Free ATP Research Articles

Potency and mechanism of p-glycoprotein chemosensitizers in rainbow trout (Oncorhynchus mykiss) hepatocytes.

The membrane efflux transporter P-glycoprotein (P-gp, [ABCB1, MDR1]) exports a wide range of xenobiotic compounds, resulting in a continuous first line of defense against toxicant accumulation at basal expression levels, and contributing to the multixenobiotic resistance (MXR) phenotype at elevated expression levels. Relatively little information exists on P-gp inhibition in fish by chemosensitizers, compounds which lower toxicity thresholds for harmful P-gp substrates in complex mixtures. The effects of four known mammalian chemosensitizers (cyclosporin A [CsA], quinidine, valspodar [PSC833], and verapamil) on the P-gp-mediated transport of rhodamine 123 (R123) and cortisol in primary cultures of rainbow trout (Oncorhynchus mykiss) hepatocytes were examined. Competitive accumulation assays using 25µM R123 or cortisol and varying concentrations of chemosensitizers (0-500µM) were used. CsA, quinidine, and verapamil inhibited R123 export (IC50 values ± SE: 132 ± 60, 83.3 ± 27.2, and 43.2 ± 13.6µM, respectively). CsA and valspodar inhibited cortisol export (IC50 values: 294 ± 106 and 92.2 ± 34.9µM, respectively). In an ATP depletion assay, hepatocytes incubated with all four chemosensitizers resulted in lower free ATP concentrations, suggesting that they act via competitive inhibition. Chemosensitizers that inhibit MXR transporters are an important class of environmental pollutant, and these results show that rainbow trout transporters are inhibited by similar chemosensitizers (and mostly at similar concentrations) as seen in mammals and other fish species.

Enhanced triacylglycerol metabolism contributes to the efficient biosynthesis of spinosad in Saccharopolyspora spinosa

Triacylglycerol (TAG) is crucial for antibiotic biosynthesis derived from Streptomyces, as it serves as an important carbon source. In this study, the supplementation of exogenous TAG led to a 3.92-fold augmentation in spinosad production. The impact of exogenous TAG on the metabolic network of Saccharopolyspora spinosa were deeply analyzed through comparative proteomics. To optimize TAG metabolism and enhance spinosad biosynthesis, the lipase-encoding genes lip886 and lip385 were overexpressed or co-expressed. The results shown that the yield of spinosad was increased by 0.8-fold and 0.4-fold when lip886 and lip385 genes were overexpressed, respectively. Synergistic co-expression of these genes resulted in a 2.29-fold increase in the yield of spinosad. Remarkably, the combined overexpression of lip886 and lip385 in the presence of exogenous TAG elevated spinosad yields by 5.5-fold, led to a drastic increase in spinosad production from 0.036 g/L to 0.234 g/L. This study underscores the modification of intracellular concentrations of free fatty acids (FFAs), short-chain acyl-CoAs, ATP, and NADPH as mechanisms by which exogenous TAG modulates spinosad biosynthesis. Overall, the findings validate the enhancement of TAG catabolism as a beneficial strategy for optimizing spinosad production and provide foundational insights for engineering secondary metabolite biosynthesis pathways in another Streptomyces.

Physiological responses and quality assessment of turbot (Scophthalmus maximus) during waterless preservation and transportation: Implications for survival and biochemical indicators

In this study, changes in the physiological parameters of the muscles of living turbot (Scophthalmus maximus) fish during waterless preservation and transportation were assessed. Results indicated that the survival rate of turbot at low temperature, waterless conditions in pure O2 was 100% at 24 h and 95% at 36 h. Crude fat, organic acids, free amino acids, ATP levels, and pH values in the fish at 12 h after revitalization were not significantly different (p < 0.05) in the waterless preservation treatment than they were in the control group. Total protein content in turbot meat of recovered fish was close to the content in the control group. Although the protein content was lower than it was in the control group, the difference was negligible. Moreover, the elasticity, chewiness, and viscosity of turbot meat after 12 h of recovery was the same as those attributes in fish in the control group. The low-temperature waterless preservation treatment induced a strong stress response in turbot fish, characterized by strong biochemical reactions that resulted in fluctuations in the level of several stress indicators. Nevertheless, the recovery period provided a partial recovery of the indicator levels. The results of this study will contribute to the development of an effective, eco-friendly method to improve the survival rate and quality of living turbot fish during waterless preservation and transport. The study provides new insights that can be used to improve the waterless preservation and transport of living turbot fish, while addressing both market needs and consumer expectations.

