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Stimulation of sympathetic nerves in the vas deferens yields biphasic contractions consisting of a rapid transient component resulting from activation of P2X1 receptors by ATP and a secondary sustained component mediated by activation of α1-adrenoceptors by noradrenaline. Noradrenaline can also potentiate the ATP-dependent contractions of the vas deferens, but the mechanisms underlying this effect are unclear. The purpose of the present study was to investigate the mechanisms underlying potentiation of transient contractions of the vas deferens induced by activation of α1-adrenoceptors. Contractions of the mouse vas deferens were induced by electric field stimulation (EFS). Delivery of brief (1s duration) pulses (4 Hz) yielded transient contractions that were inhibited tetrodotoxin (100 nM) and guanethidine (10 µM). α,β-meATP (10 µM), a P2X1R desensitising agent, reduced the amplitude of these responses by 65% and prazosin (100 nM), an α1-adrenoceptor antagonist, decreased mean contraction amplitude by 69%. Stimulation of α1-adrenoceptors with phenylephrine (3 µM) enhanced EFS and ATP-induced contractions and these effects were mimicked by the phorbol ester PDBu (1 µM), which activates PKC. The PKC inhibitor GF109203X (1 µM) prevented the stimulatory effects of PDBu on ATP-induced contractions of the vas deferens but only reduced the stimulatory effects of phenylephrine by 40%. PDBu increased the amplitude of ATP-induced currents recorded from freshly isolated vas deferens myocytes and HEK-293 cells expressing human P2X1Rs by 93%. This study indicates that: (1) potentiation of ATP-evoked contractions of the mouse vas deferens by α1-adrenoceptor activation were not fully blocked by the PKC inhibitor GF109203X and (2) that the stimulatory effect of PKC on ATP-induced contractions of the vas deferens is associated with enhanced P2X1R currents in vas deferens myocytes.

Fluctuations in the initiation rate of transcription, the first step in gene expression, ensue from the stochastic behavior of the molecular process that controls transcription. In steady state, the regulatory process is often assumed to operate reversibly, i.e., in equilibrium. However, reversibility imposes fundamental limits to information processing. For instance, the assumption of equilibrium is difficult to square with the precision with which the regulatory process executes its task in eukaryotes. Here we provide evidence - from microscopic analyses of the transcription dynamics at a single gene copy of yeast - that the regulatory process for transcription is cyclic and irreversible (out of equilibrium). The necessary coupling to reservoirs of free energy occurs via sequence-specific transcriptional activators and the recruitment, in part, of ATP-dependent chromatin remodelers. Our findings may help explain how eukaryotic cells reconcile the dual but opposing requirements for fast regulatory kinetics and high regulatory specificity.

The heart beats approximately 100,000 times per day in humans, imposing substantial energetic demands on cardiac muscle. Adenosine triphosphate (ATP) is an essential energy source for normal function of cardiac muscle during each beat, as it powers ion transport, intracellular Ca2+ handling, and actin-myosin cross-bridge cycling. Despite this, the impact of excitation-contraction coupling on the intracellular ATP concentration ([ATP]i) in myocytes is poorly understood. Here, we conducted real-time measurements of [ATP]i in ventricular myocytes using a genetically encoded ATP fluorescent reporter. Our data reveal rapid beat-to-beat variations in [ATP]i. Notably, diastolic [ATP]i was

The ability to sense chemical gradients and respond with directional motility and chemical activity is a defining feature of complex living systems. There is a strong interest among scientists to design synthetic systems that emulate these properties. Here, we realize and control such behaviors in a synthetic system by tailoring multivalent interactions of adenosine nucleotides with catalytic microbeads. We first show that multivalent interactions of the bead with gradients of adenosine mono-, di- and trinucleotides (AM/D/TP) control both the phoretic motion and a proton-transfer catalytic reaction, and find that both effects are diminished greatly with increasing valence of phosphates. We exploit this behavior by using enzymatic hydrolysis of ATP to AMP, which downregulates multivalent interactivity in situ. This produces a sudden increase in transport of the catalytic microbeads (a phoretic jump), which is accompanied by increased catalytic activity. Finally, we show how this enzymatic activity can be systematically tuned, leading to simultaneous in situ spatial and temporal control of the location of the microbeads, as well as the products of the reaction that they catalyze. These findings open up new avenues for utilizing multivalent interaction-mediated programming of complex chemo-mechanical behaviors into active systems.

Airway neuroendocrine (NE) cells have been proposed to serve as specialized sensory epithelial cells that modulate respiratory behavior by communicating with nearby nerve endings. However, their functional properties and physiological roles in the healthy lung, trachea, and larynx remain largely unknown. In this work, we show that murine NE cells in these compartments have distinct biophysical properties but share sensitivity to two commonly aspirated noxious stimuli, water and acid. Moreover, we found that tracheal and laryngeal NE cells protect the airways by releasing adenosine 5'-triphosphate (ATP) to activate purinoreceptive sensory neurons that initiate swallowing and expiratory reflexes. Our work uncovers the broad molecular and biophysical diversity of NE cells across the airways and reveals mechanisms by which these specialized excitable cells serve as sentinels for activating protective responses.

