What happens if atp synthase is inhibited

This approach takes a known inhibitor and structurally modifies it to give more potent inhibitors. This approach involves the de novo design of inhibitor scaffolds. For example, fragments are positioned in chosen sites in the ATP synthase and then joined to create full molecules. These molecules are scored and ranked for factors such as their predicted binding affinity. In virtual screening, rapid docking algorithms are used to search databases of commercially available compounds in order to identify novel molecules predicted to bind to the chosen protein target.

Our team provides you with outstanding support and meets your specific needs with a professional technology platform. If you are interested in our services, please contact us for more details. Product Service. Hydrolysis of the third phosphate group produces adenosine diphosphate ADP and inorganic phosphate Pi , along with considerable release of energy.

Other than supporting almost all the cellular functions that require energy, ATP also works as a coenzyme during phosphorylation reactions. Khakh and Burnstock, established that ATP also serves as a critical signaling molecule that allows inter and intracellular communications. The ubiquitous distribution of ATP allows for signaling functions that have a uniquely broad influence on physiological functioning [ 1 ].

Estimates show that the normal body uses 40 kg of ATP on a daily basis; hence ATP production is one of the most frequent processes occurring in the body [ 2 ]. The aggregate amount of ATP in the human body is about 0.

The energy utilized every day by an adult requires the hydrolysis of to Moles of ATP. This implies every ATP molecule must be reused or more multiple times in a day [ 3 ].

ATP cannot be stored and so its synthesis is closely linked to its consumption. These enzymes are found in the cristae and the inner membrane of mitochondria, the thylakoid membrane of chloroplasts, and the plasma membrane of bacteria [ 5 ]. Usually, there is a general understanding that ATP generation occurs in mitochondria. However, in the case of bacteria and archaea that lack mitochondria, ATP synthase is found in their plasma membrane. Additionally, ATP synthases are licensed to inhabit the chloroplast of plant cells.

Production and Utilization of Acetyl CoA: The complex macromolecules present in dietary food are processed through various metabolic pathways into Acetyl CoA. Acetyl CoA in the mitochondria is then oxidized to carbon dioxide and water through the Citric Acid Cycle and Oxidative Phosphorylation. ATP synthesis is the most widespread chemical reaction inside the biological world.

ATP synthase is the very last enzyme in oxidative phosphorylation pathway that makes use of electrochemical energy to power ATP synthesis [ 7 , 8 , 9 , 10 ]. The mitochondrial ATP synthase is a multi-subunit protein complex having an approximate molecular weight of kDa. This enzyme is the smallest known biological nanomotor and plays a crucial role in ATP generation. In plants, energy acquired from photons is transferred through photosynthetic electron transport chain ETC , which induces an electrochemical gradient to build up across the membrane.

Basically, protons are pumped across the inner mitochondrial membrane as electrons pass through the electron transfer chain. This induces a proton gradient, with a decreased pH in the intermembrane space and an increased pH in the matrix of the mitochondria. The proton gradient and membrane potential are the major forces involved in ATP synthesis. It is well established that the electrochemical potential of protons delivered by electron transfer chains across the mitochondrial, chloroplast or bacterial membrane provides the energy for ATP synthesis [ 14 ].

Cellular respiration in the mitochondria is a widely studied process that incorporates chemiosmosis for the production of ATP. Mitochondria, the chief organelles producing ATP, are absent in prokaryotic organisms. In the absence of mitochondria, archaea and bacteria maneuver chemiosmosis to produce ATP through photophosphorylation.

The electrochemical energy built through the difference in proton concentration and separation of charge across inner mitochondrial membrane translates to the proton motive force PMF. This also satisfies a main criterion stated by Mitchell for the chemiosmotic coupling to occur: the inner mitochondrial membrane must be impermeable to protons.

Thus, protons are compelled to re-enter matrix through F 0 while F 1 catalyzes the synthesis of ATP [ 16 ]. The Electron transport chain composed of four different multi-subunit complexes transfer electrons e- in a sequential manner ultimately reducing O 2 to H 2 O. Electron transfer is coupled to a vectorial proton translocation outdoor into the matrix via three of the four complexes I, III and IV.