ATP modulates self-perpetuating conformational conversion generating structurally distinct yeast prion amyloids that limit autocatalytic amplification

Prion-like self-perpetuating conformational conversion of proteins into amyloid aggregates is associated with both transmissible neurodegenerative diseases and non-Mendelian inheritance. The cellular energy currency ATP is known to indirectly regulate the formation, dissolution, or transmission of amyloid-like aggregates by providing energy to the molecular chaperones that maintain protein homeostasis. In this work, we demonstrate that ATP molecules, independent of any chaperones, modulate the formation and dissolution of amyloids from a yeast prion domain (NM domain of Saccharomyces cerevisiae Sup35) and restricts autocatalytic amplification by controlling the amount of fragmentable and seeding-competent aggregates. ATP, at (high) physiological concentrations in the presence of Mg2+, kinetically accelerates NM aggregation. Interestingly, ATP also promotes phase-separation-mediated aggregation of a human protein harboring a yeast prion-like domain. We also show that ATP disaggregates preformed NM fibrils in a dose-independent manner. Our results indicate that ATP-mediated disaggregation, unlike the disaggregation by the disaggregase Hsp104, yields no oligomers that are considered one of the critical species for amyloid transmission. Furthermore, high concentrations of ATP delimited the number of seeds by giving rise to compact, ATP-bound NM fibrils that exhibited nominal fragmentation by either free ATP or Hsp104 disaggregase to generate lower molecular weight amyloids. Additionally, (low) pathologically relevant ATP concentrations restricted autocatalytic amplification by forming structurally distinct amyloids which are found seeding-inefficient due to their reduced β-content. Our results provide key mechanistic underpinnings of concentration-dependent chemical chaperoning by ATP against prion-like transmissions of amyloids.

Activation of P2X4 receptor exacerbates acute brain injury after intracerebral hemorrhage.

IntroductionIntracerebral hemorrhage (ICH) accounts for 10%–15% of all strokes and culminates in high mortality and disability. After ICH, brain injury is initiated by the mass effect of hematoma, followed by secondary cytotoxic injury from dying brain cells, hematoma disintegration, and cascading brain immune response. However, the molecular mechanism of secondary cytotoxic brain injury in ICH is not completely understood. The sensitive purinergic receptor, P2X4 receptor (P2X4R), was known to recognize extracellular free ATP released by dying cells during tissue injury.AimsIn this study, we aim to understand the role of P2X4R in acute brain injury triggered by ICH.ResultsIn this study, we found that the sensitive purinergic receptor, P2X4R, was upregulated in the brain of patients with ICH as well as in a mouse model of ICH induced by collagenase injection. P2X4R blockage with the specific inhibitor 5‐BDBD attenuated brain injury in ICH mice by significantly reducing brain edema, blood–brain barrier leakage, neural death, and ultimately acute neurodeficits. Further study indicated that the protective effect of P2X4R inhibition is related to decreased pro‐inflammatory activity of microglia and recruitment of peripheral immune cells into the hemorrhagic brain.ConclusionsThese results suggest that the P2X4 receptor is activated by ICH stimuli which worsen brain injury following ICH.

ATP Can Efficiently Stabilize Protein through a Unique Mechanism.

Recent experiments suggested that ATP can effectively stabilize protein structure and inhibit protein aggregation when its concentration is less than 10 mM, which is significantly lower than cosolvent concentrations required in conventional mechanisms. The ultrahigh efficiency of ATP suggests a unique mechanism that is fundamentally different from previous models of cosolvents. In this work, we used molecular dynamics simulation and experiments to study the interactions of ATPs with three proteins: lysozyme, ubiquitin, and malate dehydrogenase. ATP tends to bind to the surface regions with high flexibility and high degree of hydration. These regions are also vulnerable to thermal perturbations. The bound ATPs further assemble into ATP clusters mediated by Mg2+ and Na+ ions. More interestingly, in Mg2+-free ATP solution, Na+ at higher concentration (150 mM under physiological conditions) can similarly mediate the formation of the ATP cluster on protein. The ATP cluster can effectively reduce the fluctuations of the vulnerable region and thus stabilize the protein against thermal perturbations. Both ATP binding and the considerable improvement of thermal stability of ATP-bound protein were verified by experiments.

