Low ATP Concentrations Research Articles

A New Glance on Energy Metabolism Investigation in Cultured Cells

The aim of the study was to analyze the input of cytosolic and oxidative pathways of energy metabolism in ATP production of cultured cells by using FLIM and routine biochemical techniques. Fluorescent imaging of endogenous cofactors NADH and FAD demonstrated a more pronounced oxidative redox status of fibroblasts compared to tumor cells and significant differences in metabolic processes in which FAD is involved. Analysis of glucose and lactate content and absorption showed that tumor cells not only absorb glucose more intensively from the environment, but also use it more intensively during anaerobic glycolysis. Lower energy efficiency of glycolysis and FAD oxidative path and greater energy consumption is the reason for the lower concentration of ATP in tumor cells. Presumably, the prevalence of glycolytic metabolism in tumor cells could be largely determined by their hypoxic reprogramming through the PI3K/AKT/mTOR signal pathway. The results of the study suggest that correlation between intracellular consumption of glucose and cytosolic concentration of NADH may contribute to the characteristic of energy metabolism state of cultured cells and serve as the biosensor of malignant cell transformation.

Positive allosteric modulation of P2X7 promotes apoptotic cell death over lytic cell death responses in macrophages

P2X7 is an ATP-gated ion channel that is highly expressed by leukocytes, such as macrophages. Here, P2X7 has been demonstrated to be involved in the regulation of various cell death pathways; including apoptosis, pyroptosis, necrosis, and autophagy. However, cell death induction via P2X7 is complex and is reliant upon the nature of the stimulus, the duration of the stimulus, and the cell type investigated. Previous reports state that high extracellular ATP concentrations promote osmotic lysis, but whether positive allosteric modulation of P2X7 in the presence of lower concentrations of ATP condemns cells to the same fate is unknown. In this study, we compared cell death induced by high ATP concentrations, to cell death induced by compound K, a recently identified and potent positive allosteric modulator of P2X7. Based on our observations, we propose that high ATP concentrations induce early cell swelling, loss of mitochondrial membrane potential, plasma membrane rupture, and LDH release. Conversely, positive allosteric modulation of P2X7 primarily promotes an intrinsic apoptosis pathway. This was characterised by an increase in mitochondrial Ca2+, accelerated production of mitochondrial ROS, loss of mitochondrial membrane permeability in a Bax-dependent manner, the potential involvement of caspase-1, and caspase-3, and significantly accelerated kinetics of caspase-3 activation. This study highlights the ability of positive allosteric modulators to calibrate P2X7-dependent cell death pathways and may have important implications in modulating the antimicrobial immune response and in the resolution of inflammation.

Single-molecule analysis reveals rotational substeps and chemo-mechanical coupling scheme of Enterococcus hirae V1-ATPase

V1-ATPase (V1), the catalytic domain of an ion-pumping V-ATPase, is a molecular motor that converts ATP hydrolysis–derived chemical energy into rotation. Here, using a gold nanoparticle probe, we directly observed rotation of V1 from the pathogen Enterococcus hirae (EhV1). We found that 120° steps in each ATP hydrolysis event are divided into 40 and 80° substeps. In the main pause before the 40° substep and at low ATP concentration ([ATP]), the time constant was inversely proportional to [ATP], indicating that ATP binds during the main pause with a rate constant of 1.0 × 107 m−1 s−1. At high [ATP], we observed two [ATP]-independent time constants (0.5 and 0.7 ms). One of two time constants was prolonged (144 ms) in a rotation driven by slowly hydrolyzable ATPγS, indicating that ATP is cleaved during the main pause. In another subpause before the 80° substep, we noted an [ATP]-independent time constant (2.5 ms). Furthermore, in an ATP-driven rotation of an arginine-finger mutant in the presence of ADP, −80 and −40° backward steps were observed. The time constants of the pauses before −80° backward and +40° recovery steps were inversely proportional to [ADP] and [ATP], respectively, indicating that ADP- and ATP-binding events trigger these steps. Assuming that backward steps are reverse reactions, we conclude that 40 and 80° substeps are triggered by ATP binding and ADP release, respectively, and that the remaining time constant in the main pause represents phosphate release. We propose a chemo-mechanical coupling scheme of EhV1, including substeps largely different from those of F1-ATPases.

