphorylation. Inhibitors can block e − transfer at different levels of the respiratory chain. Rotenone, amytal, and other barbiturates act at the level of complex I; antimycin A, at complex III; and cyanide, carbon monoxide, and azide, on complex IV. Inhibitors of oxidative phosphorylation include proton and K + ionophores, which suppress the mitochondrial electrical potential gradient, acting as uncoupling agents, and compounds, such as oligomycin, which interfere with the function of the F 1 F 0 ATPase. Brown fat present in infants and hibernating animals has thermogenin, a protein that inhibits ATP synthesis, uncoupling mitocondrial function and contributing to maintain body temperature. Oxidative phosphorylation is mainly controlled by ADP levels. Phosphorylation at substrate level is another way to generate ATP by phosphoryl transfer from high energy metabolites.

The intracellular pathogen Legionella pneumophila delivers more than 330 effectors into host cells by its Dot/Icm secretion system. Those effectors direct the biogenesis of the Legionella-containing vacuole (LCV) that permits its intracellular survival and replication. It has long been documented that the LCV is associated with mitochondria and a number of Dot/Icm effectors have been shown to target to this organelle. Yet, the biochemical function and host cell target of most of these effectors remain unknown. Here, we found that the Dot/Icm substrate Ceg3 (Lpg0080) is a mono-ADP-ribosyltransferase that localizes to the mitochondria in host cells where it attacks ADP/ATP translocases by ADP-ribosylation, and blunts their ADP/ATP exchange activity. The modification occurs on the second arginine residue in the -RRRMMM- element, which is conserved among all known ADP/ATP carriers from different organisms. Our results reveal modulation of host energy metabolism as a virulence mechanism for L. pneumophila.

The European corn borer Ostrinia nubilalis is a pest species, whose fifth instar larvae gradually develop cold hardiness during diapause. The physiological changes underlying diapause progression and cold hardiness development are still insufficiently understood in insects. Here, we follow a complex of changes related to energy metabolism during cold acclimation (5°C) of diapausing larvae and compare this to warm-acclimated (22°C) and non-diapause controls. Capillary electrophoresis of nucleotides and coenzymes has shown that in gradually cold-acclimated groups concentrations of ATP/ADP and, consequently, energy charge slowly decrease during diapause, while the concentration of AMP increases, especially in the first months of diapause. Also, the activity of cytochrome c oxidase (COX), as well as the concentrations of NAD+ and GMP, decline in cold-acclimated groups, until the latter part of diapause, when they recover. Relative expression of NADH dehydrogenase (nd1), coenzyme Q-cytochrome c reductase (uqcr), COX, ATP synthase (atp), ADP/ATP translocase (ant), and prohibitin 2 (phb2) is supressed in cold-acclimated larvae during the first months of diapause and gradually increases toward the termination of diapause. Contrary to this, NADP+ and UMP levels significantly increased in the first few months of diapause, after gradual cold acclimation, which is in connection with the biosynthesis of cryoprotective molecules, as well as regeneration of small antioxidants. Our findings evidence the existence of a cold-induced energy-saving program that facilitates long-term maintenance of larval diapause, as well as gradual development of cold hardiness. In contrast, warm acclimation induced faster depletion of ATP, ADP, UMP, NAD+, and NADP+, as well as higher activity of COX and generally higher expression of all energy-related genes in comparison to cold-acclimated larvae. Moreover, such unusually high metabolic activity, driven by high temperatures, lead to premature mortality in the warm-acclimated group after 2 months of diapause. Thus, our findings strongly support the importance of low temperature exposure in early diapause for gradual cold hardiness acquisition, successful maintenance of the resting state and return to active development. Moreover, they demonstrate potentially adverse effects of global climate changes and subsequent increase in winter temperatures on cold-adapted terrestrial organisms in temperate and subpolar regions.

The mitochondrial ADP/ATP carrier (AAC) performs the first and last step in oxidative phosphorylation by exchanging ADP and ATP across the mitochondrial inner membrane. Its optimal function has been shown to be dependent on cardiolipins (CLs), unique phospholipids located almost exclusively in the mitochondrial membrane. In addition, AAC exhibits an enthralling threefold pseudosymmetry, a unique feature of members of the SLC25 family. Recently, its conformation poised for binding of ATP was solved by x-ray crystallography referred to as the matrix state. Binding of the substrate leads to conformational changes that export of ATP to the mitochondrial intermembrane space. In this contribution, we investigate the influence of CLs on the structure, substrate-binding properties, and structural symmetry of the matrix state, employing microsecond-scale molecular dynamics simulations. Our findings demonstrate that CLs play a minor stabilizing role on the AAC structure. The interdomain salt bridges and hydrogen bonds forming the cytoplasmic network and tyrosine braces, which ensure the integrity of the global AAC scaffold, highly benefit from the presence of CLs. Under these conditions, the carrier is found to be organized in a more compact structure in its interior, as revealed by analyses of the electrostatic potential, measure of the AAC cavity aperture, and the substrate-binding assays. Introducing a convenient structure-based symmetry metric, we quantified the structural threefold pseudosymmetry of AAC, not only for the crystallographic structure, but also for conformational states of the carrier explored in the molecular dynamics simulations. Our results suggest that CLs moderately contribute to preserve the pseudosymmetric structure of AAC.

Specific import of ADP and export of ATP by ADP/ATP carrier (AAC) across the inner mitochondrial membrane are crucial for sustainable energy supply in all eukaryotes. However, mechanism for highly specific substrate recognition in the dynamic transport process remains largely elusive. Here, unguided MD simulations of 22 microseconds in total reveal that AAC in ground c-state uses the second basic patch (K91K95R187), tyrosine ladder (Y186Y190Y194), F191 and N115 in the upper region of the cavity to specifically recognize ADP and confer selectivity for ADP over ATP. Mutations of these residues in yeast AAC2 reduce ADP transport across the L. lactis membrane and induce defects in OXPHOS and ATP production in yeast. Sequence analyses also suggest that AAC and other adenine nucleotide transporters use the upper region of the cavity, rather than the central binding site to discriminate their substrates. Identification of the new site unveils the unusually high substrate specificity of AAC, and together with central binding site support early biochemical findings about existence of two substrate binding sites. Our results imply that using different sites for substrate recognition and conformational transition could be a smart strategy for transporters to cope with substrate recognition problem in the highly dynamic transport process.