Ispitivanje neuroprotektivnog efekta inhibicije Ro-kinaze u ćelijskom modelu neurodegeneracije izazvane 1-metil-4-fenilpiridinijumom (MPP+)

Introduction: The 1-methyl 4-phenyl 1,2,3,6-tetrahydropiridium (MPTP) induced model of neurodegeneration in Parkinson's disease (PD) is one of the most commonly used experimental models. This neurotoxic agent , or rather its metabolite MPP+, leads to inhibition of mitochondrial complex I, an increase in free radicals' production and ATP depletion, all resulting in cellular demise and death. Rho-kinase is an enzyme involved with numerous cellregulatory mechanisms, such as cytoskeleton organization, axonogenesis, vesicular transport regulation and apoptosis regulation, which are all important for cell survival. Aim: Our aim was to investigate the effects of Rho-kinase inhibition on the MPP+ induced model of neurodegeneration and the role of Akt and adenosine monophosphate-activated protein kinase (AMPK) signaling pathways in this process. Material and methods: The experiments were performed on the human neuroblastoma SHSY5Y cell line. The MTT test was used to measure the viability of the cells after the MPP+ and/ or Rho-kinase inhibitor, fasudil, treatments. Changes in activation levels, or expression of pAMPK, pAkt, AMPK and Akt, were measured using the immunoblotting method, and the protein levels were quantified by densitometry. Results: The MPP+ caused a dose-dependent decrease in cellular viability, compared to the control group (untreated cells), while fasudil treatment, prior to MPP+ exposure, improved cell viability in a dose dependant manner, compared to MPP+ treatment. Analysis of activation status of target proteins showed an increase in Akt activation after the fasudil treatment, while the AMPK activation was not significantly changed. Conclusion: Inhibition of Rho-kinase using fasudil causes a decrease in MPP+ induced cell death, which is possibly mediated by an activation of the Akt/PI3K signaling pathway.

Allosteric modulation of alternatively spliced Ca2+-activated Cl− channels TMEM16A by PI(4,5)P2 and CaMKII

Transmembrane 16A (TMEM16A, anoctamin1), 1 of 10 TMEM16 family proteins, is a Cl- channel activated by intracellular Ca2+ and membrane voltage. This channel is also regulated by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. We find that two splice variants of TMEM16A show different sensitivity to endogenous PI(4,5)P2 degradation, where TMEM16A(ac) displays higher channel activity and more current inhibition by PI(4,5)P2 depletion than TMEM16A(a). These two channel isoforms differ in the alternative splicing of the c-segment (exon 13). The current amplitude and PI(4,5)P2 sensitivity of both TMEM16A(ac) and (a) are significantly strengthened by decreased free cytosolic ATP and by conditions that decrease phosphorylation by Ca2+/calmodulin-dependent protein kinase II (CaMKII). Noise analysis suggests that the augmentation of currents is due to a rise of single-channel current (i), but not of channel number (N) or open probability (PO). Mutagenesis points to arginine 486 in the first intracellular loop as a putative binding site for PI(4,5)P2, and to serine 673 in the third intracellular loop as a site for regulatory channel phosphorylation that modulates the action of PI(4,5)P2 In silico simulation suggests how phosphorylation of S673 allosterically and differently changes the structure of the distant PI(4,5)P2-binding site between channel splice variants with and without the c-segment exon. In sum, our study reveals the following: differential regulation of alternatively spliced TMEM16A(ac) and (a) by plasma membrane PI(4,5)P2, modification of these effects by channel phosphorylation, identification of the molecular sites, and mechanistic explanation by in silico simulation.

Irisin/FNDC5: A participant in camel metabolism.

