Inhibition Of Mitochondrial ATP Synthesis Research Articles

Inhibition of the mitochondrial respiratory chain reduces catecholamine‑stimulated lipolysis via increasing lactate production in 3T3‑L1 adipocytes.

Adipose tissue serves a significant role in the regulation of energy metabolism in the body. The re‑esterification of the fatty acids generated during lipolysis is critical for efficient lipolysis. However, the effect of the intracellular energy state on lipolytic activity and fatty acid re‑esterification during lipolysis is not yet fully understood. The present study aimed to assess the effect of the intracellular energy state on lipolytic activity and fatty acid re‑esterification during lipolysis. 3T3‑L1 adipocytes were incubated with mitochondrial respiratory chain inhibitors, oligomycin A or rotenone, during isoproterenol stimulation; and glycerol, glucose and lactate concentrations in the medium were measured. Western blot analysis was performed to examine the phosphorylation levels of cAMP‑dependent protein kinase A (PKA). The results showed that inhibition of mitochondrial ATP synthesis decreased catecholamine‑stimulated lipolysis without affecting PKA signaling. The inhibition of mitochondrial respiration increased glucose uptake and lactate production, indicating that a large amount of glucose taken up into the cell was preferentially used for ATP production rather than for re‑esterification. In conclusion, the results of the present study suggested that the energy state during lipolysis may influence lipolytic activity by suppressing fatty acid re‑esterification.

Intracellular Mg2+ protects mitochondria from oxidative stress in human keratinocytes

Reactive oxygen species (ROS) are harmful for the human body, and exposure to ultraviolet irradiation triggers ROS generation. Previous studies have demonstrated that ROS decrease mitochondrial membrane potential (MMP) and that Mg2+ protects mitochondria from oxidative stress. Therefore, we visualized the spatio-temporal dynamics of Mg2+ in keratinocytes (a skin component) in response to H2O2 (a type of ROS) and found that it increased cytosolic Mg2+ levels. H2O2-induced responses in both Mg2+ and ATP were larger in keratinocytes derived from adults than in keratinocytes derived from newborns, and inhibition of mitochondrial ATP synthesis enhanced the H2O2-induced Mg2+ response, indicating that a major source of Mg2+ was dissociation from ATP. Simultaneous imaging of Mg2+ and MMP revealed that larger Mg2+ responses corresponded to lower decreases in MMP in response to H2O2. Moreover, Mg2+ supplementation attenuated H2O2-induced cell death. These suggest the potential of Mg2+ as an active ingredient to protect skin from oxidative stress.

Asteltoxins: Synthesis and Biological Studies

Asteltoxins belong to a group of polyene pyrone mycotoxins that are known to be potent inhibitors of mitochondrial ATP synthesis and ATP hydrolysis. Asteltoxin A was first isolated from the toxic maize cultures of Aspergillus stellatus. Several attempts have been made to synthesize asteltoxin A, starting with the synthesis of a bis(tetrahydrofuran) moiety that has been demonstrated previously in biosynthetic studies. This review highlights the fungal sources of asteltoxins, similar asteltoxins, biosynthetic pathways, their synthetic trials, and their biological activities. This review is the first of its kind covering the periods from 1979 to 2023.

Remnants of the Balbiani body are required for formation of RNA transport granules in Xenopus oocytes

SummaryThe Balbiani body (Bb), an organelle comprised of mitochondria, ER, and RNA, is found in the oocytes of most organisms. In Xenopus, the structure is initially positioned immediately adjacent to the nucleus, extends toward the vegetal pole, and eventually disperses, leaving behind a region highly enriched in mitochondria. This area is later transversed by RNP complexes that are being localized to the vegetal cortex. Inhibition of mitochondrial ATP synthesis prevents perinuclear formation of the transport complexes that can be reversed by a nonhydrolyzable ATP analog, indicating the nucleotide is acting as a hydrotrope. The protein composition, sensitivity to hexanediol, and coalescence in the absence of transport provide evidence that the transport RNP complexes are biocondensates. The breakdown of the Bb engenders regions of clustered mitochondria that are used not to meet extraordinary energy demands, but rather to promote a liquid-liquid phase separation.