Circadian clocks tick a rhythm with a nearly 24-hour period in various organisms. The clock proteins of cyanobacteria, KaiA, KaiB, and KaiC, compose a minimum circadian clock. The slow KaiB-KaiC complex formation, which is essential in determining the clock period, occurs when the C1 domain of KaiC binds ADP produced by ATP hydrolysis. KaiC is considered to promote this complex formation by inhibiting the backward process, ADP/ATP exchange, rather than activating the forward process, ATP hydrolysis. Remarkably, although inhibition of backward process, in general, decelerates the whole process, KaiC oppositely accelerates the complex formation. In this article, by building a novel reaction model, we investigate the molecular mechanism of the simultaneous promotion and acceleration of the complex formation, which may play a significant role in keeping the period invariant under environmental perturbations. Based on several experimental results, we assume in this model that six KaiB monomers cooperatively and rapidly binds to C1 with the stabilization of the binding-competent conformation of C1 only when C1 binds six ADP. We find the cooperative KaiB binding effectively separates the pre-binding process of C1 into a fast transformation to binding-competent C1 requiring multiple ATP hydrolyses and its slow backward transformation. Since the ADP/ATP exchange retards the forward process, its inhibition results in the acceleration of the complex formation. We also find that, in a simplified monomeric model where KaiB binds to a KaiC monomer independently of the other monomers, the ADP/ATP exchange inhibition cannot accelerate the complex formation. In summary, we conclude that the ring-shaped hexameric form of KaiC enables the acceleration of the complex formation induced by the backward process inhibition because the cooperative KaiB binding arises from the structure of KaiC.Author summaryCircadian clocks tick a rhythm with a nearly 24-hour period in various organisms. The cyanobacterial circadian clock has attracted much attention because of its simplicity, composed of only three proteins, KaiA, KaiB, and KaiC. The rate of the slow KaiB-KaiC complex formation, which plays an essential role in the period determination, is mainly regulated by the ATP hydrolysis (forward process) and the ADP/ATP exchange (backward process) of KaiC. KaiC promotes the complex formation by inhibiting the backward process rather than activating the forward process. Remarkably, although inhibition of backward process, in general, slows down the whole process, KaiC oppositely accelerates the complex formation. In this article, we investigate the molecular mechanism of this acceleration by building a novel mathematical model based on several significant experimental results. We find the cooperative binding of six KaiB to a KaiC hexamer, which arises from the ring-shaped hexameric structure of KaiC, effectively separates the pre-binding process into a fast transformation to the binding-competent KaiC requiring multiple ATP hydrolyses and its slow backward transformation. Since the ADP/ATP exchange retards the forward process, its inhibition results in the acceleration of the complex formation.

ATP/ADP depicts the bioenergetic state of Mycobacterium tuberculosis (Mtb). However, the metabolic state of Mtb during infection remains poorly defined due to the absence of appropriate tools. Perceval HR (PHR) was recently developed to measure intracellular ATP/ADP levels, but it cannot be employed in mycobacterial cells due to mycobacterial autofluorescence. Here, we reengineered the ATP/ADP sensor Perceval HR into PHR-mCherry to analyze ATP/ADP in fast- and slow-growing mycobacteria. ATP/ADP reporter strains were generated through the expression of PHR-mCherry. Using the Mtb reporter strain, we analyzed the changes in ATP/ADP levels in response to antimycobacterial agents. As expected, bedaquiline induced a decrease in ATP/ADP. Interestingly, the transcriptional inhibitor rifampicin led to the depletion of ATP/ADP levels, while the cell wall synthesis inhibitor isoniazid did not affect the ATP/ADP levels in Mtb. The usage of this probe revealed that Mtb faces depletion of ATP/ADP levels upon phagocytosis. Furthermore, we observed that the activation of macrophages with interferon gamma and lipopolysaccharides leads to metabolic stress in intracellular Mtb. Examination of the bioenergetics of mycobacteria residing in subvacuolar compartments of macrophages revealed that the bacilli residing in phagolysosomes and autophagosomes have significantly less ATP/ADP than the bacilli residing in phagosomes. These observations indicate that phagosomes represent a niche for metabolically active Mtb, while autophagosomes and phagolysosomes harbor metabolically quiescent bacilli. Interestingly, even in activated macrophages, Mtb residing in phagosomes remains metabolically active. We further observed that macrophage activation affects the metabolic state of intracellular Mtb through the trafficking of Mtb from phagosomes to autophagosomes and phagolysosomes. IMPORTANCE ATP/ADP levels guide bacterial cells, whether to replicate or to enter nonreplicating persistence. However, tools for measuring ATP/ADP levels with spatiotemporal resolution are lacking. Here, we describe a method for tracking ATP/ADP levels at the single-cell and population levels. Using this tool, we have demonstrated that the transcription inhibitor rifampicin induces metabolic stress. In contrast, the cell wall synthesis inhibitor isoniazid does not alter the metabolic state of the bacilli, suggesting that transcription is tightly intertwined with metabolism, while cell wall synthesis is not. Furthermore, we analyzed the metabolic state of mycobacteria residing in different compartments of macrophages. We observed that Mtb cells residing inside phagosomes have healthy ATP/ADP levels. In contrast, the bacteria residing inside phagolysosomes and autophagosomes face depletion of ATP. Interestingly, the activation of macrophages facilitates the trafficking of mycobacterial cells from metabolism-conducive phagosomes to metabolism-averse phagolysosomes and autophagosomes. We believe that this tool holds the key to the identification of inhibitors of mycobacterial metabolism.

Packaging of phage phi29 genome requires the ATPase gp16 and prohead RNA (pRNA). The highly conserved pRNA forms the interface between the connector complex and gp16. Understanding how pRNA interacts with gp16 under packaging conditions can shed light on the molecular mechanism of the packaging motor. Here, we present 3D models of the pRNA–gp16 complex and its conformation change in response to ATP or ADP binding. Using a combination of crystallography, small angle X-ray scattering and chemical probing, we find that the pRNA and gp16 forms a ‘Z’-shaped complex, with gp16 specifically binds to pRNA domain II. The whole complex closes in the presence of ATP, and pRNA domain II rotates open as ATP hydrolyzes, before resetting after ADP is released. Our results suggest that pRNA domain II actively participates in the packaging process.

PercevalHR (Perceval High Resolution) is an artificially designed fluorescent protein, which changes its excitation spectrum based on the ADP/ATP ratio of the environment. Here we demonstrated that PercevalHR can be used for monitoring energy status of Saccharomyces cerevisiae cells, which are affected by diauxic shift and mitochondria inhibition, in a non-invasive and non-destructive manner.