The quantification, localization, production, function, and regulation of irisin/FNDC5 in camel species have not been previously studied. The objective of this study was to detect the irisin content in Arabian camel blood and tissues and study the gene expression of FNDC5 and PGC-1α in camel skeletal muscles and white adipose tissue depots under basal conditions. To monitor if exercise influences blood and tissue irisin protein levels as well as FNDC5 and PGC-1α gene expression levels, we analyzed irisin concentrations in the serum, skeletal muscles (soleus and gastrocnemius), and white adipose tissues (hump, subcutaneous, visceral, epididymal, and perirenal) in both control (n = 6) and exercised group (n = 6) using ELISA and determined the cellular localization of irisin/FNDC5 and the mRNA levels of FNDC5 and PGC-1α in skeletal muscles and adipose tissues via immunohistochemistry and real-time PCR, respectively. The possible regulatory roles of exercise on some hormones and metabolites as well as the detection of links between serum irisin and other circulating hormones (insulin, leptin, and cortisol) and metabolites (glucose, free fatty acids, triglycerides, and ATP) were explored for the first time in camels. Our results indicated that exercise induces tissue-specific regulation of the camel irisin, FNDC5, and PGC-1α levels, which subsequently regulates the circulating irisin level. Significant associations were detected between the levels of irisin/FNDC5/PGC-1α in camels and the metabolic and hormonal responses to exercise. Our study suggested that irisin regulates, or is regulated by, glucose, FFA, insulin, leptin, and cortisol in camels. The novel results of the present study will serve as baseline data for camels.

Trimetazidine Ameliorates Myocardial Metabolic Remodeling in Isoproterenol-Induced Rats Through Regulating Ketone Body Metabolism via Activating AMPK and PPAR α.

BackgroundMetabolic remodeling plays a vital role in the development of heart failure. The trimetazidine can optimize fatty acid and glucose oxidation via inhibition of long-chain 3-ketoacyl CoA thiolase in the heart. So, trimetazidine commonly used in cardiovascular therapy as a myocardial metabolic drug. This study was conducted to assess the effects and mechanisms of trimetazidine on ketone body metabolism in heart failure rats.MethodsA rat model of heart failure was established by continuous subcutaneous injection of isoproterenol in 10 mg/kg/d. We examined body weight, heart weight index, and tested B-type natriuretic peptide by kit. We detected the structure and function of the heart. Hematoxylin-eosin staining and Masson’s trichrome staining was performed to assess myocardial tissue morphology. To evaluate apoptosis, we used Tunel staining. Metabolic substrate contents of glucose, free fatty acid, ketone bodies, lactic acid, and pyruvate and ATP levels in myocardial tissues were measured with the corresponding kit. We detected the levels of protein expressions related to myocardial substrate uptake and utilization by Western blot.ResultsTrimetazidine remarkably reduced the heart weight index and B-type natriuretic peptide levels. Besides, trimetazidine increased the level of blood pressure and decreased heart rate. Moreover, trimetazidine inhibited decreases in left ventricular ejection fraction and left ventricular fractional shortening. Further, trimetazidine decreased the levels of collagen volume fraction and promoted ATP production in myocardial tissues. Trimetazidine also reduced the levels of free fatty acid, ketone bodies, lactic acid, and increased glucose and pyruvate levels in myocardial tissues. Furthermore, trimetazidine markedly inhibited apoptosis. More importantly, the protein expression levels related to myocardial substrate uptake and utilization increased dramatically in the trimetazidine group. In particular, the protein expressions related to ketone body utilization were prominent.ConclusionsTrimetazidine could attenuate metabolic remodeling and improve cardiac function in heart failure rats. The potential mechanism for the cardioprotective effect of trimetazidine may be highly associated with its regulation of adenosine monophosphate-activated protein kinase, and peroxisome proliferator activated receptor α expressions. Along with the regulation, myocardial substrate utilization was improved, especially the utilization of ketone bodies.

Resonant-Cantilever-Detected Kinetic/Thermodynamic Parameters for Aptamer-Ligand Binding on a Liquid-Solid Interface.

Nucleic acid aptamers have been widely used as recognition elements on various biosensing interfaces, but quantitative kinetic/thermodynamic analysis for revealing the aptamer-ligand binding mechanism, which occurs on a liquid-solid interface, has not been realized due to a lack of usable biophysical tools. Herein we apply a resonant microcantilever sensor to continuously record the frequency shift according to the binding-induced mass change on the liquid-solid interface. The frequency-shift curve is used for tracing the reaction process and is fitted with classic equations to calculate a set of kinetic/thermodynamic parameters, such as rate constants (ka = 902.95 M-1 s-1, kd = 0.000141 s-1), equilibrium constants (KD = 1.55 μM), the Gibbs free energy (ΔG° = -32.57 kJ/mol), and the activation energy (Ea = 38.03 kJ/mol) for the immobilized aptamer and free ATP. This quantitative analysis method is label-free, calibration-free, and highly sensitive. The kinetic/thermodynamic parameter detection method provides new resolution to the in-depth understanding of the ligand-aptamer interaction on the liquid-solid interface for biosensing or lab-on-a-chip applications.