Liquid-liquid phase separation (LLPS) plays a pivotal role in the organization and functionality of living cells. It is imperative to understand the underlying driving forces behind LLPS and to control its occurrence. In this study, we employed coarse-grained (CG) simulations as a research tool to investigate systems comprising oligolysine and adenosine triphosphate (ATP) under conditions of various ionic concentrations and oligolysine lengths. Consistent with experimental observations, our CG simulations captured the formation of LLPS upon the addition of ATP and tendency of dissociating under high ionic concentration. The electrostatic interaction between oligolysine and ATP is of great importance in forming LLPS. An in-depth analysis on the structural properties of LLPS was conducted, where the oligolysine structure remained unchanged with increased ionic concentration and the addition of ATP led to a more pronounced curvature, aligning with the observed enhancement of α-helical secondary structure in experiments. In terms of the dynamic properties, the introduction of ATP led to a significant reduction in translational and vibrational degrees of freedom but not rotational degrees of freedom. Through keeping the total number of charged residues constant and varying their entropic effects, we constructed two systems of similar biochemical significance and further validated the entropy effects on the LLPS formation. These findings provide a deeper understanding of LLPS formation and shed lights on the development of novel bioreactor and primitive artificial cells for synthesizing key chemicals for certain diseases.

Background: Asynchronous telepsychiatry (ATP) consultations are a novel form of psychiatric consultation. Studies comparing patient and provider satisfaction for ATP with that for synchronous telepsychiatry (STP) do not exist. Methods: This mixed-methods study is a secondary analysis of patients' and primary care providers' (PCPs) satisfaction from a randomized clinical trial of ATP compared with STP. Patients and their PCPs completed satisfaction surveys, and provided unstructured feedback about their experiences with either ATP or STP. Differences in patient satisfaction were assessed using mixed-effects logistic regression models, and the qualitative data were analyzed using thematic analysis with an inductive coding framework. Results: Patient satisfaction overall was high with 84% and 97% of respondents at 6 months reported being somewhat or completely satisfied with ATP and STP, respectively. Patients in the STP group were more likely to report being completely satisfied, to recommend the program to a friend, and to report being comfortable with their care compared with ATP (all p < 0.05). However, there was no difference between the patients in ATP and STP in perceived change in clinical outcomes (p = 0.51). The PCP quantitative data were small, and thus only summarized descriptively. Conclusions: Patients expressed their overall satisfaction with both STP and ATP. Patients in ATP reported more concerns about the process, likely because feedback after ATP was slower than that after STP consultations. PCPs had no apparent preference for STP or ATP, and reported implementing the psychiatrists' recommendations for both groups when such recommendations were made, which supports our previous findings. Trial Registration: ClinicalTrials.gov NCT02084979; https://clinicaltrials.gov/ct2/show/NCT02084979.

BACKGROUND: Phenotypic frailty syndrome identifies older adults at greater risk for adverse health outcomes. Despite the critical role of mitochondria in maintaining cellular function, including energy production, the associations between muscle mitochondrial energetics and frailty have not been widely explored in a large, well-phenotyped, older population. METHODS: The Study of Muscle, Mobility and Aging (SOMMA) assessed muscle energetics in older adults (N = 879, mean age = 76.3 years, 59.2% women). 31Phosporous magnetic resonance spectroscopy measured maximal production of adenosine triphosphate (ATPmax) in vivo, while ex vivo high-resolution respirometry of permeabilized muscle fibers from the vastus lateralis measured maximal oxygen consumption supported by fatty acids and complex I- and II-linked carbohydrates (e.g., Max OXPHOSCI+CII). Five frailty criteria, shrinking, weakness, exhaustion, slowness, and low activity, were used to classify participants as robust (0, N = 397), intermediate (1-2, N = 410), or frail (≥ 3, N = 66). We estimated the proportional odds ratio (POR) for greater frailty, adjusted for multiple potential confounders. RESULTS: One-SD decrements of most respirometry measures (e.g., Max OXPHOSCI+CII, adjusted POR = 1.5, 95%CI [1.2,1.8], p = 0.0001) were significantly associated with greater frailty classification. The associations of ATPmax with frailty were weaker than those between Max OXPHOSCI+CII and frailty. Muscle energetics was most strongly associated with slowness and low physical activity components. CONCLUSIONS: Our data suggest that deficits in muscle mitochondrial energetics may be a biological driver of frailty in older adults. On the other hand, we did observe differential relationships between measures of muscle mitochondrial energetics and the individual components of frailty.

Social stress experienced in childhood is associated with adverse health later in life. Mitochondrial function has been implicated as a mechanism for how stressful life events get under the skin to influence physical well-being. Using data from the Study of Muscle, Mobility, and Aging (n = 879, 59% women), linear models examined whether adverse childhood events (i.e., physical abuse) were associated with two measures of skeletal muscle mitochondrial energetics in older adults: (i) maximal adenosine triphosphate production (ATPmax) and (ii) maximal state 3 respiration (Max OXPHOS). Forty-five percent of the sample reported experiencing one or more adverse childhood events. After adjustment, each additional event was associated with -0.08 SD (95% confidence interval = -0.13, -0.02) lower ATPmax. No association was observed with Max OXPHOS. Adverse childhood events are associated with lower ATP production in later life. Findings indicate that mitochondrial function may be a mechanism for understanding how early social stress influences health in later life.

Mitochondrial membrane potential directly powers many critical functions of mitochondria, including ATP production, mitochondrial protein import, and metabolite transport. Its loss is a cardinal feature of aging and mitochondrial diseases, and cells closely monitor membrane potential as an indicator of mitochondrial health. Given its central importance, it is logical that cells would modulate mitochondrial membrane potential in response to demand and environmental cues, but there has been little exploration of this question. We report that loss of the Sit4 protein phosphatase in yeast increases mitochondrial membrane potential, both by inducing the electron transport chain and the phosphate starvation response. Indeed, a similarly elevated mitochondrial membrane potential is also elicited simply by phosphate starvation or by abrogation of the Pho85-dependent phosphate sensing pathway. This enhanced membrane potential is primarily driven by an unexpected activity of the ADP/ATP carrier. We also demonstrate that this connection between phosphate limitation and enhancement of mitochondrial membrane potential is observed in primary and immortalized mammalian cells as well as in Drosophila. These data suggest that mitochondrial membrane potential is subject to environmental stimuli and intracellular signaling regulation and raise the possibility for therapeutic enhancement of mitochondrial function even in defective mitochondria.