Inhibition of Mitochondrial ATP Synthesis and Regulation of Oxidative Stress Based on {SbW8O30} Determined by Single‐Cell Proteomics Analysis

AbstractThe 10‐nuclear heteroatom cluster modified {SbW8O30} was successfully synthesized and exhibited inhibitory activity (IC50=0.29 μM). Based on proteomics analysis, Na4Ni2Sb2W2‐SbW8 inhibited ATP production by affecting the expression of 16 related proteins, hindering metabolic functions in vivo and cell proliferation due to reactive oxygen species (ROS) stress. In particular, the low expression of FAD/FMN‐binding redox enzymes (relative expression ratio of the experimental group to the control=0.43843) could be attributed to the redox mechanism of Na4Ni2Sb2W2‐SbW8, which was consistent with the effect of polyoxometalates (POMs) and FMN‐binding proteins on ATP formation. An electrochemical study showed that Na4Ni2Sb2W2‐SbW8 combined with FMN to form Na4Ni2Sb2W2‐SbW8‐2FMN complex through a one‐electron process of the W atoms. Na4Ni2Sb2W2‐SbW8 acted as catalase and glutathione peroxidase to protect the cell from ROS stress, and the inhibition rates were 63.3 % at 1.77 μM of NADPH and 86.06 % at 10.62 μM of 2‐hydroxyterephthalic acid. Overall, our results showed that POMs can be specific oxidative/antioxidant regulatory agents.

Inhibition of Mitochondrial ATP Synthesis and Regulation of Oxidative Stress Based on {SbW8 O30 } Determined by Single-Cell Proteomics Analysis.

The 10-nuclear heteroatom cluster modified {SbW8 O30 } was successfully synthesized and exhibited inhibitory activity (IC50 =0.29 μM). Based on proteomics analysis, Na4 Ni2 Sb2 W2 -SbW8 inhibited ATP production by affecting the expression of 16 related proteins, hindering metabolic functions in vivo and cell proliferation due to reactive oxygen species (ROS) stress. In particular, the low expression of FAD/FMN-binding redox enzymes (relative expression ratio of the experimental group to the control=0.43843) could be attributed to the redox mechanism of Na4 Ni2 Sb2 W2 -SbW8 , which was consistent with the effect of polyoxometalates (POMs) and FMN-binding proteins on ATP formation. An electrochemical study showed that Na4 Ni2 Sb2 W2 -SbW8 combined with FMN to form Na4 Ni2 Sb2 W2 -SbW8 -2FMN complex through a one-electron process of the W atoms. Na4 Ni2 Sb2 W2 -SbW8 acted as catalase and glutathione peroxidase to protect the cell from ROS stress, and the inhibition rates were 63.3 % at 1.77 μM of NADPH and 86.06 % at 10.62 μM of 2-hydroxyterephthalic acid. Overall, our results showed that POMs can be specific oxidative/antioxidant regulatory agents.

Targeting breast cancer metabolism with a novel inhibitor of mitochondrial ATP synthesis.

Inhibitors of mitochondrial respiration and ATP synthesis may promote the selective killing of respiration-competent cancer cells that are critical for tumor progression. We previously reported that CADD522, a small molecule inhibitor of the RUNX2 transcription factor, has potential for breast cancer treatment. In the current study, we show that CADD522 inhibits mitochondrial oxidative phosphorylation by decreasing the mitochondrial oxygen consumption rate (OCR) and ATP production in human breast cancer cells in a RUNX2-independent manner. The enzyme activity of mitochondrial ATP synthase was inhibited by CADD522 treatment. Importantly, results from cellular thermal shift assays that detect drug-induced protein stabilization revealed that CADD522 interacts with both α and β subunits of the F1-ATP synthase complex. Differential scanning fluorimetry also demonstrated interaction of α subunits of the F1-ATP synthase to CADD522. These results suggest that CADD522 might target the enzymatic F1 subunits in the ATP synthase complex. CADD522 increased the levels of intracellular reactive oxygen species (ROS), which was prevented by MitoQ, a mitochondria-targeted antioxidant, suggesting that cancer cells exposed to CADD522 may elevate ROS from mitochondria. CADD522-increased mitochondrial ROS levels were enhanced by exogenously added pro-oxidants such as hydrogen peroxide or tert-butyl hydroperoxide. Conversely, CADD522-mediated cell growth inhibition was blocked by N-acetyl-l-cysteine, a general ROS scavenger. Therefore, CADD522 may exert its antitumor activity by increasing mitochondrial driven cellular ROS levels. Collectively, our data suggest in vitro proof-of-concept that supports inhibition of mitochondrial ATP synthase and ROS generation as contributors to the effectiveness of CADD522 in suppression of tumor growth.