Mitochondrial-targeted methionine sulfoxide reductase overexpression increases the production of oxidative stress in mitochondria from skeletal muscle.

Objective:Mitochondrial dysfunction comprises part of the etiology of myriad health issues, particularly those that occur with advancing age. Methionine sulfoxide reductase A (MsrA) is a ubiquitous protein oxidation repair enzyme that specifically and catalytically reduces a specific epimer of oxidized methionine: methionine sulfoxide. In this study, we tested the ways in which mitochondrial bioenergetic functions are affected by increasing MsrA expression in different cellular compartments.Methods:In this study, we tested the function of isolated mitochondria, including free radical generation, ATP production, and respiration, from the skeletal muscle of two lines of transgenic mice with increased MsrA expression: mitochondria-targeted MsrA overexpression or cytosol-targeted MsrA overexpression.Results:Surprisingly, in the samples from mice with mitochondrial-targeted MsrA overexpression, we found dramatically increased free radical production though no specific defect in respiration, ATP production, or membrane potential. Among the electron transport chain complexes, we found the activity of complex I was specifically reduced in mitochondrial MsrA transgenic mice. In mice with cytosolic-targeted MsrA overexpression, we found no significant alteration made to any of these parameters of mitochondrial energetics.Conclusions:There is also a growing amount of evidence that MsrA is a functional requirement for sustaining optimal mitochondrial respiration and free radical generation. MsrA is also known to play a partial role in maintaining normal protein homeostasis by specifically repairing oxidized proteins. Our studies highlight a potential novel role for MsrA in regulating the activity of mitochondrial function through its interaction with the mitochondrial proteome.

Overexpression of native IF1 downregulates glucose-stimulated insulin secretion by pancreatic INS-1E cells

We have previously reported that transient knock-down of ATPase inhibitory factor 1 (IF1) by siRNA upregulates ATP levels and subsequently augments insulin secretion in model pancreatic β-cells INS-1E. Here we investigated how long-term IF1-overexpression impacts pancreatic β-cell bioenergetics and insulin secretion. We generated INS-1E cell line stably overexpressing native IF1. We revealed that IF1 overexpression leads to a substantial decrease in ATP levels and reduced glucose-stimulated insulin secretion. A decrease in total cellular ATP content was also reflected in decreased free ATP cytosolic and mitochondrial levels, as monitored with ATeam biosensor. Consistently, cellular respiration of IF1-overexpressing cells was decreased. 3D structured illumination microscopy (SIM) revealed a higher amount of insulin granules with higher volume in IF1-overexpressing cells. Similar effects occurred when cells were incubated at low glucose concentrations. Noteworthy, activation of PKA by dibutyryl cAMP entirely abolished the inhibitory effect of IF1 overexpression on ATP production and insulin secretion. Mitochondrial network morphology and cristae ultrastructure in INS-1E overexpressing IF1 remained mostly unchanged. Finally, we show that INS-1E cells decrease their IF1 protein levels relative to ATP synthase α-subunit in response to increased glucose. In conclusion, IF1 actively downregulates INS-1E cellular metabolism and reduces their ability to secrete insulin.