Effects of histidine load on ammonia, amino acid, and adenine nucleotide concentrations in rats.

The unique capability of proton buffering is the rationale for using histidine (HIS) as a component of solutions for induction of cardiac arrest and myocardial protection in cardiac surgery. In humans, infusion of cardioplegic solution may increase blood plasma HIS from ~ 70 to ~ 21,000µM. We examined the effects of a large intravenous dose of HIS on ammonia and amino acid concentrations and energy status of the body. Rats received 198mM HIS intravenously (20ml/kg) or vehicle. Samples of blood plasma, urine, liver, and soleus (SOL) and extensor digitorum longus (EDL) muscles were analysed at 2 or 24h after treatment. At 2h after HIS load, we found higher HIS concentration in all examined tissues, higher urea and ammonia concentrations in blood and urine, lower ATP content and higher AMP/ATP ratio in the liver and muscles, higher concentrations of almost all examined amino acids in urine, and lower glycine concentration in blood plasma, liver, and muscles when compared with controls. Changes in other amino acids were tissue dependent, markedly increased alanine and glutamate in the blood and the liver. At 24h, the main findings were lower ATP concentrations in muscles, lower concentrations of branched-chain amino acids (BCAA; valine, leucine, and isoleucine) in blood plasma and muscles, and higher carnosine content in SOL when compared with controls. It is concluded that a load of large HIS dose results in increased ammonia levels and marked alterations in amino acid and energy metabolism. Pathogenesis is discussed in the article.

High-resolution detection of ATP release from single cultured mouse dorsal horn spinal cord glial cells and its modulation by noradrenaline.

Human embryonic kidney 293 (HEK293) cells stably transfected with the rat P2X2 receptor subunit were preincubated with 200nM progesterone (HEK293-P2X2-PROG), a potent positive allosteric modulator of homomeric P2X2 receptors, and used to detect low nanomolar concentrations of extracellular ATP. Fura-2-loaded HEK293-P2X2-PROG cells were acutely plated on top of cultured DH glial cells to quantify ATP release from single DH glial cells. Application of the α1 adrenoceptor agonist phenylephrine (PHE, 20μM) or of a low K+ (0.2mM) solution evoked reversible increases in the intracellular calcium concentration ([Ca2+]i) in the biosensor cells. A reversible increase in [Ca2+]i was also detected in half of the biosensor cells following the interruption of general extracellular perfusion. All increases in [Ca2+]i were blocked in the presence of the P2X2 antagonist PPADS or after preloading the glial cells with the calcium chelator BAPTA, indicating that they were due to calcium-dependent ATP release from the glial cells. ATP release induced by PHE was blocked by -L-phenylalanine 2-naphtylamide (GPN) that permeabilizes secretory lysosomes and bafilomycin A1 (Baf A1), an inhibitor of the H+-pump of acidic secretory vesicles. By contrast, ATP release induced by application of a low-K+ solution was abolished by Baf A1 but not by GPN. Finally, spontaneous ATP release observed after interrupting general perfusion was insensitive to both GPN and Baf A1 pretreatment. Our results indicate that ATP is released in a calcium-dependent manner from two distinct vesicular pools and one non-vesicular pool coexisting in DH glial cells and that noradrenaline and PHE selectively target the secretory lysosome pool.

Inhibition by fluoxetine of LH-stimulated cyclic AMP synthesis in tumor Leydig cells partly involves AMPK activation.