An increasing number of commonly prescribed drugs are known to interfere with mitochondrial function, causing cellular toxicity, but the underlying mechanisms are largely unknown. Although often not considered, mitochondrial transport proteins form a significant class of potential mitochondrial off-targets. So far, most drug interactions have been reported for the mitochondrial ADP/ATP carrier (AAC), which exchanges cytosolic ADP for mitochondrial ATP. Here, we show inhibition of cellular respiratory capacity by only a subset of the 18 published AAC inhibitors, which questions whether all compound do indeed inhibit such a central metabolic process. This could be explained by the lack of a simple, direct model system to evaluate and compare drug-induced AAC inhibition. Methods: For its development, we have expressed and purified human AAC1 (hAAC1) and applied two approaches. In the first, thermostability shift assays were carried out to investigate the binding of these compounds to human AAC1. In the second, the effect of these compounds on transport was assessed in proteoliposomes with reconstituted human AAC1, enabling characterization of their inhibition kinetics. Results: Of the proposed inhibitors, chebulinic acid, CD-437 and suramin are the most potent with IC50-values in the low micromolar range, whereas another six are effective at a concentration of 100 μM. Remarkably, half of all previously published AAC inhibitors do not show significant inhibition in our assays, indicating that they are false positives. Finally, we show that inhibitor strength correlates with a negatively charged surface area of the inhibitor, matching the positively charged surface of the substrate binding site. Conclusion: Consequently, we have provided a straightforward model system to investigate AAC inhibition and have gained new insights into the chemical compound features important for inhibition. Better evaluation methods of drug-induced inhibition of mitochondrial transport proteins will contribute to the development of drugs with an enhanced safety profile.

Las bacterias causantes del huanglongbing (HLB), Canditatus Liberibacter spp., son bacterias obligadas al floema de los cítricos y a diferentes sistemas del insecto vector, Diaphorina citri, por lo tanto, el enfoque genómico ha sido útil para estudiar sus mecanismos de patogenicidad. Dicho enfoque ha permitido identificar una copia homóloga del gen codificante de la enzima ATP/ADP translocasa, la cual tiene la capacidad de importar ATP y nucleótidos desde el hospedante, causando un parasitismo de energía considerable. Esta enzima se ha relacionado con la actividad endoparasítica de patógenos animales y humanos más que con fitopatógenos. El presente trabajo analizó la relación evolutiva entre la secuencia de aminoácidos de la ATP/ADP translocasa entre diferentes especies de Ca. Liberibacter y grupos como Ricketssia sp. y Chlamydia sp. Análisis filogenéticos muestran que la variación en la secuencia del gen codificante de la enzima está delimitada en clados correspondientes a las especies de Ca. Liberibacter, sugiriendo que la variación en la enzima responde a un proceso coevolutivo con los hospederos. Asimismo, la filogenia muestra que el ancestro común más cercano a Ca. Liberibacter podría ser un endosimbionte no patogénico del género Ca. Midichloria. Análisis de conservación de la secuencia de aminoácidos muestran que existen varias posiciones en la secuencia que podrían estar relacionadas con la variación específica de esta enzima dentro de Ca. Liberibacter. Este trabajo presenta la hipótesis de que el origen evolutivo de la capacidad de parasitismo energético del género Ca. Liberibacter, causantes del HLB, es un endosimbionte no patogénico.

ATP-sensitive potassium (KATP) channels couple cellular metabolism to excitability, making them ideal candidate sensors for hypoxic vasodilation. However, it is still unknown whether cellular nucleotide levels are affected sufficiently to activate vascular KATPchannels during hypoxia. To address this fundamental issue, we measured changes in the intracellular ATP:ADP ratio using the biosensors Perceval/PercevalHR, and membrane potential using the fluorescent probe DiBAC4(3) in human coronary artery smooth muscle cells (HCASMCs). ATP:ADP ratio was significantly reduced by exposure to hypoxia. Application of metabolic inhibitors for oxidative phosphorylation also reduced ATP:ADP ratio. Hyperpolarization caused by inhibiting oxidative phosphorylation was blocked by either 10 µM glibenclamide or 60 mM K+. Hyperpolarization caused by hypoxia was abolished by 60 mM K+but not by individual K+channel inhibitors. Taken together, these results suggest hypoxia causes hyperpolarization in part by modulating K+channels in SMCs.

Cryptosporidiumparvum is a clinically important eukaryotic parasite that causes the disease cryptosporidiosis, which manifests with gastroenteritis-like symptoms. The protist has mitosomes, which are organelles of mitochondrial origin that have only been partially characterized. The genome encodes a highly reduced set of transport proteins of the SLC25 mitochondrial carrier family of unknown function. Here, we have studied the transport properties of one member of the C. parvum carrier family, demonstrating that it resembles the mitochondrial ADP/ATP carrier of eukaryotes. However, this carrier has a broader substrate specificity for nucleotides, transporting adenosine, thymidine, and uridine di- and triphosphates in contrast to its mitochondrial orthologues, which have a strict substrate specificity for ADP and ATP. Inspection of the putative translocation pathway highlights a cysteine residue, which is a serine in mitochondrial ADP/ATP carriers. When the serine residue is replaced by cysteine or larger hydrophobic residues in the yeast mitochondrial ADP/ATP carrier, the substrate specificity becomes broad, showing that this residue is important for nucleotide base selectivity in ADP/ATP carriers.

IF1 (ATPIF1) is a nuclear DNA-encoded protein with an activity in the inhibition of catalytic activity of F1Fo-ATP synthase (ATPase), an enzyme for ATP synthesis in mitochondria. A role of IF1 remains unknown in the metabolic disorder in obesity. In this study, IF1 was examined in the diet-induced obese (DIO) mice and a decrease in IF1 protein was observed in several tissues including the skeletal muscle, liver and intestine in the absence of mRNA alteration. Significance of the reduction was investigated in the IF1-KO mice, in which insulin sensitivity was improved in the absence of body weight alteration on Chow diet. On a high fat diet (HFD), the IF1-KO mice gain more body weight as a result of enhanced fat tissue growth. The energy expenditure and locomotion activity were decreased in the KO mice without an alteration in food intake. The increase in insulin sensitivity remained in the obese KO mice. The colon tissue exhibited a resistance to the HFD-induced atrophy with less cell apoptosis and more secretion of GLP-1. Mitochondria exhibited an enhanced ATP production and maximal oxygen consumption without an alteration in the respiratory chain proteins. However, the ATP level was reduced in the fasting condition in the muscle as well as the liver. Mitophagy was enhanced with elevated accumulation of PINK1 and Parkin proteins in the mitochondria. The protein abundance of ADP/ATP translocase 2 (ANT2) was decreased in the inner membrane of mitochondria to account for the reduced apoptosis and enhanced mitophagy. The data suggest that the IF1 reduction in obesity leads to reprogramming of mitochondrial metabolism in a compensatory response to maintain the insulin sensitivity through down-regulation of ANT2 protein.