Respiratory chain dysfunction can decrease ATP and increase reactive oxygen species (ROS) levels. Despite the importance of these metabolic parameters to a wide range of cellular functions and disease, we lack an integrated understanding of how they are differentially regulated. To address this question, we adapted a CRISPRi- and FACS-based platform to compare the effects of respiratory gene knockdown on ROS to their effects on ATP. Focusing on genes whose knockdown is known to decrease mitochondria-derived ATP, we showed that knockdown of genes in specific respiratory chain complexes (I, III, and CoQ10 biosynthesis) increased ROS, whereas knockdown of other low ATP hits either had no impact (mitochondrial ribosomal proteins) or actually decreased ROS (complex IV). Moreover, although shifting metabolic conditions profoundly altered mitochondria-derived ATP levels, it had little impact on mitochondrial or cytosolic ROS. In addition, knockdown of a subset of complex I subunits-including NDUFA8, NDUFB4, and NDUFS8-decreased complex I activity, mitochondria-derived ATP, and supercomplex level, but knockdown of these genes had differential effects on ROS. Conversely, we found an essential role for ether lipids in the dynamic regulation of mitochondrial ROS levels independent of ATP. Thus, our results identify specific metabolic regulators of cellular ATP and ROS balance that may help dissect the roles of these processes in disease and identify therapeutic strategies to independently target energy failure and oxidative stress.

Epulopiscium spp. are the largest known heterotrophic bacteria; a large cigar-shaped individual is a million times the volume of Escherichia coli. To better understand the metabolic potential and relationship of Epulopiscium sp. type B with its host Naso tonganus, we generated a high-quality draft genome from a population of cells taken from a single fish. We propose the name Candidatus Epulopiscium viviparus to describe populations of this best-characterized Epulopiscium species. Metabolic reconstruction reveals more than 5% of the genome codes for carbohydrate active enzymes, which likely degrade recalcitrant host-diet algal polysaccharides into substrates that may be fermented to acetate, the most abundant short-chain fatty acid in the intestinal tract. Moreover, transcriptome analyses and the concentration of sodium ions in the host intestinal tract suggest that the use of a sodium motive force (SMF) to drive ATP synthesis and flagellar rotation is integral to symbiont metabolism and cellular biology. In natural populations, genes encoding both F-type and V-type ATPases and SMF generation via oxaloacetate decarboxylation are among the most highly expressed, suggesting that ATPases synthesize ATP and balance ion concentrations across the cell membrane. High expression of these and other integral membrane proteins may allow for the growth of its extensive intracellular membrane system. Further, complementary metabolism between microbe and host is implied with the potential provision of nitrogen and B vitamins to reinforce this nutritional symbiosis. The few features shared by all bacterial behemoths include extreme polyploidy, polyphosphate synthesis, and thus far, they have all resisted cultivation in the lab.

The recent increase in the use of nicotine products by teenagers has revealed an urgent need to better understand the impact of nicotine on the adolescent brain. Here, we sought to examine the actions of extracellular ATP as a neurotransmitter and to investigate whether ATP and nicotinic signaling interact during adolescence. With the GRABATP (G-protein-coupled receptor activation-based ATP sensor), we first demonstrated that nicotine induces extracellular ATP release in the medial habenula, a brain region involved in nicotine aversion and withdrawal. Using patch-clamp electrophysiology, we then demonstrated that activation of the ATP receptors P2X or P2Y1 increases the neuronal firing of cholinergic neurons. Surprisingly, contrasting interactive effects were observed with nicotine exposure. For the P2X receptor, activation had no observable effect on acute nicotine-mediated activity, but during abstinence after 10 d of nicotine exposure, coexposure to nicotine and the P2X agonist potentiated neuronal activity in female, but not male, neurons. For P2Y1 signaling, a potentiated effect of the agonist and nicotine was observed with acute exposure, but not following extended nicotine exposure. These data reveal a complex interactive effect between nicotinic and ATP signaling in the adolescent brain and provide mechanistic insights into extracellular ATP signaling with sex-specific alterations of neuronal responses based on prior drug exposure.SIGNIFICANCE STATEMENT In these studies, it was discovered that nicotine induces extracellular ATP release in the medial habenula and subsequent activation of the ATP purinergic receptors increases habenular cholinergic neuronal firing in the adolescent brain. Interestingly, following extended nicotine exposure, nicotine was found to alter the interplay between purinergic and nicotinic signaling in a sex-specific manner. Together, these studies provide a novel understanding for the role of extracellular ATP in mediating habenular activity and reveal how nicotine exposure during adolescence alters these signaling mechanisms, which has important implications given the high incidence of e-cigarette/vape use by youth.

The proximal tubule (PT) is a nephron segment that is responsible for the majority of solute and water reabsorption in the kidney. Each of its sub-segments have specialized functions; however, little is known about the genes and proteins that determine the oxidative phosphorylation capacity of the PT sub-segments. This information is critical to understanding kidney function and will provide a comprehensive landscape of renal cell adaptations to injury, physiologic stressors, and development. This study analyzed three immortalized murine renal cell lines (PT S1, S2, and S3 segments) for protein content and compared them to a murine fibroblast cell line. All three proximal tubule cell lines generate ATP predominantly by oxidative phosphorylation while the fibroblast cell line is glycolytic. The proteomic data demonstrates that the most significant difference in proteomic signatures between the cell lines are proteins known to be localized in the nucleus followed by mitochondrial proteins. Mitochondrial metabolic substrate utilization assays were performed using the proximal tubule cell lines to determine substrate utilization kinetics thereby providing a physiologic context to the proteomic dataset. This data will allow researchers to study differences in nephron-specific cell lines, between epithelial and fibroblast cells, and between actively respiring cells and glycolytic cells. SIGNIFICANCE: Proteomic analysis of proteins expressed in immortalized murine renal proximal tubule cells was compared to a murine fibroblast cell line proteome. The proximal tubule segment specific cell lines: S1, S2 and S3 are all grown under conditions whereby the cells generate ATP by oxidative phosphorylation while the fibroblast cell line utilizes anaerobic glycolysis for ATP generation. The proteomic studies allow for the following queries: 1) comparisons between the proximal tubule segment specific cell lines, 2) comparisons between polarized epithelia and fibroblasts, 3) comparison between cells employing oxidative phosphorylation versus anaerobic glycolysis and 4) comparisons between cells grown on clear versus opaque membrane supports. The data finds major differences in nuclear protein expression and mitochondrial proteins. This proteomic data set will be an important baseline dataset for investigators who need immortalized renal proximal tubule epithelial cells for their research.