Fluoxetine (FLX), a widely used antidepressant primarily acting as a selective serotonin reuptake inhibitor (SSRI), has been shown to exhibit other mechanisms of action in various cell types. Consequently, it might have unexpected adverse effects not related to its intended use, possibly in the endocrine regulation of reproduction. We show in the present report that after a 1-hour preincubation of MLTC-1 Leydig cells with FLX, the intracellular cyclic adenosine monophosphate (cAMP) responses to Luteinizing Hormone (LH) and forskolin (FSK) are reduced through AMPK-dependent and -independent pathways respectively. FLX at low concentrations (12.5μM and 25μM) induced this inhibition without triggering AMPK phosphorylation, while higher FLX concentrations (50μM and 100μM) induced AMPK phosphorylation and lowered ATP concentration similarly to Metformin. Pretreatment with the specific AMPK inhibitor Compound C (CpdC), significantly diminished the inhibition of cAMP synthesis caused by high concentration of FLX. Moreover, as expected FLX also caused a decline of steroidogenesis which is under the control of cAMP. Taken together, these findings demonstrate that the inhibition of cAMP synthesis by FLX is dose-dependent and occurs in MLTC-1 cells through two mechanisms, AMPK-independent and AMPK-dependent, at low and high concentrations, respectively. FLX also inhibited hormone-induced steroidogenesis in MLTC-1 cells and mouse testicular Leydig cells, suggesting similar mechanisms in both cell types.

Birth and Resuscitation of (p)ppGpp Induced Antibiotic Tolerant Persister Cells

Transient antibiotic treatment typically eradicates most sensitive bacteria except a few survivors called persisters. The second messenger (p)ppGpp plays a key role in persister formation in Escherichia coli populations but the underlying mechanisms have remained elusive. In this study we induced (p)ppGpp synthesis by modulating tRNA charging and then directly observed the stochastic appearance, antibiotic tolerance, and resuscitation of persister cells using live microscopy. Different physiological parameters of persister cells as well as their regularly growing ancestors and sisters were continuously monitored using fluorescent reporters. Our results confirmed previous findings that high (p)ppGpp levels are critical for persister formation, but the phenomenon remained strikingly stochastic without any correlation between (p)ppGpp levels and antibiotic tolerance on the single-cell level. We could not confirm previous notions that persisters exhibit markedly low concentrations of intracellular ATP or were linked to post-transcriptional effects of (p)ppGpp through the activation of small genetic elements known as toxin-antitoxin (TA) modules. Instead, we suggest that persister cell formation under regular conditions is driven by the transcriptional response to increased (p)ppGpp levels.

Single-Molecule Analysis of Spliceosome Activation Kinetics Reveals Multiple Intermediate States

During pre-mRNA splicing, the spliceosome needs to be activated in an ATP-dependent manner in order to carry out the catalytic steps of the splicing reaction. During activation, the protein composition of the spliceosome changes dramatically and includes loss of the B complex proteins (Spp381, Snu23 and Prp38) and Snu66. Using colocalization single molecule spectroscopy (CoSMoS), we show that Spp381, Snu23, and Snu66 release is dependent on ATP by carrying out reactions at both high and low ATP concentrations. By comparing the characteristic dwell times for these proteins at high ATP, we found that Spp381 and Snu23 behave similar to each other and with kinetics distinct from Snu66. Specifically, at both ATP concentrations, Snu66 shows much longer-lived populations than Spp381 and Snu23. This suggests that Snu66 is bound more stably to the spliceosome than the other two. These results are consistent with previous biochemical and structural studies suggesting that Spp381, Snu23, and Prp38 form a protein subcomplex of the spliceosome. Together, our results suggest that this subcomplex and Snu66 likely have two different roles in the activation step and give rise to multiple activation intermediates as they are sequentially released from the spliceosome.

A model for the chemomechanical coupling of myosin-V molecular motors.

Herein, a model for the chemomechanical coupling of dimeric myosin-V motors is presented. Based on this model and the proposal that the rate constants of the ATPase activity of the two heads are independent of an external force in a range smaller than the stall force, we analytically studied the dynamics of the motor, such as the stepping ratio, dwell time between two mechanical steps, and velocity, under varying force and ATP concentrations. The theoretical results well reproduce the diverse available single-molecule experimental data. In particular, the experimental data showing that at a low ATP concentration, the dwell time and velocity have less force dependency than at a high ATP concentration is explained quantitatively. Moreover, the dependency of the chemomechanical coupling ratio on the force and ATP concentration was studied.