Peroxiredoxin 6 (PRDX6) is one antioxidant enzyme which could control the levels of reactive oxygen species and to avoid oxidative damage of sperm. In this study, we aimed to investigate the position change of PRDX6 in human sperm under oxidative stress during cryopreservation. Semen samples were obtained from 98 healthy donors and 27 asthenozoospermic donors. The plasma membrane protein and cytoplasmic protein of sperm samples were extracted and analyzed after cryopreservation. Western blot and immunofluorescence were used to measure the expressions of PRDX6. Liquid chromatography mass spectrometric (LC-MS/MS) analysis was performed to confirm the component of sperm membrane complex. Western blot showed that the detection rate of PRDX6 in plasma membranes with low sperm motility (≤20%) was significantly higher than that with high sperm motility (≥40%). Western blot and Immunofluorescence revealed that cryopreservation and thawing induced the position change of the PRDX6 from cytoplasm to sperm membrane. LC-MS/MS analysis showed that PRDX6, ADP/ATP translocase 4 (ANT4) and glyceraldehyde-3-phosphte dehydrogenase (GAPDHS) were present in the components of membrane complex after cryopreservation. The present study indicated that the presence of PRDX6 in sperm plasma membrane was related to sperm motility. GAPDHS and ANT4 may be involved the position change of the PRDX6 from cytoplasm to sperm membrane under oxidative stress during cryopreservation.

Cell migration is a dynamic process that entails extensive protein synthesis and recycling, structural remodeling, and a considerable bioenergetic demand. Autophagy is one of the pathways that maintain cellular homeostasis. Time-lapse imaging of autophagosomes and ATP/ADP levels in migrating cells in the rostral migratory stream of mice revealed that decrease in ATP levels force cells into the stationary phase and induce autophagy. Genetic impairment of autophagy in neuroblasts using either inducible conditional mice or CRISPR/Cas9 gene editing decreased cell migration due to the longer duration of the stationary phase. Autophagy is modulated in response to migration-promoting and inhibiting molecular cues and is required for the recycling of focal adhesions. Our results show that autophagy and energy consumption act in concert in migrating cells to dynamically regulate the pace and periodicity of the migratory and stationary phases in order to sustain neuronal migration.HighlightsADP levels dynamically change during cell migrationA decrease in ATP levels leads to cell pausing and autophagy induction via AMPKAutophagy is required to sustain neuronal migration by recycling focal adhesionsAutophagy level is dynamically modulated by migration-promoting and inhibiting cues

The structural assembly and conformation of yeast’s major ADP/ATP carrier is dependent on the signature lipid of mitochondria., The phospholipid cardiolipin has pleiotropic structural and functional roles that are collectively essential for mitochondrial biology. Yet, the molecular details of how this lipid supports the structure and function of proteins and protein complexes are poorly understood. To address this property of cardiolipin, we use the mitochondrial adenosine 5′-diphosphate/adenosine 5′-triphosphate carrier (Aac) as a model. Here, we have determined that cardiolipin is critical for both the tertiary and quaternary assembly of the major yeast Aac isoform Aac2 as well as its conformation. Notably, these cardiolipin-provided structural roles are separable. In addition, we show that multiple copies of Aac2 engage in shared complexes that are largely dependent on the presence of assembled respiratory complexes III and IV or respiratory supercomplexes. Intriguingly, the assembly state of Aac2 is sensitive to its transport-related conformation. Together, these results expand our understanding of the numerous structural roles provided by cardiolipin for mitochondrial membrane proteins.

Validation and verification of cleaning and inspection methods are essential to prevent the spread of allergens via cross-contact. Among the hygiene monitoring tests used on-site, the ATP test is rapid and provides quantifiable results. Nevertheless, because a wide variety of foods contain significant amount of ADP and/or AMP due to the degradation of ATP, the ATP+ADP+AMP (A3) test is preferred for detecting food debris. Hence, the A3 test may be valuable in screening food debris that may contain residual allergens. In this study, the detection limits of the A3 test for 40 foods that are regulated in several countries as allergenic were compared with those of the other hygiene monitoring tests used on-site: the conventional ATP test with similar sensitivity for ATP, the protein swab test that detects as little as 50 μg of protein, and the lateral flow immunoassay (LFI). The A3 test demonstrated lower detection limits than did the ATP test. The detection sensitivity of the A3 test was greater than that of the protein swab test except for its use on gelatin (extracted protein). The cleaning validation performance using a stainless steel model in fish and meat revealed that the A3 test is efficient in verifying the levels of remaining food debris. Although LFI displayed the best sensitivities for 10 of 14 foods, it is not commercially available for some specific allergens; however, the A3 test can detect such food debris. Moreover, the detection limits of the A3 test were preferable or comparable to those of LFI for crustacean shellfish and for processed grains, with the exception of wheat flour and buckwheat. A field study in a food processing plant demonstrated that the amount of both A3 and milk protein (enzyme-linked immunosorbent assay) considerably decreased as the cleaning steps progressed. Therefore, the A3 test is effective in detecting the risk for allergen cross-contact after inadequate cleaning. HIGHLIGHTS

We studied and compared in detail two published crystallographic structures of ADP/ATP carrier (AAC) protein in matrix open (m-state) and cytoplasmic open (c-state) state with and without cardiolipin (CDL) molecules embedded in the DOPC bilayer. We simulated and analyzed both states and obtained results show that both states are stable and water impermeable. In comparison with c-state, m-state is more occluded and has longer water impermeable area. Protein in both states, especially in the m- state, is more similar to crystallographic structure and less flexible in simulations when CDL molecules are present. This stabilization of m-state with CDL molecules sheds new light on AAC protein stability, representing a crucial factor for this structure which is significantly less stable compared with c- state.