Transmission of Mechanical Information by Purinergic Signaling

The skeleton constantly interacts and adapts to the physical world. We have previously reported that physiologically relevant mechanical forces lead to small repairable membrane injuries in bone-forming osteoblasts, resulting in release of ATP and stimulation of purinergic (P2) calcium responses in neighboring cells. The goal of this study was to develop a theoretical model describing injury-related ATP and ADP release, their extracellular diffusion and degradation, and purinergic responses in neighboring cells. After validation using experimental data for intracellular free calcium elevations, ATP, and vesicular release after mechanical stimulation of a single osteoblast, the model was scaled to a tissue-level injury to investigate how purinergic signaling communicates information about injuries with varying geometries. We found that total ATP released, peak extracellular ATP concentration, and the ADP-mediated signaling component contributed complementary information regarding the mechanical stimulation event. The total amount of ATP released governed spatial factors, such as the maximal distance from the injury at which purinergic responses were stimulated. The peak ATP concentration reflected the severity of an individual cell injury, allowing to discriminate between minor and severe injuries that released similar amounts of ATP because of differences in injury repair, and determined temporal aspects of the response, such as signal propagation velocity. ADP-mediated signaling became relevant only in larger tissue-level injuries, conveying information about the distance to the injury site and its geometry. Thus, we identified specific features of extracellular ATP and ADP spatiotemporal signals that depend on tissue mechanoresilience and encode the severity, scope, and proximity of the mechanical stimulus.

Synthetic energy sensor AMPfret deciphers adenylate-dependent AMPK activation mechanism

AMP-activated protein kinase AMPK senses and regulates cellular energy state. AMPK activation by increasing AMP and ADP concentrations involves a conformational switch within the heterotrimeric complex. This is exploited here for the construction of a synthetic sensor of cellular energetics and allosteric AMPK activation, AMPfret. Based on engineered AMPK fused to fluorescent proteins, the sensor allows direct, real-time readout of the AMPK conformational state by fluorescence resonance energy transfer (FRET). AMPfret faithfully and dynamically reports the binding of AMP and ADP to AMPK γ-CBS sites, competed by Mg2+-free ATP. FRET signals correlate with activation of AMPK by allosteric mechanisms and protection from dephosphorylation, attributed here to specific CBS sites, but does not require activation loop phosphorylation. Moreover, AMPfret detects binding of pharmacological compounds to the AMPK α/β-ADaM site enabling activator screening. Cellular assays demonstrate that AMPfret is applicable in vivo for spatiotemporal analysis of energy state and allosteric AMPK activation.

Mg2+ -free ATP regulates the processivity of native cytoplasmic dynein.

Cytoplasmic dynein, a microtubule-based motor protein, is responsible for many cellular functions ranging from cargo transport to cell division. The various functions are carried out by a single isoform of cytoplasmic dynein, thus requiring different forms of motor regulation. A possible pathway to regulate motor function was revealed in optical trap experiments. Switching motor function from single steps to processive runs could be achieved by changing Mg2+ and ATP concentrations. Here, we confirm by single molecule total internal reflection fluorescence microscopy that a native cytoplasmic dynein dimer is able to switch to processive runs of more than 680 consecutive steps or 5.5 μm. We also identified the ratio of Mg2+ -free ATP to Mg.ATP as the regulating factor and propose a model for dynein processive stepping.

МЕТОД АТФ-БИОЛЮМИНЕСЦЕНЦИИ ДЛЯ ОПЕРАТИВНОГО КОНТРОЛЯ КАЧЕСТВА САНАЦИИ СОСКОВОЙ РЕЗИНЫ ДОИЛЬНЫХ АППАРАТОВ

The article presents materials on determining the possibility of using ATP-bioluminescence to control the quality of the nipple rubber after washing and disinfection. The degree of ATP bioluminescence, including indicators of total (free and bacterial) ATP, was determined using a luminometer HY-Lite®2 (Merk, Germany) and special test systems. It was found that on the inner surface of the nipple rubber, depending on the service life, QMAFAnM increases and, accordingly, ATP-bioluminescence rise. It was also noted that during the period of operation, a gray coating of milk stone forms on the nipple rubber, which irritates the skin of the nipples of the udder during milking and can disrupt its protective functions. Milk stone on the nipple rubber contributes to the formation of biofilms by pathogenic microorganisms that cause mastitis and impair the quality of raw milk. Measuring of ATP bioluminescence on the inner surface of the nipple rubber quickly allows you to monitor the effectiveness of washing and disinfection and in time take corrective measures in accordance with the principles of HACCP.

Mitochondrial potassium channels - an overview.