Identification of chemical compounds as an inhibitor of mitochondrial ATP synthesis, leading to an increased stress resistance and an extended lifespan in C. elegans

It is well known that the disruption of the mitochondrial respiratory components prolongs lifespan in many species. The mitochondrial stress response can lead to an increased survival rate through the restoration of the cellular homeostasis. Therefore, developing pharmacological interventions that induce mitochondrial stress response may be desirable to delay the onset of age-related diseases and promote a healthy life. In this study, we present chemical compounds, revealed by systematic screening of chemical libraries, which inhibit mitochondrial ATP synthesis in mammalian cells. Our study demonstrates that these compounds alter the body length and promote the oxidative stress response which leads to an increased longevity in Caenorhabditis elegans. Thus, our study identifies chemical compounds that may have potential therapeutic applications through affecting the mitochondrial function.

Deployment of an attractive toxic sugar bait system (ATSB) with insecticide, for adult Tribolium castaneum (Coleoptera: Tenebrionidae)

Tribolium castaneum is a critical stored-grain pest of nuts and cereals. Insecticides have traditionally been used to control it; however, their overuse can result in the development of insecticide resistance. Effective bait pesticides could be useful tools for slowing down the rate of insecticide-resistance development and improving the management of stored grain pests. Attractive toxic sugar bait (ATSB) systems are used with mosquitoes and flies; they lure pests with liquid foods that contain sugars but also an oral toxin. It is unknown whether the ATSB system is applicable to T. castaneum adults who prefer dry food products with reduced water content. The objective of this study is to develop an optimal ATSB system for T. castaneum control. Thus far, it found that T. castaneum consumed a gypsum block. The dietary intake of T. castaneum adult beetles was facilitated with added mannitol into gypsum block. Combining insecticides that are oral toxins of the pests with these facilitating sugars results in the death of the pest. Spirotetramat causes a significant reduction in fatty acid biosynthesis by inhibiting acetyl-CoA carboxylase. In this investigation, we have demonstrated that spirotetramat, at low concentrations, was lethal to T. castaneum adults, when used with the ATSB system, combined with mannitol. The lethality of chlorfenapyr, an inhibitor of mitochondrial ATP synthesis, to adult T. castaneum did not increase when used in the ATSB system with mannitol. These results show that the active ingredient used in the ATSB system is integral for beetle control. Increasing the oral delivery rate of insecticides using carbohydrates leads to increased lethality rates of stored-grain pests.

Bioassay-guided isolation of three anthelmintic compounds from Warburgia ugandensis Sprague subspecies ugandensis, and the mechanism of action of polygodial