The mitochondrial ATP-Mg/Pi carrier is responsible for the calcium-dependent regulation of adenosine nucleotide concentrations in the mitochondrial matrix, which allows mitochondria to respond to changing energy requirements of the cell. The carrier is expressed in mitochondria of fungi, plants and animals and belongs to the family of mitochondrial carriers. The carrier is unusual as it consists of three separate domains: (i) an N-terminal regulatory domain with four calcium-binding EF-hands similar to calmodulin, (ii) a loop domain containing an amphipathic α-helix and (iii) a mitochondrial carrier domain related to the mitochondrial ADP/ATP carrier. This striking example of three domains coming together from different origins to provide new functions represents an interesting quirk of evolution. In this review, we outline how the carrier was identified and how its physiological role was established with a focus on human isoforms. We exploit the sequence and structural information of the domains to explore the similarities and differences to their closest counterparts; mitochondrial ADP/ATP carriers and proteins with four EF-hands. We discuss how their combined function has led to a mechanism for calcium-regulated transport of adenosine nucleotides. Finally, we compare the ATP-Mg/Pi carrier with the mitochondrial aspartate/glutamate carrier, the only other mitochondrial carrier regulated by calcium, and we will argue that they have arisen by convergent rather than divergent evolution. © 2018 The Authors. IUBMB Life published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 70(12):1222-1232, 2018.

ATP/ADP isopentenyltransferase (IPTs) genes encode key enzymes involved in cytokinin synthesis. In this study, the functions of ATP/ADP PpIPTs in peach were investigated. According to the genome sequence, we have found and verified that there are four members of this gene family in peach, namely, PpIPT1, PpIPT3, PpIPT5, and PpIPT7. Overexpression of each of these genes in Arabidopsis resulted in increased levels of cytokinins in the transgenic plants, confirming their roles in cytokinin synthesis. Numerous altered phenotypes were observed in the transgenic plants, including vigorous growth and enhanced salt resistance. ATP/ADP PpIPTs were expressed in tissues throughout the plant, but the expression patterns differed between the genes. Only PpIPT3 was upregulated within 2 h after the application of nitrate to N-deprived peach seedlings, and the increase was resistant to pre-treatment of a specific nitrate metabolism inhibitor. Results showed that ATP/ADP PpIPT expression levels decreased significantly in pulp within 2 weeks after flowering and remained low. However, pulp cytokinin levels were quite high during this time. Only PpIPT5 in seed increased significantly within 2 weeks after flowering, which was consistent with cytokinin levels during early fruit development, suggesting that PpIPT5 in seed is the key gene for cytokinin biosynthesis during early fruit development. ATP/ADP PpIPT expression also increased significantly during later fruit development in seed.

Mitofusin-2 (MFN2) is a GTPase in the outer mitochondrial membrane involved in the regulation of mitochondrial fusion and bioenergetics. MFN2 also plays a role in mitochondrial fusion induced by changes in the intracellular redox state. Adding oxidized glutathione (GSSG), the core cellular stress indicator, to mitochondrial preparations stimulates mitochondrial fusion by inducing disulphide bond-mediated oligomer formation of MFN2 and its homolog MFN1 which involve cysteine 684 (C684) of MFN2. Mitochondrial hyperfusion represents an adaptive stress response that confers transient protection by increasing mitochondrial ATP production but how this depends on the thiol switch C684 in MFN2 has not been investigated. We now studied mitochondrial function using high-resolution respirometry in cells stably expressing wildtype or C684A MFN2 in MFN2-deficient fibroblasts in response to alterations of the redox state. Empty vector and untransfected cells served as controls. A single treatment of cells with 100 μM hydrogen peroxide 24 h before analysis had no effect on wildtype cells, but normalized the otherwise increased respiration of knockout cells and significantly increased respiration in C684A cells. In line with this, treating permeabilized cells for 10 min with 1 mM GSH greatly reduced respiration only in C684A cells. Our data indicate that mutation of this cysteine which forms disulphide bridges in an oxidative state, apparently renders MFN2 more susceptible to alterations of the redox environment. It remains to be investigated whether other posttranslational modifications like glutathionylation might play an additional role.

How the absence of the major mitochondrial ADP/ATP carrier in yeast, Aac2p, results in a specific defect in cytochrome c oxidase (COX; complex IV) activity is a long-standing mystery. Aac2p physically associates with respiratory supercomplexes, which include complex IV, raising the possibility that its activity is dependent on its association with Aac2p. Here, we have leveraged a transport-dead pathogenic AAC2 point mutant to determine the basis for the reduced COX activity in the absence of Aac2p. The steady-state levels of complex IV subunits encoded by the mitochondrial genome are significantly reduced in the absence of Aac2p function, whether its association with respiratory supercomplexes is preserved or not. This diminution in COX amounts is not caused by a reduction in the mitochondrial genome copy number or the steady-state level of its transcripts, and does not reflect a defect in complex IV assembly. Instead, the absence of Aac2p activity, genetically or pharmacologically, results in an aberrant pattern of mitochondrial translation. Interestingly, compared with the complete absence of Aac2p, the complex IV–related defects are greater in mitochondria expressing the transport-inactive Aac2p mutant. Our results highlight a critical role for Aac2p transport in mitochondrial translation whose disturbance uniquely impacts cytochrome c oxidase.

The nucleotides ATP and ADP regulate many aspects of endothelial cell (EC) biology, including intracellular calcium concentrations, focal adhesion activation, cytoskeletal organization, and cellular motility. In vivo, ECs are constantly under flow, and the concentration of ATP/ADP on the EC surface is determined by the combined effects of nucleotide convective and diffusive transport as well as hydrolysis by ectonucleotidases on the EC surface. In addition, experiments have demonstrated that flow induces ATP release from the cells. Previously computational models have incorporated the above effects and thus described concentration at the EC surface. However, it remains unclear what physical processes are responsible for nucleotide regulation. While some EC responses to flow have been shown to be directly driven by shear stress, others appear to also involve a non-negligible contribution of transport. In the present work, we develop a mathematical model and perform numerical simulations to investigate the relative contributions of shear stress and transport to nucleotide concentration at the EC surface, with the effect of cell density. Because in vitro experiments are performed by using confluent cells in some cases and subconfluent cells in other cases, we also investigate the effect of cell density on the results. The outcomes of the simulations demonstrate a complex interplay between shear stress and transport such that transport has a significant contribution at certain shear stress values but not at others. The effect of transport on nucleotide concentration increases with cell density. The present findings enhance our understanding of the mechanisms that govern the regulation of such molecules at the EC surface under flow. The implications of these findings for downstream responses such as cellular motility merit future investigation.