Age-Dependent Decrease in Requirement for Glycolysis in Hematopoietic Stem Cells

Metabolic process governs cell cycle and differentiation status of hematopoietic stem cells (HSCs) through regulating the balance between oxidative phosphorylation and glycolysis. Cell cycle and self-renewal capacity dynamically changes during developmental and aging process; in early stage of life, HSCs are highly cycling and self-renew, whereas adult HSCs exhibit quiescent in cell-cycle, and gradually decline in its function as they age. How functional alteration of HSCs is coupled with metabolic program is an important issue for understanding the lifecourse of HSCs. Phosphoglycerate mutase 1 (Pgam1) is an enzyme catalyzing conversion of 3-phosphoglycerate into 2-phosphoglycerate. By deleting Pgam1, ATP production through glycolysis was abrogated while pentose-phosphate pathway (PPP) was spared, allowing us to examine dependence on glycolytic energy production without affecting nucleotide and NADPH production through PPP. Following inducible Pgam1 deletion in mice at various ages, the number of HSCs was measured. At an early life stage (5-week old), Pgam1 deletion resulted in decreased number of HSCs, suggesting an essential role of glycolysis for HSC maintenance. Notably, the number of HSCs in Pgam1-deleted mice restored in an age-dependent manner, with 2-year old mice exhibiting almost comparable number of HSCs with wild type mice. By contrast, upon HSC transplantation, the repopulation capacity of Pgam1-deficient HSCs from any stages of life was uniformly abrogated, indicating that glycolytic energy production is essential for stressed condition in which HSCs are forced to proliferate. Reverse transplantation of wild type HSCs into Pgam1-floxed recipients followed by conditional deletion in non-hematopoietic cells showed no phenotypes in hematopoietic lineages, suggesting a cell-autonomous requirement of Pgam1 for blood cells.To further determine the relevance of glycolysis with maintenance/proliferation balance, we performed in vitro culture utilizing newly-developed quiescence-maintaining condition with or without glucose. The number of HSCs under the quiescence-maintaining condition was irrelevant to the presence or absence of glucose, while cell number under the proliferating condition was markedly decreased in glucose-deprived condition. Furthermore, lymphoid-primed multipotential progenitors (LMPP) with marker phenotype of Flt3+ linage markers- Sca-1+ c-Kit+ cells, which show high cycling status in vivo, were highly dependent on glucose for survival in vitro. Collectively, proliferating cells require glucose irrespective to its cell type, while quiescent HSCs are less dependent on glycolytic energy production in line with age-dependent decrease of requirement for glycolysis in vivo.ATP concentration directly reflects ATP production/consumption rate inside cells. We utilized ATP-biosensor (GO-ATeam2)-transgenic mice to monitor ATP concentration in live HSCs under various metabolic environments in vitro. HSCs cultured in the proliferating condition exhibited gradual decrease in ATP concentration during 7-day culture, whereas in the quiescence-maintaining condition ATP was kept high. In the steady state, cycling multipotent progenitors (MPPs) exhibited lower ATP concentration than quiescent HSCs. These results suggest that quiescent state is characterized by lower ATP production/consumption rate than the proliferating cells. We then compared ATP concentration in young and old HSCs using the same system. The decrease of ATP under the proliferating condition was less prominent in old HSCs, while ATP concentration was comparable in the quiescence-maintaining condition. The result indicates the lower ATP production/consumption rate in old HSCs, presumably explaining lower dependence on glycolytic energy production in the steady state hematopoiesis.Altogether, we concluded that the dependence on glycolysis-driven ATP production derived from glucose decreased with age, underlying mechanism of which is explained by lower ATP consumption rate in old HSCs. DisclosuresOkamoto:Chugai Pharmaceutical Co., Ltd.: Research Funding; Eisai Co.,Ltd.: Research Funding; Bristol-Myers Squibb K.K.: Honoraria, Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Toyama Chemical Co., Ltd.: Research Funding; Alexion Pharmaceuticals, Inc.:: Research Funding; Nippon Shinyaku Co., Ltd: Research Funding; Shionogi & Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Asahi Kasei Pharma Corp.:: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Teijin Pharma Limited: Research Funding; Kyowa Hakko Kirin Co.: Research Funding; Pfizer Inc.: Honoraria, Research Funding; JCR Pharmaceuticals Co., Ltd.: Research Funding.