Protons gather and create an electrochemical gradient throughout the inner mitochondrial membrane. This osmotic potential is used to power ATP synthesis when protons re-enter the mitochondrial matrix through ATP synthase [ 13 ]. The equation for reaction catalyzed is:. There are only slight variations in its structure in the chloroplast and in the mitochondria. The chloroplast ATPase has two isoforms and in the mitochondria it has additional subunits.

Besides these differences, ATPases are structurally and functionally similar. The F 0 part, bound to inner mitochondrial membrane is involved in proton translocation, whereas the F 1 part found in the mitochondrial matrix is the water soluble catalytic domain. F 1 is the first factor recognized and isolated from bovine heart mitochondria and is involved in oxidative phosphorylation.

F 0 was named so as it is a factor that conferred oligomycin sensitivity to soluble F 1 [ 18 ]. Schematic subunit composition of ATP synthase. The structure of enzyme ATP synthase mimics an assembly of two motors with a shared common rotor shaft and stabilized by a peripheral stator stalk. Bacterial F 0 has the simplest subunit structure consisting a 1 , b 2 and c subunits.

Other additional subunits such as subunit e, f, g, and A6L extending over the membrane cohort with F 0 [ 5 , 10 , 20 ]. Paul Boyer proposed a simple catalytic scheme, commonly known as the binding change mechanism, which predicted that F-ATPase implements a rotational mechanism in the catalysis of ATP [ 21 ]. The movement of subunits within the ATP synthase complex plays essential roles in both transport and catalytic mechanisms. Another subsequent change in conformation brings about the release of ATP.

These conformational changes are accomplished by rotating the inner core of the enzyme. The core itself is powered by the proton motive force conferred by protons crossing the mitochondrial membrane.

In this mini review, we will briefly summarize recent findings regarding the regulation of the expression and activity of IF1, playing special emphasis to the results obtained in conditional tissue-specific transgenic mice overexpressing IF1 or an active mutant version of the protein. More detailed reviews on other aspects in this subject can be found elsewhere Garcia-Bermudez and Cuezva, ; Esparza-Molto et al.

Based on in situ hybridization databases, it has been suggested that IF1 is ubiquitously expressed in mammalian tissues Faccenda et al. However, analysis of the expression of the protein in several human tissues revealed that its expression varies greatly, with high expression restricted to heart, liver, kidney, stomach, and brain and negligible expression in breast, colon, lung, and ovary Sanchez-Cenizo et al.

In contrasts, IF1 is highly overexpressed in human colon, lung, breast and ovarian carcinomas Sanchez-Cenizo et al. Consistent with a relevant role played by IF1 in cellular metabolic reprograming, pulse-chase Sanchez-Arago et al. Moreover, changes in the expression of IF1 during cellular differentiation are accompanied by profound changes in the rate of IF1 turnover Sanchez-Arago et al. Hence, these findings support that regulation of IF1 expression is tissue-specific and controlled at posttranscriptional levels Figure 1A.

Studies addressing the basal expression and mechanisms that regulate IF1 levels in human and mouse tissues are required in order to deepen into the characterization of its role in cellular physiology and in the regulation of the ATP synthase. Posttranscriptional regulation of the expression and activity of IF1. A The expression of the short-lived IF1 protein is tissue-specific and regulated at posttranscriptional levels Sanchez-Arago et al. Inhibition of the enzyme triggers the raise in mitochondrial membrane potential and the production of mitochondrial ROS Sanchez-Cenizo et al.

Overexpression of IEX-1 resulted in the downregulation of IF1 by targeting the protein to degradation by an unidentified protease Figure 1A ; Shen et al. A short-interfering RNA-based screen aimed at the identification of potential proteases involved in the degradation of IF1 in human colon cancer cells and hMSCs failed to provide candidates for the degradation of IF1 Sanchez-Arago et al. Consistent with the stringent tissue-specific posttranscriptional regulation of IF1 expression, it has been described that conditional knockout mice devoid of the RNA binding protein LRPPRC leucine rich pentatricopeptide repeat containing protein in heart have impaired ATP synthase activity as a result of a deficient assembly of the enzyme Mourier et al.