Parasitic helminths continue to pose problems in human and veterinary medicine, as well as in agriculture. Resistance to current anthelmintics has prompted the search for new drugs. Anthelmintic metabolites from medicinal plants could be good anthelmintic drug candidates. However, the compounds active against nematodes have not been identified in most medicinal plants with anthelmintic activity. In this study, we aimed to identify the active compounds against helminths in Warburgia ugandensis Sprague subspecies ugandensis (Canellaceae) and study the underlying mechanism of action. A bioassay-guided isolation of anthelmintic compounds from the plant was performed using a Caenorhabditis elegans (C. elegans) test model with a WMicrotracker instrument to monitor motility. Three active compounds were purified and identified by nuclear magnetic resonance and high resolution MS: warburganal (IC50: 28.2 ± 8.6 μM), polygodial (IC50: 13.1 ± 5.3 μM) and alpha-linolenic acid (ALA, IC50: 70.1 ± 17.5 μM). A checkerboard assay for warburganal and ALA as well as polygodial and ALA showed a fractional inhibitory concentration index of 0.41 and 0.37, respectively, suggesting that polygodial and ALA, as well as warburganal and ALA, have a synergistic effect against nematodes. A preliminary structure–activity relationship study for polygodial showed that the α,β-unsaturated 1,4-dialdehyde structural motif is essential for the potent activity. None of a panel of C. elegans mutant strains, resistant against major anthelmintic drug classes, showed significant resistance to polygodial, implying that polygodial may block C. elegans motility through a mechanism which differs from that of currently marketed drugs. Further measurements showed that polygodial inhibits mitochondrial ATP synthesis of C. elegans in a dose-dependent manner (IC50: 1.8 ± 1.0 μM). Therefore, we believe that the underlying mechanism of action of polygodial is probably inhibition of mitochondrial ATP synthesis. In conclusion, polygodial could be a promising anthelmintic drug candidate worth considering for further development.

General anesthetics cause mitochondrial dysfunction and reduction of intracellular ATP levels.

General anesthetics are indispensable for effective clinical care. Although, the mechanism of action of general anesthetics remains controversial, lipid bilayers and proteins have been discussed as their targets. In this study, we focused on the relationship between cellular ATP levels and general anesthetics. The ATP levels of nematodes and cultured mammalian cells were decreased by exposure to three general anesthetics: isoflurane, pentobarbital, and 1-phenoxy-2-propanol. Furthermore, these general anesthetics abolished mitochondrial membrane potential, resulting in the inhibition of mitochondrial ATP synthesis. These results suggest that the observed decrease of cellular ATP level is a common phenomenon of general anesthetics.

Dynamic energy dependency of Chlamydia trachomatis on host cell metabolism during intracellular growth: Role of sodium-based energetics in chlamydial ATP generation

Chlamydia trachomatis is an obligate intracellular human pathogen responsible for the most prevalent sexually-transmitted infection in the world. For decades C. trachomatis has been considered an "energy parasite" that relies entirely on the uptake of ATP from the host cell. The genomic data suggest that C. trachomatis respiratory chain could produce a sodium gradient that may sustain the energetic demands required for its rapid multiplication. However, this mechanism awaits experimental confirmation. Moreover, the relationship of chlamydiae with the host cell, in particular its energy dependence, is not well understood. In this work, we are showing that C. trachomatis has an active respiratory metabolism that seems to be coupled to the sodium-dependent synthesis of ATP. Moreover, our results show that the inhibition of mitochondrial ATP synthesis at an early stage decreases the rate of infection and the chlamydial inclusion size. In contrast, the inhibition of the chlamydial respiratory chain at mid-stage of the infection cycle decreases the inclusion size but has no effect on infection rate. Remarkably, the addition of monensin, a Na+/H+ exchanger, completely halts the infection. Altogether, our data indicate that chlamydial development has a dynamic relationship with the mitochondrial metabolism of the host, in which the bacterium mostly depends on host ATP synthesis at an early stage, and at later stages it can sustain its own energy needs through the formation of a sodium gradient.

Glycolysis is the primary bioenergetic pathway for cell motility and cytoskeletal remodeling in human prostate and breast cancer cells

The ability of a cancer cell to detach from the primary tumor and move to distant sites is fundamental to a lethal cancer phenotype. Metabolic transformations are associated with highly motile aggressive cellular phenotypes in tumor progression. Here, we report that cancer cell motility requires increased utilization of the glycolytic pathway. Mesenchymal cancer cells exhibited higher aerobic glycolysis compared to epithelial cancer cells while no significant change was observed in mitochondrial ATP production rate. Higher glycolysis was associated with increased rates of cytoskeletal remodeling, greater cell traction forces and faster cell migration, all of which were blocked by inhibition of glycolysis, but not by inhibition of mitochondrial ATP synthesis. Thus, our results demonstrate that cancer cell motility and cytoskeleton rearrangement is energetically dependent on aerobic glycolysis and not oxidative phosphorylation. Mitochondrial derived ATP is insufficient to compensate for inhibition of the glycolytic pathway with regard to cellular motility and CSK rearrangement, implying that localization of ATP derived from glycolytic enzymes near sites of active CSK rearrangement is more important for cell motility than total cellular ATP production rate. These results extend our understanding of cancer cell metabolism, potentially providing a target metabolic pathway associated with aggressive disease.