Profilin facilitates PKCε activation by accelerating ATP supply

Profilin catalyzes the exchange of actin-bound ADP to ATP. The present study investigated the role of profilin in PKCε activation. Profilin associated with PKCε in differentiated PC-12 cells under the basal conditions, which was inhibited by the PKC inhibitor GF109203X. The selective PKCε activator DCP-LA markedly increased the association, which was clearly prevented by GF109203X. The basal PKC activity in PC-12 cells was attenuated by knocking-down profilin, while the basal activities of PKA and CaMKII were not affected. DCP-LA enhanced the PKC activity to approximately 3.5 folds of the basal levels, and the effect was suppressed by knocking-down profilin. In the cell-free system, PKCε was not activated by profilin alone. DCP-LA activated PKCε in an ATP concentration (2–500 μM)-dependent manner, and addition of profilin shifted the ATP concentration/DCP-LA-induced PKCε activity relation curve to the left (the direction of lower ATP concentrations). Taken together, the results of the present study indicate that profilin binds to activated PKCε and facilitates PKCε activation by accelerating ATP supply to PKCε.

The properties of Msh2–Msh6 ATP binding mutants suggest a signal amplification mechanism in DNA mismatch repair

DNA mismatch repair (MMR) corrects mispaired DNA bases and small insertion/deletion loops generated by DNA replication errors. After binding a mispair, the eukaryotic mispair recognition complex Msh2–Msh6 binds ATP in both of its nucleotide-binding sites, which induces a conformational change resulting in the formation of an Msh2–Msh6 sliding clamp that releases from the mispair and slides freely along the DNA. However, the roles that Msh2–Msh6 sliding clamps play in MMR remain poorly understood. Here, using Saccharomyces cerevisiae, we created Msh2 and Msh6 Walker A nucleotide–binding site mutants that have defects in ATP binding in one or both nucleotide-binding sites of the Msh2–Msh6 heterodimer. We found that these mutations cause a complete MMR defect in vivo. The mutant Msh2–Msh6 complexes exhibited normal mispair recognition and were proficient at recruiting the MMR endonuclease Mlh1–Pms1 to mispaired DNA. At physiological (2.5 mm) ATP concentration, the mutant complexes displayed modest partial defects in supporting MMR in reconstituted Mlh1–Pms1-independent and Mlh1–Pms1-dependent MMR reactions in vitro and in activation of the Mlh1–Pms1 endonuclease and showed a more severe defect at low (0.1 mm) ATP concentration. In contrast, five of the mutants were completely defective and one was mostly defective for sliding clamp formation at high and low ATP concentrations. These findings suggest that mispair-dependent sliding clamp formation triggers binding of additional Msh2–Msh6 complexes and that further recruitment of additional downstream MMR proteins is required for signal amplification of mispair binding during MMR.

Coronary Smooth Muscle Cell Calcium Dynamics: Effects of Bifurcation Angle on Atheroprone Conditions.

This work investigates the effect of arterial bifurcation angulation on atherosclerosis development through in-silico simulations of coupled cell dynamics. The computational model presented here combines cellular pathways, fluid dynamics, and physiologically-realistic vessel geometries as observed in the human vasculature. The coupled cells model includes endothelial cells (ECs) and smooth muscle cells (SMCs) with ion dynamics, hetero and homotypic coupling, as well as electro-diffusive coupling. Three arterial bifurcation surface models were used in the coupled cells simulations. All three simulations showed propagating waves of Ca2+ in both the SMC and EC layers, following the introduction of a luminal agonist, in this case ATP. Immediately following the introduction of ATP concentration Ca2+ waves propagate from the area of high ATP toward the areas of low ATP concentration, forming complex patterns where waves interact with eachother, collide and fade. These dynamic phenomena are repeated with a series of waves of slower velocity. The underlying motivation of this research was to examine the macro-scale phenomena, given that the characteristic length scales of atherosclerotic plaques are much larger than a single cell. The micro-scale dynamics were modeled on macro-scale arterial bifurcation surfaces containing over one million cells. The results of the simulations presented here suggest that susceptibility to atherosclerosis development depends on the bifurcation angulation. In conjunction with findings reported in the literature, the simulation results demonstrate that arterial bifurcations containing wider angles have a more prominent influence on the coupled cells pathways associated with the development of atherosclerosis, by means of disturbed flow and lower SMC Ca2+ concentrations. The discussion of the results considers the findings of this research within the context of the potential link between information transport through frequency encoding of Ca2+ wave dynamics and development of atheroprone conditions.