Mitochondria play a fundamental role in ATP synthesis within the majority of mammalian cells. Potassium channels present in the inner mitochondrial membrane are fine regulators of mitochondrial function, based on inner membrane K+ permeability. These channels are regulated by a plethora of factors and conditions in a way similar to plasma membrane potassium channels. Regulators of mitochondrial potassium channels include the membrane potential, calcium ions, free fatty acids and ATP levels within the cells. Recently, it was shown that these channels are regulated by the respiratory chain, stretching of the membrane and phosphorylation. The essential interest that has driven studies of mitochondrial potassium channels for nearly 25 years is their role in cytoprotection and in cell death. Mitochondrial potassium channels have been described in neurons, astrocytoma, cardiac and skeletal muscles, fibroblasts, keratinocytes and endothelial cells. In this overview, we summarize the current knowledge of mitochondrial potassium channels. This summary will be done with a special focus on studies performed over the last 20 years in the Laboratory of Intracellular Ion Channels at the Nencki Institute. These include studies on the electrophysiological and pharmacological properties of mitochondrial potassium channels and on their regulation by endogenous intracellular substances. Additionally, the regulation of mitochondrial potassium channels by the respiratory chain and by stretching of the inner mitochondrial membrane will be reviewed. Properties of mitochondrial potassium channels in various organisms will also be summarized.

Triple resonance EPR spectroscopy determines the Mn2+ coordination to ATP

Mn2+ often serves as a paramagnetic substitute to Mg2+, providing means for exploring the close environment of Mg2+ in many biological systems where it serves as an essential co-factor. This applies to proteins with ATPase activity, where the ATP hydrolysis requires the binding of Mg2+-ATP to the ATPase active site. In this context, it is important to distinguish between the Mn2+ coordination mode with free ATP in solution as compared to the protein bound case. In this work, we explore the Mn2+ complexes with ATP, the non-hydrolysable ATP analog, AMPPNP, and ADP free in solution. Using W-band 31P electron-nuclear double resonance (ENDOR) we obtained information about the coordination to the phosphates, whereas from electron-electron double resonance (ELDOR) – detected NMR (EDNMR) we determined the coordination to an adenosine nitrogen. The coordination to these ligands has been reported earlier, but whether the nitrogen and phosphate coordination is within the same nucleotide molecules or different ones is still under debate. By applying the correlation technique, THYCOS (triple hyperfine correlation spectroscopy), and measuring 15N-31P correlations we establish that in Mn-ATP in solution both phosphates and a nitrogen are coordinated to the Mn2+ ion. We also carried out DFT calculations to substantiate this finding. In addition, we expanded the understanding of the THYCOS experiment by comparing it to 2D-EDNMR for 55Mn-31P correlation experiments and through simulations of THYCOS and 2D-EDNMR spectra with 15N-31P correlations.

Prostate cancer chemoprevention by sulforaphane in a preclinical mouse model is associated with inhibition of fatty acid metabolism.

Increased de novo synthesis of fatty acids is a rather unique and targetable mechanism of human prostate cancer. We have shown previously that oral administration of sulforaphane (SFN) significantly inhibits the incidence and/or burden of prostatic intraepithelial neoplasia and well-differentiated adenocarcinoma in TRansgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice. The present study used cellular models of prostate cancer and archived plasma/adenocarcinoma tissues and sections from the TRAMP study to demonstrate inhibition of fatty acid synthesis by SFN treatment in vitro and in vivo. Treatment of androgen-responsive (LNCaP) and castration-resistant (22Rv1) human prostate cancer cells with SFN (5 and 10 μM) resulted in downregulation of protein and mRNA levels of acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FASN), but not ATP citrate lyase. Protein and mRNA levels of carnitine palmitoyltransferase 1A (CPT1A), which facilitates fatty acid uptake by mitochondria for β-oxidation, were also decreased following SFN treatment in both cell lines. Immunohistochemistry revealed a significant decrease in expression of FASN and ACC1 proteins in prostate adenocarcinoma sections of SFN-treated TRAMP mice when compared with controls. SFN administration to TRAMP mice resulted in a significant decrease in plasma and/or prostate adenocarcinoma levels of total free fatty acids, total phospholipids, acetyl-CoA and ATP. Consistent with these results, number of neutral lipid droplets was lower in the prostate adenocarcinoma sections of SFN-treated TRAMP mice than in control tumors. Collectively, these observations indicate that prostate cancer chemoprevention by SFN in TRAMP mice is associated with inhibition of fatty acid metabolism.