Mitochondria and Melanosomes Establish Physical Contacts Modulated by Mfn2 and Involved in Organelle Biogenesis

To efficiently supply ATP to sites of high-energy demand and finely regulate calcium signaling, mitochondria adapt their metabolism, shape, and distribution within the cells, including relative positioning with respect to other organelles. However, physical contacts between mitochondria and the secretory/endocytic pathway have been demonstrated so far only with the ER, through structural and functional interorganellar connections. Here we show by electron tomography that mitochondria physically contact melanosomes, specialized lysosome-related organelles of pigment cells, through fibrillar bridges resembling the protein tethers linking mitochondria and the ER. Mitofusin (Mfn) 2, which bridges ER to mitochondria, specifically localizes also to melanosome-mitochondrion contacts, and its knockdown significantly reduces the interorganellar connections. Contacts are associated tothe melanogenesis process, as indicated by the fact that they are reduced in a model of aberrant melanogenesis whereas they are enhanced both where melanosome biogenesis takesplace in the perinuclear area and when it is actively stimulated by OA1, a G protein-coupled receptor implicated in ocular albinism and organellogenesis. Consistently, Mfn2 knockdown prevents melanogenesis activation by OA1, and the pharmacological inhibition of mitochondrial ATP synthesis severely reduces contact formation and impairs melanosome biogenesis, by affecting in particular the developing organelles showing the highest frequency of contacts. Altogether, our findings reveal the presence ofan unprecedented physical and functional connection between mitochondria and the secretory/endocytic pathway that goes beyond the ER-mitochondria linkage and is spatially and timely associated to secretory organelle biogenesis.

Requirement of a dopaminergic neuronal phenotype for toxicity of low concentrations of 1-methyl-4-phenylpyridinium to human cells

LUHMES cells are conditionally-immortalized non-transformed human fetal cells that can be differentiated to acquire a dopaminergic neuron-like phenotype under appropriate growth conditions. After differentiation by GDNF and cyclic adenosine monophosphate, LUHMES were sensitive to 1-methyl-4-phenylpyridinium (MPP +) toxicity at ≤ 5 μM, but resistant to the parental compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The high homogeneity and purity of the cultures allowed the detection of metabolic changes during the degeneration. Cellular ATP dropped in two phases after 24 and 48 h; cellular glutathione (GSH) decreased continuously, paralleled by an increase in lipid peroxidation. These events were accompanied by a time-dependent degeneration of neurites. Block of the dopamine transporter by GBR 12909 or mazindol completely abrogated MPP + toxicity. Inhibition of de novo dopamine synthesis by α-methyl- l-tyrosine or 3-iodo- l-tyrosine attenuated toxicity, but did not reduce the initial drop in ATP. Inhibition of mixed lineage kinases by CEP1347 completely prevented the MPP +-induced loss of viability and intracellular GSH, but failed to attenuate the initial drop of ATP. For the quantitative assessment of neurite degeneration, an automated imaging-based high content screening approach was applied and confirmed the findings made by pharmacological interventions in this study. Our data indicate that inhibition of mitochondrial ATP synthesis is not sufficient to trigger cell death in MPP +-treated LUHMES.