Oxidative phosphorylation and glycolysis are the dominant ATP-generating pathways in mammalian metabolism. The balance between these two pathways is often shifted to execute cell-specific functions in response to stimuli that promote activation, proliferation, or differentiation. However, measurement of these metabolic switches has remained mostly qualitative, making it difficult to discriminate between healthy, physiological changes in energy transduction or compensatory responses due to metabolic dysfunction. We therefore present a broadly applicable method to calculate ATP production rates from oxidative phosphorylation and glycolysis using Seahorse XF Analyzer data and empirical conversion factors. We quantify the bioenergetic changes observed during macrophage polarization as well as cancer cell adaptation to in vitro culture conditions. Additionally, we detect substantive changes in ATP utilization upon neuronal depolarization and T cell receptor activation that are not evident from steady-state ATP measurements. This method generates a single readout that allows the direct comparison of ATP produced from oxidative phosphorylation and glycolysis in live cells. Additionally, the manuscript provides a framework for tailoring the calculations to specific cell systems or experimental conditions.

Anaerobic fungi (Neocallimastigomycetes) found in the guts of herbivores are biomass deconstruction specialists with a remarkable ability to extract sugars from recalcitrant plant material. Anaerobic fungi, as well as many species of anaerobic bacteria, deploy multi-enzyme complexes called cellulosomes, which modularly tether together hydrolytic enzymes, to accelerate biomass hydrolysis. While the majority of genomically encoded cellulosomal genes in Neocallimastigomycetes are biomass degrading enzymes, the second largest family of cellulosomal genes encode spore coat CotH domains, whose contribution to fungal cellulosome and/or cellular function is unknown. Structural bioinformatics of CotH proteins from the anaerobic fungus Piromyces finnis shows anaerobic fungal CotH domains conserve key ATP and Mg2+ binding motifs from bacterial Bacillus CotH proteins known to act as protein kinases. Experimental characterization further demonstrates ATP hydrolysis activity in the presence and absence of substrate from two cellulosomal P. finnis CotH proteins when recombinantly produced in E. coli. These results present foundational evidence for CotH activity in anaerobic fungi and provide a path towards elucidating the functional contribution of this protein family to fungal cellulosome assembly and activity.

Life is based on energy conversion. In particular, in the nervous system, significant amounts of energy are needed to maintain synaptic transmission and homeostasis. To a large extent, neurons depend on oxidative phosphorylation in mitochondria to meet their high energy demand. For a comprehensive understanding of the metabolic demands in neuronal signaling, accurate models of ATP production in mitochondria are required. Here, we present a thermodynamically consistent model of ATP production in mitochondria based on previous work. The significant improvement of the model is that the reaction rate constants are set such that detailed balance is satisfied. Moreover, using thermodynamic considerations, the dependence of the reaction rate constants on membrane potential, pH, and substrate concentrations are explicitly provided. These constraints assure that the model is physically plausible. Furthermore, we explore different parameter regimes to understand in which conditions ATP production or its export are the limiting steps in making ATP available in the cytosol. The outcomes reveal that, under the conditions used in our simulations, ATP production is the limiting step and not its export. Finally, we performed spatial simulations with nine 3-D realistic mitochondrial reconstructions and linked the ATP production rate in the cytosol with morphological features of the organelles.

Renal ischemia/reperfusion (I/R) injury contributes to the development of acute kidney injury (AKI). Kidney is the second organ rich in mitochondrial content next to the heart. Mitochondrial damage substantially contributes for AKI development. Mitophagy eliminates damaged mitochondria from the cells to maintain a healthy mitochondrial population, which plays an important role in AKI. Pannexin 1 (PANX1) channel transmembrane proteins are known to drive inflammation and release of adenosine triphosphate (ATP) during I/R injury. However, the specific role of PANX1 on mitophagy regulation in renal I/R injury remains elusive. In this study, we find that serum level of PANX1 is elevated in patients who developed AKI after cardiac surgery, and the level of PANX1 is positively correlated with serum creatinine and urea nitrogen levels. Using the mouse model of renal I/R injury in vivo and cell-based hypoxia/reoxygenation (H/R) model in vitro, we prove that genetic deletion of PANX1 mitigate the kidney tubular cell death, oxidative stress and mitochondrial damage after I/R injury through enhanced mitophagy. Mechanistically, PANX1 disrupts mitophagy by influencing ATP-P2Y-mTOR signal pathway. These observations provide evidence that PANX1 could be a potential biomarker for AKI and a therapeutic target to alleviate AKI caused by I/R injury.