Mitochondria are fundamental structures that fulfil important and diverse functions within cells, including cellular respiration and iron-sulfur cluster biogenesis. Mitochondrial function is reliant on the organelles proteome, which is maintained and adjusted depending on cellular requirements. The majority of mitochondrial proteins are encoded by nuclear genes and must be trafficked to, and imported into the organelle following synthesis in the cytosol. These nuclear-encoded mitochondrial precursors utilise dynamic and multimeric translocation machines to traverse the organelles membranes and be partitioned to the appropriate mitochondrial subcompartment. Yeast model systems have been instrumental in establishing the molecular basis of mitochondrial protein import machines and mechanisms, however unique players and mechanisms are apparent in higher eukaryotes. Here, we review our current knowledge on mitochondrial protein import in human cells and how dysfunction in these pathways can lead to disease.

The experiment was conducted to identify the mitochondrial protein responsible for enhancing glycolytic flux. We hypothesized that mitochondrial F1-ATPase promotes ATP hydrolysis and thereby the flux through glycolysis. Porcine longissimus muscle mitochondria were incorporated into an in vitro system designed to recapitulate postmortem glycolysis with or without Na-azide to specifically inhibit the β-subunit of mitochondrial F1-ATPase that catalyzes ATP hydrolysis. Addition of mitochondria enhanced ATP hydrolysis, glycogen degradation, lactate accumulation, and pH decline in the in vitro system. However, the majority of mitochondria-mediated enhancement in glycolytic flux was abolished in the presence of Na-azide. To investigate further, myofibrillar and mitochondrial proteins were added to the in vitro system after 240min from the initiation of the reaction. Greater pH decline and lactate accumulation were observed in system containing mitochondrial protein compared to their myofibrillar counterpart. In conclusion, mitochondrial F1-ATPase is capable of increasing glycolytic flux through promoting greater ATP hydrolysis at lower pH.

ATP and ADP are the major energy source in metabolism of cells, and furthermore ATP works as an important extracellular signaling material. ATP bioimaging is required to understand their metabolism. In this paper, a label-free ATP/ADP image sensor was fabricated using a 128 × 128 (16 k) pixel array semiconductor CCD-type pH image sensor and Apyrase. The principle is based on measuring protons produced by the enzyme reaction between Apyrase and ATP or ADP. In order to put a uniform potential response of the sensor into practice, two different methods, (3-APTES and CEST), which chemically fixed N-terminal of Apyrase with the sensor were examined. The sensor modified by the CEST method had a quite clean surface microscopically and demonstrated a fine real-time image monitoring the ATP concentration. The potential response of the image sensor was characterized; effect of buffer solution, calibration curves of ATP and ADP, durability, the limit of detection (LOD) for ATP, and the response of time. The potentia...

Membrane organelles comprise both proteins and lipids. Remodeling of these membrane structures is controlled by interactions between specific proteins and lipids. Mitochondrial structure and function depend on regulated fusion and the division of both the outer and inner membranes. Here we discuss recent advances in the regulation of mitochondrial dynamics by two critical phospholipids, phosphatidic acid (PA) and cardiolipin (CL). These two lipids interact with the core components of mitochondrial fusion and division (Opa1, mitofusin, and Drp1) to activate and inhibit these dynamin-related GTPases. Moreover, lipid-modifying enzymes such as phospholipases and lipid phosphatases may organize local lipid composition to spatially and temporarily coordinate a balance between fusion and division to establish mitochondrial morphology.

Mitochondrial function is coupled to metabolic and survival pathways through both direct signaling cascades and dynamic changes in mitochondrial morphology. For example, a hyperfused mitochondrial reticulum is activated upon cellular stress and is protective against cell death. As part of a genome-wide small inhibitory ribonucleic acid screen, we identified the central redox regulator, Keap1, as a novel regulator of mitochondrial morphology. Here, we aimed to determine the mechanism through which redox signaling and Keap1 mediate changes in mitochondrial morphology.We found that the Nrf2 transcription factor is required for mitochondrial hyperfusion induced by knockdown of Keap1. Nrf2, which is negatively regulated by Keap1, mediates the cell's response to stress by controlling the expression of several hundred genes, including proteasome expression. We next showed that increased proteasome activity, a result of increased Nrf2 activity, is responsible for the degradation of the mitochondrial fission protein Drp1, which occurs in an ubiquitin-independent manner.Our study described a novel pathway by which Nrf2 activation, known to occur in response to increased oxidative stress, decreases mitochondrial fission and contributes to a hyperfused mitochondrial network.This study has identified the Keap1-Nrf2 nexus and modulation of proteasomal activity as novel avenues to inhibit mitochondrial fission. These findings are important, because inhibiting mitochondrial fission is a promising therapeutic approach to restore the balance between fission and fusion, which is attractive for an increasing number of disorders linked to mitochondrial dysfunction. Antioxid. Redox Signal. 27, 1447-1459.

While Indium's toxicity to organs is realized, its effects on mitochondria are still under investigation. Mitochondrial permeability transition (MPT) is widely accepted in mitochondrial dysfunction approaches and its importance in metal-induced mitochondrial degradation has been proposed. Since mitochondria are respiratory organelles, their interaction with free In3+ is analyzed to access structural and functional changes. Spectral methods and multimode plate reader was used to detect mitochondrial swelling, membrane potential, membrane fluidity, and inner membrane permeability. Flow cytometry was employed to detect mitochondrial reactive oxygen species (ROS) generation and transmission electron microscopy to image mitochondria. And oxygen electrode was used to measure respiratory rate, microcalorimetry to monitor long-term real-time mitochondrial metabolism. In3+ at a concentration up to 1mM induces mitochondrial swelling, membrane depolarization and inhibits the protons transportation. In3+-induced mitochondrial swelling and membrane depolarization is protected by MPT inhibitors and -SH protectors, but the influence on protons transportation is not protected. In addition, In3+ is able to accelerate the ROS production and inhibit the electron transition and respiratory chain while it stimulates long-term metabolism. Our findings show that In3+ induces MPT by inhibiting the proton channels located in the inner mitochondrial membrane and by stimulating mitochondrial oxidative stress.