166 POSTER Inhibitors of mitochondrial ATP synthesis show preferential cytotoxicity to pancreatic cancer cells under glucose-deprived conditions

166 POSTER Inhibitors of mitochondrial ATP synthesis show preferential cytotoxicity to pancreatic cancer cells under glucose-deprived conditions

The acute lethality of acrylonitrile is not due to brain metabolic arrest

Acrylonitrile (AN) is an organic compound produced in large quantities by the chemical industry and is acutely toxic. One mechanism proposed to explain the toxicity of AN is metabolism by P450 into cyanide (CN). Although blood and brain levels of CN in rats following an LD90 dose of AN are consistent with acute toxicity, blocking CN formation with P450 inhibitors does not prevent lethality. Another mechanism implicated in toxicity is covalent binding of AN to cysteine residues in tissue proteins. Previous work in our laboratory has shown that AN can irreversibly inactivate the catalytically active cysteine-149 in glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Inactivation of GAPDH by AN would be expected to impair glycolytic ATP production and, when coupled to the inhibition of mitochondrial ATP synthesis by the AN metabolite CN, would result in metabolic arrest, particularly in brain. In this study we have measured the high energy metabolites phosphocreatine (PCr), ATP, ADP and AMP by HPLC and compared their levels in the brains of rats treated with an LD90 dose of AN, when respiration ceased, vs. controls. Two methods of rapid brain freezing in liquid nitrogen were used: funnel freezing (FF) and head immersion (HI). AN administration resulted in large decreases in PCr of 74% (FF) and 80% (HI) but relatively minor decreases in ATP of 5% (FF) and 21% (HI) and Energy Charge of 6% (FF) and 10% (HI). Thus, although substantial depletion of PCr was observed, possibly due to inhibition of creatine kinase by AN, we found no evidence that brain ATP is depleted when respiration ceases in AN-intoxicated rats.

ATP hydrolysis-driven H+ translocation is stimulated by sulfate, a strong inhibitor of mitochondrial ATP synthesis

Sulfate is a partial inhibitor at low and a non-essential activator at high [ATP] of the ATPase activity of F(1). Therefore, a catalytically-competent ternary F(1) x ATP x sulfate complex can be formed. In addition, the ANS fluorescence enhancement driven by ATP hydrolysis in submitochondrial particles is also stimulated by sulfate, clearly showing that the ATP hydrolysis in its presence is coupled to H(+) translocation. However, sulfate is a strong linear inhibitor of the mitochondrial ATP synthesis. The inhibition was competitive (K (i) = 0.46 mM) with respect to Pi and mixed (K (i) = 0.60 and K'(i) = 5.6 mM) towards ADP. Since it is likely that sulfate exerts its effects by binding at the Pi binding subdomain of the catalytic site, we suggest that the catalytic site involved in the H(+) translocation driven by ATP hydrolysis has a more open conformation than the half-closed one (beta(HC)), which is an intermediate in ATP synthesis. Accordingly, ATP hydrolysis is not necessarily the exact reversal of ATP synthesis.

Happily at Work

Happily at Work

Acrylonitrile irreversibly inactivates glyceraldehyde-3-phosphate dehydrogenase by alkylating the catalytically active cysteine 149

Acrylonitrile (AN) is a vinyl monomer used in the production of synthetic fibers, rubber and plastics. AN is acutely toxic but its mechanism of toxicity remains to be established. AN is metabolized to cyanide in vivo but cyanide production alone cannot explain acute AN toxicity. Previous work in our laboratory has shown that AN can alkylate highly reactive cysteine residues in proteins. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a critical enzyme involved in glycolysis, has a catalytically active cysteine 149 in its active site. We report that AN irreversibly inhibits GAPDH with second-order rate constants, at pH 7.4, of 3.7 and 9.2 M −1 s −1 at 25 and 37 °C, respectively. A combination of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) and electrospray ionization–mass spectrometry–mass spectrometry (ESI–MS–MS) was used to show that AN inactivates GAPDH by covalently binding to cysteine 149 in the active site of the enzyme. Inactivation of GAPDH by AN would be expected to impair glycolytic ATP production and when coupled with the inhibition of mitochondrial ATP synthesis by the AN metabolite cyanide would result in metabolic arrest. The brain can withstand metabolic arrest for only a few minutes thus these combined actions may account for the acute toxicity of AN in vivo.