Interestingly, the upregulation of IF1 in heart of these mice takes place in the absence of relevant changes in the availability of IF1 mRNA and it is accompanied by the appearance of IF1-bound in subassembled ATP synthase complexes Mourier et al. Moreover, it is known that LRPPRC associates with both nuclear and mitochondrial mRNAs and as such is a candidate for coordinating nuclear and mitochondrial gene expression at posttranscriptional levels Mili and Pinol-Roma, Remarkably, recent findings have stressed the role of IF1 bound to the key inhibited intermediate F1-c8 complex in the assembly pathway of the mammalian ATP synthase He et al.

This suggestion is in agreement with the appearance of IF1-bound in subassemblies of ATP synthase complexes Mourier et al. It seems obvious that additional studies are required to unveil the posttranscriptional mechanisms that regulate the tissue-specific biogenesis of mitochondrial OXPHOS complexes and specifically of those affecting the ATP synthase and its inhibitor, both in health and in disease.

Above neutral pH, IF1 forms inactive tetramers by occlusion of its inhibitory N-terminal disordered domain. One important residue of IF1 is histidine 49, being the mutant H49K protein active as inhibitor even at pH above neutrality Schnizer et al.

In addition to the pH regulated activity of IF1, we have recently described that the regulation of its activity as inhibitor of the ATP synthase also involves protein phosphorylation Figure 1B ; Garcia-Bermudez et al. In fact, the development and expression of the phospho-deficient and phospho-mimetic serine mutants of IF1 demonstrated that phosphorylation of IF1 in serine 39 S39 renders a protein unable to bind the ATP synthase what results in an increase of the enzyme activity Figure 1B ; Garcia-Bermudez et al.

Only the dephosphorylated IF1 or the phospho-deficient S39A is able to bind and inhibit both the synthase and hydrolase activities of the enzyme in different physiological situations that result in the reprogramming of cellular energy metabolism to an enhanced glycolysis Figure 1B ; Garcia-Bermudez et al. In this regard, dephosphorylated IF1 is present in hypoxic cells and the fraction of IF1 bound to the ATP synthase is significantly increased in this situation Garcia-Bermudez et al.

Interestingly, metabolic reprogramming to an enhanced aerobic glycolysis in human carcinomas also correlates with the expression of dephosphorylated IF1 Garcia-Bermudez et al. In contrast, cells in G1, the high-energy demanding phase of the cell cycle, display phosphorylated IF1 and an increased ATP synthase activity Garcia-Bermudez et al.

The phosphorylation status of IF1 depends on the cell type analyzed Garcia-Bermudez et al. Administration of the agonist clenbuterol promoted a significant increase in the fraction of phosphorylated IF1 present in heart concurrently with an increase in the ATP synthase activity in mitochondria.

These findings also suggested that in some high-energy demanding tissues, there is a fraction of IF1-bound to the enzyme, maintaining a pool of inactive ATP synthase in order to facilitate the tissue response to a sudden physiological increase in energy requests Garcia-Bermudez et al. Clearly, there is lack of knowledge regarding the tissue content and state of phosphorylation of IF1 in relation to the content of the ATP synthase in mammalian tissues.

A sAC has been described inside mitochondria Acin-Perez et al. In fact, that would be in agreement with the observed phosphorylation of IF1 in response to treatment of the cells with forskolin Garcia-Bermudez et al. Examples of mitochondrial proteins phosphorylated by the activity of PKA in advance of its import are already available De Rasmo et al.

Attenuation of the cAMP signal on targeted proteins is exerted by the large family of phosphodiesterases Maurice et al. The mechanisms regulating the dephosphorylation of IF1 are presently unknown.

Overall, IF1 inhibits both the synthetic and hydrolytic activities of the ATP synthase as long as the inhibitor protein is bound to the enzyme Garcia-Bermudez et al. Under pathophysiological conditions triggered by a mitochondrial deficiency in oxygen availability and matrix acidification, it has been suggested that IF1 can bind the ATP synthase to prevent its hydrolase activity Rouslin and Pullman, ; Campanella et al.