Single mutations in the ε subunit from thermophilic Bacillus PS3 generate a high binding affinity site for ATP.

The ε subunit from ATP synthases acts as an ATP sensor in the bacterial cell to prevent ATP hydrolysis and thus the waste of ATP under conditions of low ATP concentration. However, the ATP binding affinities from various bacterial organisms differ markedly, over several orders of magnitude. For example, the ATP synthases from thermophilic Bacillus PS3 and Escherichia coli exhibit affinities of 4 µM and 22 mM, respectively. The recently reported R103A/R115A double mutant of Bacillus PS3 ATP synthase demonstrated an increased binding affinity by two orders of magnitude with respect to the wild type. Here, we used atomic-resolution molecular dynamics simulations to determine the role of the R103A and R115A single mutations. These lead us to predict that both single mutations also cause an increased ATP binding affinity. Evolutionary analysis reveals R103 and R115 substitutions in the ε subunit from other bacillic organisms, leading us to predict they likely have a higher ATP binding affinity than previously expected.

Calcium binding to a remote site can replace magnesium as cofactor for mitochondrial Hsp90 (TRAP1) ATPase activity

The Hsp90 molecular chaperones are ATP-dependent enzymes that maintain protein homeostasis and regulate many essential cellular processes. Higher eukaryotes have organelle-specific Hsp90 paralogs that are adapted to each subcellular environment. The mitochondrial Hsp90, TNF receptor–associated protein 1 (TRAP1), supports the folding and activity of electron transport components and is increasingly appreciated as a critical player in mitochondrial signaling. Calcium plays a well-known and important regulatory role in mitochondria where it can accumulate to much higher concentrations than in the cytoplasm. Surprisingly, we found here that calcium can replace magnesium, the essential enzymatic cofactor, to support TRAP1 ATPase activity. Anomalous X-ray diffraction experiments revealed a calcium-binding site within the TRAP1 nucleotide-binding pocket located near the ATP α-phosphate and completely distinct from the magnesium-binding site adjacent to the β- and γ-phosphates. In the presence of magnesium, ATP hydrolysis by TRAP1, as with other Hsp90s, was noncooperative, whereas calcium binding resulted in cooperative hydrolysis by the two protomers within the Hsp90 dimer. The structural data suggested a mechanism for this cooperative behavior. Because of the cooperativity, at high ATP concentrations, ATPase activity was higher with calcium, whereas the converse was observed at low ATP concentrations. Integrating these observations, we propose a model in which the divalent cation choice can control switching between noncooperative and cooperative TRAP1 ATPase mechanisms in response to varying ATP concentrations. This switching may facilitate coordination between cellular energetics, mitochondrial signaling, and protein homeostasis via alterations in the TRAP1 ATP-driven cycle and its consequent effects on different mitochondrial clients.

The Pannexin-1 Channel Inhibitor Probenecid Attenuates Skeletal Muscle Cellular Energy Crisis and Histopathological Injury in a Rabbit Endotoxemia Model