Mitochondrial structure and function are influenced by the unique phospholipid composition of its membranes. While mitochondria contain all the major classes of phospholipids, recent studies have highlighted specific roles of the non-bilayer forming phospholipids phosphatidylethanolamine (PE) and cardiolipin (CL) in the assembly and activity of mitochondrial respiratory chain (MRC) complexes. The non-bilayer phospholipids are cone-shaped molecules that introduce curvature stress in the bilayer membrane and have been shown to impact mitochondrial fusion and fission. In addition to their overlapping roles in these mitochondrial processes, each non-bilayer phospholipid also plays a unique role in mitochondrial function; for example, CL is specifically required for MRC supercomplex formation. Recent discoveries of mitochondrial PE and CL trafficking proteins and prior knowledge of their biosynthetic pathways have provided targets for precisely manipulating non-bilayer phospholipid levels in the mitochondrial membranes in vivo. Thus, the genetic mutants of these pathways could be valuable tools in illuminating molecular functions and biophysical properties of non-bilayer phospholipids in driving mitochondrial bioenergetics and dynamics.

Mitochondrial fatty acid synthesis (mtFAS) is a highly conserved pathway essential for mitochondrial biogenesis. The mtFAS process is required for mitochondrial respiratory chain assembly and function, synthesis of the lipoic acid cofactor indispensable for the function of several mitochondrial enzyme complexes and essential for embryonic development in mice. Mutations in human mtFAS have been reported to lead to neurodegenerative disease. The source of malonyl-CoA for mtFAS in mammals has remained unclear. We report the identification of a conserved vertebrate mitochondrial isoform of ACC1 expressed from an ACACA transcript splicing variant. A specific knockdown (KD) of the corresponding transcript in mouse cells, or CRISPR/Cas9-mediated inactivation of the putative mitochondrial targeting sequence in human cells, leads to decreased lipoylation and mitochondrial fragmentation. Simultaneous KD of ACSF3, encoding a mitochondrial malonyl-CoA synthetase previously implicated in the mtFAS process, resulted in almost complete ablation of protein lipoylation, indicating that these enzymes have a redundant function in mtFAS. The discovery of a mitochondrial isoform of ACC1 required for lipoic acid synthesis has intriguing consequences for our understanding of mitochondrial disorders, metabolic regulation of mitochondrial biogenesis and cancer.

According to previous studies, ADP/ATP carrier (AAC) can possibly exist as a monomer or in a dimer state in the inner mitochondrial membrane; however, the question on its functional oligomeric state is still open. The aim of the present work is to establish the external factors that could control the functional oligomeric state of AAC (i.e., monomer or dimer). The study is based on the results of our previous work, which revealed that the volume regulation system of mitochondria (MVRS) affects the oxidative phosphorylation (OXPHOS) system: MVRS could transfer OXPHOS system functioning in a state of supercomplex. Consequently, one may expect that the volume regulation system could also control the functional state of AAC during phosphorylation. Here, on rat liver mitochondria we show that, depending on the incubation medium tonicity, AAC functions in two different ways: either as a monomer (in hypotonic and isotonic media) or as a dimer (in a hypertonic medium). Thus, the transition between the monomeric and dimeric forms of AAC is regulated by MVRS, as well as by functioning of OXPHOS. We conclude that the structural reorganization of AAC is associated with the entire OXPHOS reorganization into a supercomplex. It was also found that dimerization of AAC can occur not only due to the action of MVRS (in hypotonic media) but also under hypoxic conditions.

Ecto-F1-ATPase is a complex related to mitochondrial ATP synthase which has been identified as a plasma membrane receptor for apolipoprotein A-I (apoA-I), the major protein of high-density lipoprotein (HDL), and has been shown to contribute to HDL endocytosis in several cell types. On hepatocytes, apoA-I binding to ecto-F1-ATPase stimulates extracellular ATP hydrolysis into ADP, which subsequently activates a P2Y13-mediated HDL endocytosis pathway. Interestingly, other mitochondrial proteins have been found to be expressed at the plasma membrane of several cell types. Among these, adenine nucleotide translocase (ANT) is an ADP/ATP carrier but its role in controlling extracellular ADP levels and F1-ATPase-mediated HDL endocytosis has never been investigated. Here we confirmed the presence of ANT at the plasma membrane of human hepatocytes. We then showed that ecto-ANT activity increases or reduces extracellular ADP level, depending on the extracellular ADP/ATP ratio. Interestingly, ecto-ANT co-localized with ecto-F1-ATPase at the hepatocyte plasma membrane and pharmacological inhibition of ecto-ANT activity increased extracellular ADP level when ecto-F1-ATPase was activated by apoA-I. This increase in the bioavailability of extracellular ADP accordingly translated into an increase of HDL endocytosis on human hepatocytes. This study thus uncovered a new location and function of ANT for which activity at the cell surface of hepatocytes modulates the concentration of extracellular ADP and regulates HDL endocytosis.

B cell leukemia/lymphoma-2 (Bcl-2) suppresses apoptosis by binding the BH3 domain of proapoptotic factors and thereby regulating mitochondrial membrane potential (MMP). This study aimed to investigate the role of Bcl-2 in controlling the mitochondrial pathway of apoptosis during hydroquinone (HQ)-induced TK6 cytotoxicity. In this study, HQ, one metabolite of benzene, decreased the MMP in a concentration-dependent manner and induced the generation of reactive oxygen species (ROS), the activation of the DNA damage marker γ-H2AX, and production of the DNA damage-responsive enzyme poly(ADP-ribose)polymerase-1 (PARP-1). Exposure of TK6 cells to HQ leads to an increase in Bcl-2 and co-localization with PARP-1 in the cytoplasm. Inhibition of Bcl-2 using the BH3 mimetic, ABT-737, suppressed the PARP-1 nuclear to cytoplasm translocation and sensitized TK6 cells to HQ-induced apoptosis through depolarization of the MMP. Western blot analysis indicated that ABT-737 combined with HQ increased the levels of cleaved PARP and γ-H2AX, but significantly decreased the level of P53. Thus, ABT-737 can influence PARP-1 translocation and induce apoptosis via mitochondria-mediated apoptotic pathway, independently of P53. In addition, we found that knockdown of PARP-1 attenuated the HQ-induced production of cleaved PARP and P53. These results identify Bcl-2 as a protective mediator of HQ-induced apoptosis and show that upregulation of Bcl-2 helps to localize PARP-1 to the cytoplasm and stabilize MMP. Environ. Mol. Mutagen. 59:49-59, 2018. © 2017 Wiley Periodicals, Inc.