Coevolved genetic interactions within populations can be disrupted by hybridization resulting in loss of fitness in hybrid individuals (i.e., hybrid breakdown). However, the extent to which variation in fitness-related traits among hybrids is inherited across generations remains unclear, and variation in these traits may be sex-specific in hybrids due to differential effects of genetic incompatibilities in females and males. Here we present two experiments investigating variation in developmental rate among reciprocal interpopulation hybrids of the intertidal copepod Tigriopus californicus. Developmental rate is a fitness-related trait in this species that is affected by interactions between mitochondrial-encoded and nuclear-encoded genes in hybrids that result in variation in mitochondrial ATP synthesis capacities. First, we show that F2 -hybrid developmental rate is equivalent in two reciprocal crosses and is unaffected by sex, suggesting that breakdown of developmental rate is likely experienced equally by females and males. Second, we demonstrate that variation in developmental rate among F3 hybrids is heritable; times to copepodid metamorphosis of F4 offspring of fast-developing F3 parents (12.25 ± 0.05 days, μ ± SEM) were significantly faster than those of F4 offspring of slow-developing parents (14.58 ± 0.05 days). Third, we find that ATP synthesis rates in these F4 hybrids are unaffected by the developmental rates of their parents, but that mitochondria from females synthesize ATP at faster rates than mitochondria from males. Taken together, these results suggest that sex-specific effects vary among fitness-related traits in these hybrids, and that effects likely associated with hybrid breakdown display substantial inheritance across hybrid generations.

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart disease (CHD) with a likely oligogenic etiology, but our understanding of the genetic complexities and pathogenic mechanisms leading to HLHS is limited. We performed whole genome sequencing (WGS) on 183 HLHS patient-parent trios to identify candidate genes, which were functionally tested in the Drosophila heart model. Bioinformatic analysis of WGS data from an index family of a HLHS proband born to consanguineous parents prioritized 9 candidate genes with rare, predicted damaging homozygous variants. Of them, cardiac-specific knockdown (KD) of mitochondrial MICOS complex subunit dCHCHD3/6 resulted in drastically compromised heart contractility, diminished levels of sarcomeric actin and myosin, reduced cardiac ATP levels, and mitochondrial fission-fusion defects. These defects were similar to those inflicted by cardiac KD of ATP synthase subunits of the electron transport chain (ETC), consistent with the MICOS complexs role in maintaining cristae morphology and ETC assembly. Five additional HLHS probands harbored rare, predicted damaging variants in CHCHD3 or CHCHD6. Hypothesizing an oligogenic basis for HLHS, we tested 60 additional prioritized candidate genes from these patients for genetic interactions with CHCHD3/6 in sensitized fly hearts. Moderate KD of CHCHD3/6 in combination with Cdk12 (activator of RNA polymerase II), RNF149 (goliath, E3 ubiquitin ligase), or SPTBN1 (β-Spectrin, scaffolding protein) caused synergistic heart defects, suggesting the likely involvement of diverse pathways in HLHS. Further elucidation of novel candidate genes and genetic interactions of potentially disease-contributing pathways is expected to lead to a better understanding of HLHS and other CHDs.

RAD51C is an enigmatic predisposition gene for breast, ovarian, and prostate cancer. Currently, missing structural and related functional understanding limits patient mutation interpretation to homology-directed repair (HDR) function analysis. Here we report the RAD51C-XRCC3 (CX3) X-ray co-crystal structure with bound ATP analog and define separable RAD51C replication stability roles informed by its three-dimensional structure, assembly, and unappreciated polymerization motif. Mapping of cancer patient mutations as a functional guide confirms ATP-binding matching RAD51 recombinase, yet highlights distinct CX3 interfaces. Analyses of CRISPR/Cas9-edited human cells with RAD51C mutations combined with single-molecule, single-cell and biophysics measurements uncover discrete CX3 regions for DNA replication fork protection, restart and reversal, accomplished by separable functions in DNA binding and implied 5' RAD51 filament capping. Collective findings establish CX3 as a cancer-relevant replication stress response complex, show how HDR-proficient variants could contribute to tumor development, and identify regions to aid functional testing and classification of cancer mutations.

Environmental temperatures have pervasive effects on the performance and tolerance of ectothermic organisms, and thermal tolerance limits likely play key roles underlying biogeographic ranges and responses to environmental change. Mitochondria are central to metabolic processes in eukaryotic cells, and these metabolic functions are thermally sensitive; however, potential relationships between mitochondrial function, thermal tolerance limits and local thermal adaptation in general remain unresolved. Loss of ATP synthesis capacity at high temperatures has recently been suggested as a mechanistic link between mitochondrial function and upper thermal tolerance limits. Here we use a common-garden experiment with seven locally adapted populations of intertidal copepods (Tigriopus californicus), spanning approximately 21.5° latitude, to assess genetically based variation in the thermal performance curves of maximal ATP synthesis rates in isolated mitochondria. These thermal performance curves displayed substantial variation among populations with higher ATP synthesis rates at lower temperatures (20-25 °C) in northern populations than in southern populations. In contrast, mitochondria from southern populations maintained ATP synthesis rates at higher temperatures than the temperatures that caused loss of ATP synthesis capacity in mitochondria from northern populations. Additionally, there was a tight correlation between the thermal limits of ATP synthesis and previously determined variation in upper thermal tolerance limits among populations. This suggests that mitochondria may play an important role in latitudinal thermal adaptation in T. californicus, and supports the hypothesis that loss of mitochondrial performance at high temperatures is linked to whole-organism thermal tolerance limits in this ectotherm.