This study aimed to investigate the effect of probenecid (Pro) as an inhibitor of the pannexin-1 (Panx-1) channel-mediated release of intracellular ATP to the extracellular compartment on inflammation, cellular energy crisis, and organ injury in a rabbit sepsis model induced by Escherichia coli lipopolysaccharides (LPS). A total of 24 anesthetized and ventilated rabbits were randomly assigned to receive one of four treatments: infusion of LPS without Pro (LPS group), infusion of LPS with Pro (LPS + Pro group), sham operation without Pro (normal group), and sham operation with Pro (normal + Pro group). The LPS group had significantly higher serum ATP levels, serum inflammatory factor levels (TNF-α, IL-6, and IL-1β), and lower ATP concentrations and ATP/ADP ratios in the skeletal muscle tissue than the normal group. Compared to that at baseline, the expression of Panx-1 in peripheral blood cells increased significantly after the infusion of LPS (fluorescence intensity of Panx-1: T0 (baseline) vs. T1 (post-LPS) = 10 ± 1.2 vs. 84 ± 48, P < 0.0001; paired differences 73 ± 46, P = 0.024). Moreover, the LPS group exhibited higher expression of Panx-1 in the skeletal muscle tissue than the normal group. The serum ATP level was significantly positively correlated with IL-1β (R = 0.602, P = 0.001), IL-6 (R = 0.381, P = 0.033), and TNF-α (R = 0.514, P = 0.005) in 24 paired measurements. Compared to the LPS group, the LPS + Pro group had significantly lower levels of inflammatory factors (TNF-α, IL-6, and IL-1β) and serum ATP. In the skeletal muscle tissue, the LPS + Pro group also had a higher ATP concentration (1.1 ± 0.15 vs. 1.33 ± 0.17, P = 0.041) and ATP/ADP ratio (0.37 ± 0.03 vs. 0.51 ± 0.06, P = 0.002) and a lower histopathological damage score (4.67 ± 0.52 vs. 3 ± 0.63, P= 0.004). An overexpression of Panx-1 channel might be responsible for the strong inflammatory response, high serum ATP level, and skeletal muscle cellular energy crisis and histopathological damages in sepsis. Inhibiting Panx-1 channel-mediated release of intracellular ATP could decrease the above-mentioned injuries, and Panx-1 might be a potential therapeutic target in sepsis.

Iron overload promotes mitochondrial fragmentation in mesenchymal stromal cells from myelodysplastic syndrome patients through activation of the AMPK/MFF/Drp1 pathway

Iron overload (IO) has been reported to contribute to mesenchymal stromal cell (MSC) damage, but the precise mechanism has yet to be clearly elucidated. In this study, we found that IO increased cell apoptosis and lowered cell viability in MSCs, accompanied by extensive mitochondrial fragmentation and autophagy enhancement. All these effects were reactive oxygen species (ROS) dependent. In MSCs with IO, the ATP concentrations were significantly reduced due to high ROS levels and low electron respiratory chain complex (ETC) II/III activity. Reduced ATP phosphorylated AMP-activated protein kinase (AMPK). Activation of AMPK kinase complexes triggered mitochondrial fission. Moreover, gene knockout of AMPK via CRISPR/Cas9 reduced cell apoptosis, enhanced cell viability and attenuated mitochondrial fragmentation and autophagy caused by IO in MSCs. Further, AMPK-induced mitochondrial fragmentation of MSCs with IO was mediated via phosphorylation of mitochondrial fission factor (MFF), a mitochondrial outer-membrane receptor for the GTPase dynamin-related protein 1 (Drp1). Gene knockdown of MFF reversed AMPK-induced mitochondrial fragmentation in MSCs with IO. In addition, MSCs from IO patients with myelodysplastic syndrome (MDS) showed increased cell apoptosis, decreased cell viability, higher ROS levels, lower ATP concentrations and increased mitochondrial fragmentation compared with MSCs from non-IO patients. In addition, iron chelation or antioxidant weakened the activity of the AMPK/MFF/Drp1 pathway in MDS-MSCs with IO from several patients, accompanied by attenuation of mitochondrial fragmentation and autophagy. Taken together, the AMPK/MFF/Drp1 pathway has an important role in the damage to MDS-MSCs caused by IO.

Unidirectional growth of heavy meromyosin clusters along actin filaments revealed by real-time fluorescence microscopy.