However, recent findings argue against the operation of the ATP synthase in reverse under hypoxic conditions Sgarbi et al. In fact, these authors have shown that the hydrolase activity of the ATP synthase is not operative unless mitochondria are challenged by the addition of an uncoupler, an extreme anoxia-mimicking condition Sgarbi et al. In addition, it should be taken into consideration that any biochemical manifestation of the effect of IF1 on ATP synthase activities largely depend on the molar ratio that exists between the inhibitor protein and the ATP synthase in the mitochondria of that particular cell, because the mass-action ratio also controls the interaction of both proteins Sanchez-Cenizo et al.

Several findings fostered the idea that IF1, besides regulating the production of ATP, exerts additional functions in mitochondrial physiology Bernardi et al. It is accepted that the amount and site of production of mtROS, which are essential signaling molecules, defines the nuclear response of the cell to different cues Martinez-Reyes and Cuezva, ; Yun and Finkel, ; Shadel and Horvath, This process, coined as retrograde signaling from mitochondria to the nucleus, finally determines cellular responses by controlling the expression of nuclear genes that facilitate adaptation of the organism to different physiological cues or cytotoxic agents Quiros et al.

In this regard, there is growing evidence supporting that a mild mitochondrial stress can protect cells from subsequent insults, a concept termed mitohormesis Figure 1B ; Ristow, ; Yun and Finkel, ; Chandel, ; Quiros et al.

The induction of mitohormesis can be accomplished by different ways that affect mitochondrial function such as by inhibitors of the electron transport chain, mitochondrial translation, mtROS generators, etc. In the following, we will review the mitohormetic responses affecting health in transgenic mice by overexpressing IF1 to inhibit in vivo the activity of OXPHOS in neurons Formentini et al.

Mitohormetic responses targeting the ATP synthase and affecting lifespan have been recently reviewed elsewhere Esparza-Molto et al. IF1-mediated inhibition of the ATP synthase and signaling pathways of mitohormesis. The scheme shows the conditional tissue-specific transgenic mouse models developed and summarized in this mini review.

Overall, the main effect of mitohormesis is to warranty cell survival, metabolism, the antioxidant, and immune responses of the organism to allow adaptation to different stressful conditions. The numbers in brackets indicate the corresponding mouse model.

Mice expressing the pH-insensitive constitutively active mutant H49K version of human IF1 IF1-H49K in neurons Figure 2 revealed the inhibition of the ATP synthase as assessed in total brain extracts, isolated brain mitochondria, and primary cultures of cortical neurons without affecting the expression of proteins from different OXPHOS complexes Formentini et al.

Consistent with the inhibition of the synthase, transgenic mice revealed a significant reduction in brain ATP concentrations, the activation of the metabolic sensor AMPK Figure 2 and the concurrent increased expression of glycolytic proteins Formentini et al.

Interestingly, and when compared to control littermates, the inhibition of the ATP synthase also resulted in the inhibition of the activity of Complex IV of the respiratory chain by preventing the assembly of monomers of Complex IV into supercomplexes Formentini et al.

Primary cultures of cortical neurons of transgenic mice confirmed the IF1-mediated inhibition of the ATP synthase, the functional and proteomic reprogramming of neurons to an enhanced aerobic glycolysis and importantly, the increased production of mtROS and carbonylation of cellular proteins when compared to neurons of control littermates Formentini et al.

Despite the changes observed in transgenic mice, we noted no major phenotypic differences when compared to controls unless the animals were challenged by a cytotoxic insult Formentini et al. Phenotypic analysis of mice after injection of quinolinic acid into the left striatal region of the brain to induce neurotoxicity Schwarcz et al. In other words, transgenic mice were partially protected from damage, indicating that metabolic preconditioning afforded by the inhibition of the ATP synthase protects neurons from the oxidative insult Figure 2.