SARS-CoV-2 RNA dependent RNA polymerase (RdRp) serves as a highly promising antiviral drug target such as for a Remdesivir nucleotide analogue (RDV-TP or RTP). In this work, we mainly used alchemical all-atom simulations to characterize relative binding free energetics between the nucleotide analogue RTP and natural cognate substrate ATP upon initial binding and pre-catalytic insertion into the active site of SARS-CoV-2 RdRp. Natural non-cognate substrate dATP and mismatched GTP were also examined for computation control. We first identified significant differences in dynamical responses between nucleotide initial binding and subsequent insertion configurations to the open and closed active sites of the RdRp, respectively, though the RdRp protein conformational changes between the active site's open and closed states are subtle. Our alchemical simulations indicated that upon initial binding (active site open), RTP and ATP show similar binding free energies to the active sites while in the insertion state (active site closed), ATP is more stabilized (∼-2.4 kcal mol-1) than RTP in free energetics. Additional analyses show, however, that RTP is more stabilized in binding energetics than ATP, in both the insertion and initial binding states, with RTP more stabilized due to the electrostatic energy in the insertion state and due to vdW energy in the initial binding state. Hence, it appears that natural cognate ATP still excels at association stability with the RdRp active site due to that ATP maintains sufficient flexibilities e.g., in base pairing with the template, which exemplifies an entropic contribution to the cognate substrate stabilization. These findings highlight the importance of substrate flexibilities in addition to energetic stabilization in antiviral nucleotide analogue design.

The maintenance of cellular function relies on the close regulation of adenosine triphosphate (ATP) synthesis and hydrolysis. ATP hydrolysis by mitochondrial ATP Synthase (CV) is induced by loss of proton motive force and inhibited by the mitochondrial protein ATPase inhibitor (ATPIF1). The extent of CV hydrolytic activity and its impact on cellular energetics remains unknown due to the lack of selective hydrolysis inhibitors of CV. We find that CV hydrolytic activity takes place in coupled intact mitochondria and is increased by respiratory chain defects. We identified (+)-Epicatechin as a selective inhibitor of ATP hydrolysis that binds CV while preventing the binding of ATPIF1. In cells with Complex-III deficiency, we show that inhibition of CV hydrolytic activity by (+)-Epichatechin is sufficient to restore ATP content without restoring respiratory function. Inhibition of CV-ATP hydrolysis in a mouse model of Duchenne Muscular Dystrophy is sufficient to improve muscle force without any increase in mitochondrial content. We conclude that the impact of compromised mitochondrial respiration can be lessened using hydrolysis-selective inhibitors of CV.

Proteolytic complexes in Mycobacterium tuberculosis (Mtb), the deadliest bacterial pathogen, are major foci in tuberculosis drug development programs. The Clp proteases, which are essential for Mtb viability, are high-priority targets. These proteases function through the collaboration of ClpP1P2, a barrel-shaped heteromeric peptidase, with associated ATP-dependent chaperones like ClpX and ClpC1 that recognize and unfold specific substrates in an ATP-dependent fashion. The critical interaction of the peptidase and its unfoldase partners is blocked by the competitive binding of acyldepsipeptide antibiotics (ADEPs) to the interfaces of the ClpP2 subunits. The resulting inhibition of Clp protease activity is lethal to Mtb. Here, we report the surprising discovery that a fragment of the ADEPs retains anti-Mtb activity yet stimulates rather than inhibits the ClpXP1P2-catalyzed degradation of proteins. Our data further suggest that the fragment stabilizes the ClpXP1P2 complex and binds ClpP1P2 in a fashion distinct from that of the intact ADEPs. A structure-activity relationship study of the bioactive fragment defines the pharmacophore and points the way toward the development of new drug leads for the treatment of tuberculosis.

The vacuolar H+-ATPase is an enzymatic complex that functions in an ATP-dependent manner to pump protons across membranes and acidify organelles, thereby creating the proton/pH gradient required for membrane trafficking by several different types of transporters. We describe heterozygous point variants in ATP6V0C, encoding the c-subunit in the membrane bound integral domain of the vacuolar H+-ATPase, in 27 patients with neurodevelopmental abnormalities with or without epilepsy. Corpus callosum hypoplasia and cardiac abnormalities were also present in some patients. In silico modelling suggested that the patient variants interfere with the interactions between the ATP6V0C and ATP6V0A subunits during ATP hydrolysis. Consistent with decreased vacuolar H+-ATPase activity, functional analyses conducted in Saccharomyces cerevisiae revealed reduced LysoSensor fluorescence and reduced growth in media containing varying concentrations of CaCl2. Knockdown of ATP6V0C in Drosophila resulted in increased duration of seizure-like behaviour, and the expression of selected patient variants in Caenorhabditis elegans led to reduced growth, motor dysfunction and reduced lifespan. In summary, this study establishes ATP6V0C as an important disease gene, describes the clinical features of the associated neurodevelopmental disorder and provides insight into disease mechanisms.

RecA-mediated homologous recombination (HR) is a key mechanism for genome maintenance and plasticity in bacteria. It proceeds through RecA assembly into a dynamic filament on ssDNA, the presynaptic filament, which mediates DNA homology search and ordered DNA strand exchange. Here, we combined structural, single molecule and biochemical approaches to characterize the ATP-dependent assembly mechanism of the presynaptic filament of RecA from Streptococcus pneumoniae (SpRecA), in comparison to the Escherichia coli RecA (EcRecA) paradigm. EcRecA polymerization on ssDNA is assisted by the Single-Stranded DNA Binding (SSB) protein, which unwinds ssDNA secondary structures that block EcRecA nucleofilament growth. We report by direct microscopic analysis of SpRecA filamentation on ssDNA that neither of the two paralogous pneumococcal SSBs could assist the extension of SpRecA nucleopolymers. Instead, we found that the conserved RadA helicase promotes SpRecA nucleofilamentation in an ATP-dependent manner. This allowed us to solve the atomic structure of such a long native SpRecA nucleopolymer by cryoEM stabilized with ATPγS. It was found to be equivalent to the crystal structure of the EcRecA filament with a marked difference in how RecA mediates nucleotide orientation in the stretched ssDNA. Then, our results show that SpRecA and EcRecA HR activities are different, in correlation with their distinct ATP-dependent ssDNA binding modes.