Regulated inter-mitochondrial fusion/fission is essential for maintaining optimal mitochondrial respiration and control of apoptosis and autophagy. In mammals, mitochondrial fusion is controlled by outer membrane GTPases MFN1 and MFN2 and by inner membrane (IM) GTPase OPA1. Disordered mitochondrial fusion/fission contributes to various pathologies, and MFN2 or OPA1 mutations underlie neurodegenerative diseases. Here, we show that the WBSCR16 protein is primarily associated with the outer face of the inner mitochondrial membrane and is important for mitochondrial fusion. We provide evidence of a WBSCR16/OPA1 physical interaction in the intact cell and of a WBSCR16 function as an OPA1-specific guanine nucleotide exchange factor (GEF). Homozygosity for a Wbscr16 mutation causes early embryonic lethality, whereas neurons of mice heterozygous for the mutation have mitochondria with reduced membrane potential and increased susceptibility to fragmentation upon exposure to stress, suggesting roles for WBSCR16 deficits in neuronal pathologies.

In the past, divergent results have been reported based on different methods and conditions used for enzymatic activity measurements of cytochrome c oxidase (CytOx). Here, we analyze in detail and show comparable and reproducible polarographic activity measurements of ATP-dependent inhibition of CytOx kinetics in intact and non-intact rat heart mitochondria and mitoplasts. We found that this mechanism is always present in isolated rat heart mitochondria and mitoplasts; however, it is measurable only at high ATP/ADP ratios using optimal protein concentrations. In the kinetics assay, measurement of this mechanism is independent of presence or absence of Tween-20 and the composition of measuring buffer. Furthermore, the effect of atractyloside on intact rat heart mitochondria confirms that (i) ATP inhibition occurs under uncoupled conditions [in the presence of carbonly cyanide m-chlorophenyl hydrazone (CCCP)] when the classical respiratory control is absent and (ii) high ATP/ADP ratios in the matrix as well as in the cytosolic space are required for full ATP inhibition of CytOx. Additionally, ATP inhibition measured in intact mitochondria extends in the presence of oligomycin, thus indicating further that the problem to measure the inhibitory effect of ATP on CytOx is apparently due to the lack of very high ATP/ADP ratios in isolated mitochondria.

Mitochondrial membrane fragments from human platelets and monkey skeletal muscles were successfully immobilized onto immobilized artificial membrane chromatographic support for the first time, resulting in mitochondrial membrane affinity chromatography (MMAC) columns. These columns were validated by characterization of translocator protein (TSPO), where multiple concentrations of dipyridamole were run and the binding affinities (Kd) determined. Further, the relative ranking data of TSPO ligands was consistent with previously reported rankings for both, the platelet (MMAC-Platelet) and the skeletal muscle (MMAC-Muscle) column (dipyridamole > PK11195 > protoporphyrin IX > rotenone). The functional immobilization of the F-ATPase/ATP synthase was demonstrated on MMAC-Muscle column. Online hydrolysis of ATP to ADP and synthesis of ATP from ADP were both demonstrated on the MMAC-Muscle column. Hydrolysis of ATP to ADP was inhibited by oligomycin A with an IC50 of 40.2 ± 13.5 nM (~60% reduction in ATP hydrolysis, p

Mitochondrial division is critical for the maintenance and regulation of mitochondrial function, quality and distribution. This process is controlled by cytosolic actin-based constriction machinery and dynamin-related protein 1 (Drp1) on mitochondrial outer membrane (OMM). Although mitochondrial physiology, including oxidative phosphorylation, is also important for efficient mitochondrial division, morphological alterations of the mitochondrial inner-membrane (IMM) have not been clearly elucidated. Here we report spontaneous and repetitive constriction of mitochondrial inner compartment (CoMIC) associated with subsequent division in neurons. Although CoMIC is potentiated by inhibition of Drp1 and occurs at the potential division spots contacting the endoplasmic reticulum, it appears on IMM independently of OMM. Intra-mitochondrial influx of Ca2+ induces and potentiates CoMIC, and leads to K+-mediated mitochondrial bulging and depolarization. Synergistically, optic atrophy 1 (Opa1) also regulates CoMIC via controlling Mic60-mediated OMM–IMM tethering. Therefore, we propose that CoMIC is a priming event for efficient mitochondrial division., The role of morphological alterations in the mitochondrial inner-membrane in regulating mitochondrial division are unknown. Here, the authors describe spontaneous and repetitive constriction of the mitochondrial inner compartment, and suggest this acts as a priming event for efficient mitochondrial division.

The plastidic ATP/ADP transporter (AATP) imports adenosine triphosphate (ATP) from the cytosol into plastids, resulting in the increase of the ATP supply to facilitate anabolic synthesis in heterotrophic plastids of dicotyledonous plants. The regulatory role of GmAATP from soybean in increasing starch accumulation has not been investigated. In this study, a gene encoding the AATP protein, named GmAATP, was successfully isolated from soybean. Transient expression of GmAATP in Arabidopsis protoplasts and Nicotiana benthamiana leaf epidermal cells revealed the plastidic localization of GmAATP. Its expression was induced by exogenous sucrose treatment in soybean. The coding region of GmAATP was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis to obtain transgenic plants. Constitutive expression of GmAATP significantly increased the sucrose and starch accumulation in the transgenic plants. Real-time quantitative PCR (qRT-PCR) analysis showed that constitutive expression of GmAATP up-regulated the expression of phosphoglucomutase (AtPGM), ADP-glucose pyrophosphorylase (AGPase) small subunit (AtAGPase-S1 and AtAGPase-S2), AGPase large subunit (AtAGPase-L1 and AtAGPase-L2), granule-bound starch synthase (AtGBSS I and AtGBSS II), soluble starch synthases (AtSSS I, AtSSS II, AtSSS III, and AtSSS IV), and starch branching enzyme (AtSBE I and AtSBE II) genes involved in starch biosynthesis in the transgenic Arabidopsis plants. Meanwhile, enzymatic analyses indicated that the major enzymes (AGPase, GBSS, SSS, and SBE) involved in the starch biosynthesis exhibited higher activities in the transgenic plants compared to the wild type (WT). These findings suggest that GmAATP may improve starch content of Arabidopsis by up-regulating the expression of the related genes and increasing the activities of the major enzymes involved in starch biosynthesis. All these results suggest that GmAATP could be used as a candidate gene for developing high starch-accumulating plants as alternative energy crops.