This finding that was also confirmed in cultures of cortical neurons primed to death by glutamate addition Formentini et al. Three different locomotor tests further confirmed the better performance of transgenic mice over controls in neurological examinations, also coinciding with a better maintenance of the cellular redox state in the affected hemisphere of mice expressing IF1-H49K Formentini et al. In less sensitive cells, the inhibitors of glycolytic ATP production promoted their effect. This effect was assumed to underlay an anti-apoptotic action of oligomycin during apoptosis induced by such anticancer drugs as etoposide and dexamethasone Eguchi et al.

Other mechanisms of anti-apoptotic action of oligomycin have also been suggested. It was reported to block dimerization of Bax a pro-apoptotic Bcl-2 family member and following cytochrome c release involved in the calphostin C-induced apoptosis Ikemoto et al. Inhibition of cytochrome c release by oligomycin was also inherent in some Bax-independent apoptoses Matsuyama et al.

In the present study, effects of two inducers of apoptosis were investigated, namely Sts and TNF. Apoptosis induced by Sts, a nonselective inhibitor of protein kinases, resembles the great variety of stress-induced apoptoses. Signaling pathways of this type usually involve mitochondria as well as Bax, which oligomerizes and binds to mitochondria Hsu et al. The following Bid-induced mitochondrial events depend on Bak, a close relative of Bax which, in contrast to Bax, is always bound to the outer mitochondrial membrane Wei et al.

The data on a possible role of the PTP in Sts- and TNF-induced apoptoses are controversial partially because of a complex pattern of accompanying disturbances in mitochondrial energetics, which may differ in different cells.

In our experiments, we used HeLa cells. These cells, as was found in our group, are resistant to pro- and antiapoptotic effects of the inhibitors of mitochondrial respiratory chain and uncouplers of oxidative phosphorylation Shchepina et al. It is found that oligomycin arrests the TNF-, but not Sts- induced apoptosis. To test the effect of inhibitors of oxidative phosphorylation on apoptosis, we choose the HeLa carcinoma cell line.

These cells as a number of other rapidly growing, non-differentiated tumor cell lines have high levels of both respiration and aerobic glycolysis.

In this case, no signs of apoptosis were detected and Bcl-2 did not protect against the cell death not shown. These data indicate that the mitochondrial and glycolytic ATP productions are interchangable for survival of HeLa cells. Apoptotic cell death induced by Sts or TNF: effects of mitochondrial inhibitors. Results of five experiments.

For additions, see a — c. Results of three experiments. Oligomycin, respiratory inhibitors and uncouplers did not affect apoptotic cell death and the release of cytochrome c from mitochondria into cytosol. Overexpression of Bcl-2 in HeLa cells prevented the release of cytochrome c and cell death induced by Sts. These effects of Bcl-2 were insensitive to mitochondrial inhibitors Figures 2b and 3 c. Measurements of oxygen consumption showed that expression of Bcl-2 did not change effects of oligomycin, uncouplers and respiratory inhibitors on the respiration rate Figure 1.

Chromatin condensation and fragmentation a , and release of cytochrome c from mitochondria into cytosol b , c during apoptosis in HeLa and HeLa Bcl Immunostaining of cytochrome c with 7H8. Arrows indicate apoptotic cells. A total of cells were analysed in each sample. Horse cytochrome c was used as a marker Cyt c.

Concentrations of additions as in Figure 2. TNF is shown to induce typical apoptosis in HeLa cells only in combination with emetine, inhibitor of protein synthesis Sidoti-de Fraisse et al. Apoptotic changes in cellular morphology and condensation of chromatin were observed.

They correlated with the cytochrome c release into cytosol and were prevented by Bcl The inhibitors of respiration and the uncouplers did not interfere with cytochrome c release, apoptosis and the protective action of Bcl-2 Figures 2c and 3.

In contrast to the effects of Sts, the TNF-induced release of cytochrome c and apoptosis were strongly inhibited by oligomycin Figures 2c and 3. These data indicate that oligomycin inhibits the TNF signaling after formation of the death-inducing-signaling complex DISC composed of the TNF receptor, adapter proteins and procaspase On the other hand, the late steps of the apoptotic cascade presumably after the release of cytochrome c were unaffected by oligomycin.