ContextThe azide anion (N3-) is highly toxic. It exists most commonly as sodium azide, which is used widely and is readily available, raising the potential for occupational incidents and use as a weapon of mass destruction. Azide-poisoned patients present with vomiting, seizures, hypotension, metabolic acidosis, and coma; death can occur. No specific azide antidote exists, with treatment being solely supportive. Azide inhibits mitochondrial cytochrome c oxidase and is likely oxidized to nitric oxide in vivo. Cytochrome c oxidase inhibition depletes intracellular adenosine triphosphate and increases oxidative stress, while increased nitric oxide causes hypotension and exacerbates oxidative damage. Here, we tested whether the cobalamin (vitamin B12) analog cobinamide, a strong and versatile antioxidant that also neutralizes nitric oxide, can reverse azide toxicity in mammalian cells, Drosophila melanogaster, and mice.ResultsWe found cobinamide bound azide with a moderate affinity (Ka 2.87 × 105 M-1). Yet, cobinamide improved growth, increased intracellular adenosine triphosphate, and reduced apoptosis and malondialdehyde, a marker of oxidative stress, in azide-exposed cells. Cobinamide rescued Drosophila melanogaster and mice from lethal exposure to azide and was more effective than hydroxocobalamin. Azide likely generated nitric oxide in the mice, as evidenced by increased serum nitrite and nitrate, and reduced blood pressure and peripheral body temperature in the animals; the reduced temperature was likely due to reflex vasoconstriction in response to the hypotension. Cobinamide improved recovery of both blood pressure and body temperature.ConclusionWe conclude cobinamide likely acted by neutralizing both oxidative stress and nitric oxide, and that it should be given further consideration as an azide antidote.

Mitochondrial depolarization can initiate reversal activity of ATP synthase, depleting ATP by its hydrolysis. We have recently shown that increased ATP hydrolysis contributes to ATP depletion leading to a maladaptation in mitochondrial disorders, where maximal hydrolytic capacity per CV content is increasing. However, despite its importance, ATP hydrolysis is not a commonly studied parameter because of the limitations of the currently available methods. Methods that measure CV hydrolytic activity indirectly require the isolation of mitochondria and involve the introduction of detergents, preventing their utilization in clinical studies or any high-throughput analyses. Here, we describe a novel approach to assess maximal ATP hydrolytic capacity and maximal respiratory capacity in a single assay in cell lysates, PBMCs, and tissue homogenates that were previously frozen. The methodology described here has the potential to be used in clinical samples to determine adaptive and maladaptive adjustments of CV function in diseases, with the added benefit of being able to use frozen samples in a high-throughput manner and to explore ATP hydrolysis as a drug target for disease treatment.

The circadian system of the cyanobacterium Synechococcus elongatus PCC 7942 relies on a three-protein nanomachine (KaiA, KaiB, and KaiC) that undergoes an oscillatory phosphorylation cycle with a period of ~24 h. This core oscillator can be reconstituted in vitro and is used to study the molecular mechanisms of circadian timekeeping and entrainment. Previous studies showed that two key metabolic changes that occur in cells during the transition into darkness, changes in the ATP/ADP ratio and redox status of the quinone pool, are cues that entrain the circadian clock. By changing the ATP/ADP ratio or adding oxidized quinone, one can shift the phase of the phosphorylation cycle of the core oscillator in vitro. However, the in vitro oscillator cannot explain gene expression patterns because the simple mixture lacks the output components that connect the clock to genes. Recently, a high-throughput in vitro system termed the in vitro clock (IVC) that contains both the core oscillator and the output components was developed. Here, we used IVC reactions and performed massively parallel experiments to study entrainment, the synchronization of the clock with the environment, in the presence of output components. Our results indicate that the IVC better explains the in vivo clock-resetting phenotypes of wild-type and mutant strains and that the output components are deeply engaged with the core oscillator, affecting the way input signals entrain the core pacemaker. These findings blur the line between input and output pathways and support our previous demonstration that key output components are fundamental parts of the clock.

Muscle contraction is performed by arrays of contractile proteins in the sarcomere. Serious heart diseases, such as cardiomyopathy, can often be results of mutations in myosin and actin. Direct characterization of how small changes in the myosin-actin complex impact its force production remains challenging. Molecular dynamics (MD) simulations, although capable of studying protein structure-function relationships, are limited owing to the slow timescale of the myosin cycle as well as a lack of various intermediate structures for the actomyosin complex. Here, employing comparative modeling and enhanced sampling MD simulations, we show how the human cardiac myosin generates force during the mechanochemical cycle. Initial conformational ensembles for different myosin-actin states are learned from multiple structural templates with Rosetta. This enables us to efficiently sample the energy landscape of the system using Gaussian accelerated MD. Key myosin loop residues, whose substitutions are related to cardiomyopathy, are identified to form stable or metastable interactions with the actin surface. We find that the actin-binding cleft closure is allosterically coupled to the myosin motor core transitions and ATP-hydrolysis product release from the active site. Furthermore, a gate between switch I and switch II is suggested to control phosphate release at the prepowerstroke state. Our approach demonstrates the ability to link sequence and structural information to motor functions.

Thermogenesis by uncoupling protein 1 (UCP1) is one of the primary mechanisms by which brown adipose tissue (BAT) increases energy expenditure. UCP1 resides in the inner mitochondrial membrane (IMM), where it dissipates membrane potential independent of adenosine triphosphate (ATP) synthase. Here, we provide evidence that phosphatidylethanolamine (PE) modulates UCP1-dependent proton conductance across the IMM to modulate thermogenesis. Mitochondrial lipidomic analyses revealed PE as a signature molecule whose abundance bidirectionally responds to changes in thermogenic burden. Reduction in mitochondrial PE by deletion of phosphatidylserine decarboxylase (PSD) made mice cold intolerant and insensitive to β3 adrenergic receptor agonist-induced increase in whole-body oxygen consumption. High-resolution respirometry and fluorometry of BAT mitochondria showed that loss of mitochondrial PE specifically lowers UCP1-dependent respiration without compromising electron transfer efficiency or ATP synthesis. These findings were confirmed by a reduction in UCP1 proton current in PE-deficient mitoplasts. Thus, PE performs a previously unknown role as a temperature-responsive rheostat that regulates UCP1-dependent thermogenesis.