Heavy meromyosin (HMM) forms clusters along actin filaments under low ATP concentrations. Here, we observed the growth of HMM clusters under low concentrations of ATP in real time using fluorescence microscopy. When actin filaments were loosely immobilized on positively charged lipid bilayers, clusters of HMM-GFP were readily formed. Time-lapse observation revealed that the clusters grew unidirectionally. When we used a mixture of actin filaments and copolymers of actin and acto-S1dC, a chimeric protein of actin and the myosin motor domain, HMM-GFP preferentially formed clusters along the copolymers. We thus suggest that binding of myosin motors carrying ADP and Pi induces unidirectional conformational changes in actin filaments and allosterically recruits more myosin binding. In contrast, when actin filaments and copolymers were anchored to glass substrate via stable biotin-avidin linkage, higher concentrations of HMM-GFP were required to form clusters than on the lipid bilayer. Moreover, actin filaments and copolymers were not discriminated regarding preferential cluster formation. This is presumably because the myosin-induced cooperative conformational changes in actin filaments involve changes in the helical twist. Consistent with this, cofilin clusters, which supertwist the helix, were readily formed along loosely immobilized actin filaments, but not along those anchored via biotin-avidin linkage.

Myosin V: Chemomechanical-coupling ratchet with load-induced mechanical slip

A chemomechanical-network model for myosin V is presented on the basis of both the nucleotide-dependent binding affinity of the head to an actin filament (AF) and asymmetries and similarity relations among the chemical transitions due to an intramolecular strain of the leading and trailing heads. The model allows for branched chemomechanical cycles and takes into account not only two different force-generating mechanical transitions between states wherein the leading head is strongly bound and the trailing head is weakly bound to the AF but also load-induced mechanical-slip transitions between states in which both heads are strongly bound. The latter is supported by the fact that ATP-independent high-speed backward stepping has been observed for myosin V, although such motility has never been for kinesin. The network model appears as follows: (1) the high chemomechanical-coupling ratio between forward step and ATP hydrolysis is achieved even at low ATP concentrations by the dual mechanical transitions; (2) the forward stepping at high ATP concentrations is explained by the front head-gating mechanism wherein the power stroke is triggered by the inorganic-phosphate (Pi) release from the leading head; (3) the ATP-binding or hydrolyzed ADP.Pi-binding leading head produces a stable binding to the AF, especially against backward loading.

Mixomics analysis of Bacillus subtilis: effect of oxygen availability on riboflavin production

BackgroundRiboflavin, an intermediate of primary metabolism, is one kind of important food additive with high economic value. The microbial cell factory Bacillus subtilis has already been proven to possess significant importance for the food industry and have become one of the most widely used riboflavin-producing strains. In the practical fermentation processes, a sharp decrease in riboflavin production is encountered along with a decrease in the dissolved oxygen (DO) tension. Influence of this oxygen availability on riboflavin biosynthesis through carbon central metabolic pathways in B. subtilis is unknown so far. Therefore the unveiled effective metabolic pathways were still an unaccomplished task till present research work.ResultsIn this paper, the microscopic regulation mechanisms of B. subtilis grown under different dissolved oxygen tensions were studied by integrating 13C metabolic flux analysis, metabolomics and transcriptomics. It was revealed that the glucose metabolic flux through pentose phosphate (PP) pathway was lower as being confirmed by smaller pool sizes of metabolites in PP pathway and lower expression amount of ykgB at transcriptional level. The latter encodes 6-phosphogluconolactonase (6-PGL) under low DO tension. In response to low DO tension in broth, the glucose metabolic flux through Embden–Meyerhof–Parnas (EMP) pathway was higher and the gene, alsS, encoding for acetolactate synthase was significantly activated that may result due to lower ATP concentration and higher NADH/NAD+ ratio. Moreover, ResE, a membrane-anchored protein that is capable of oxygen regulated phosphorylase activity, and ResD, a regulatory protein that can be phosphorylated and dephosphorylated by ResE, were considered as DO tension sensor and transcriptional regulator respectively.ConclusionsThis study shows that integration of transcriptomics, 13C metabolic flux analysis and metabolomics analysis provides a comprehensive understanding of biosynthesized riboflavin’s regulatory mechanisms in B. subtilis grown under different dissolved oxygen tension conditions. The two-component system, ResD–ResE, was considered as the signal receiver of DO tension and gene regulator that led to differences between biomass and riboflavin production after triggering the shifts in gene expression, metabolic flux distributions and metabolite pool sizes.