Tissues derive ATP from two pathways-glycolysis and the tricarboxylic acid (TCA) cycle coupled to the electron transport chain. Most energy in mammals is produced via TCA metabolism1. In tumours, however, the absolute rates of these pathways remain unclear. Here we optimize tracer infusion approaches to measure the rates of glycolysis and the TCA cycle in healthy mouse tissues, Kras-mutant solid tumours, metastases and leukaemia. Then, given the rates of these two pathways, we calculate total ATP synthesis rates. We find that TCA cycle flux is suppressed in all five primary solid tumour models examined and is increased in lung metastases of breast cancer relative to primary orthotopic tumours. As expected, glycolysis flux is increased in tumours compared with healthy tissues (the Warburg effect2,3), but this increase is insufficient to compensate for low TCA flux in terms of ATP production. Thus, instead of being hypermetabolic, as commonly assumed, solid tumours generally produce ATP at a slower than normal rate. In mouse pancreatic cancer, this is accommodated by the downregulation of protein synthesis, one of this tissue's major energy costs. We propose that, as solid tumours develop, cancer cells shed energetically expensive tissue-specific functions, enabling uncontrolled growth despite a limited ability to produce ATP.

Phosphates and polyphosphates play ubiquitous roles in biology as integral structural components of cell membranes and bone, or as vehicles of energy storage via adenosine triphosphate and phosphocreatine. The solution phase space of phosphate species appears more complex than previously known. We present nuclear magnetic resonance (NMR) and cryogenic transmission electron microscopy (cryo-TEM) experiments that suggest phosphate species including orthophosphates, pyrophosphates, and adenosine phosphates associate into dynamic assemblies in dilute solutions that are spectroscopically "dark." Cryo-TEM provides visual evidence of the formation of spherical assemblies tens of nanometers in size, while NMR indicates that a majority population of phosphates remain as unassociated ions in exchange with spectroscopically invisible assemblies. The formation of these assemblies is reversibly and entropically driven by the partial dehydration of phosphate groups, as verified by diffusion-ordered spectroscopy (DOSY), indicating a thermodynamic state of assembly held together by multivalent interactions between the phosphates. Molecular dynamics simulations further corroborate that orthophosphates readily cluster in aqueous solutions. This study presents the surprising discovery that phosphate-containing molecules, ubiquitously present in the biological milieu, can readily form dynamic assemblies under a wide range of commonly used solution conditions, highlighting a hitherto unreported property of phosphate's native state in biological solutions.

Type 2 diabetes (T2D) has a complex pathophysiology which makes modeling the disease difficult. We aimed to develop a novel model for simulating T2D in vitro, including hyperglycemia, hyperlipidemia, and variably elevated insulin levels targeting muscle cells. We investigated insulin resistance (IR), cellular respiration, mitochondrial morphometry, and the associated function in different T2D-mimicking conditions in rodent skeletal (C2C12) and cardiac (H9C2) myotubes. The physiological controls included 5 mM of glucose with 20 mM of mannitol as osmotic controls. To mimic hyperglycemia, cells were exposed to 25 mM of glucose. Further treatments included insulin, palmitate, or both. After short-term (24 h) or long-term (96 h) exposure, we performed radioactive glucose uptake and mitochondrial function assays. The mitochondrial size and relative frequencies were assessed with morphometric analyses using electron micrographs. C2C12 and H9C2 cells that were treated short- or long-term with insulin and/or palmitate and HG showed IR. C2C12 myotubes exposed to T2D-mimicking conditions showed significantly decreased ATP-linked respiration and spare respiratory capacity and less cytoplasmic area occupied by mitochondria, implying mitochondrial dysfunction. In contrast, the H9C2 myotubes showed elevated ATP-linked and maximal respiration and increased cytoplasmic area occupied by mitochondria, indicating a better adaptation to stress and compensatory lipid oxidation in a T2D environment. Both cell lines displayed elevated fractions of swollen/vacuolated mitochondria after T2D-mimicking treatments. Our stable and reproducible in vitro model of T2D rapidly induced IR, changes in the ATP-linked respiration, shifts in energetic phenotypes, and mitochondrial morphology, which are comparable to the muscles of patients suffering from T2D. Thus, our model should allow for the study of disease mechanisms and potential new targets and allow for the screening of candidate therapeutic compounds.

Research has found that genes specific to microglia are among the strongest risk factors for Alzheimer's disease (AD) and that microglia are critically involved in the etiology of AD. Thus, microglia are an important therapeutic target for novel approaches to the treatment of AD. High-throughput in vitro models to screen molecules for their effectiveness in reversing the pathogenic, pro-inflammatory microglia phenotype are needed. In this study, we used a multi-stimulant approach to test the usefulness of the human microglia cell 3 (HMC3) cell line, immortalized from a human fetal brain-derived primary microglia culture, in duplicating critical aspects of the dysfunctional microglia phenotype. HMC3 microglia were treated with cholesterol (Chol), amyloid beta oligomers (AβO), lipopolysaccharide (LPS), and fructose individually and in combination. HMC3 microglia demonstrated changes in morphology consistent with activation when treated with the combination of Chol + AβO + fructose + LPS. Multiple treatments increased the cellular content of Chol and cholesteryl esters (CE), but only the combination treatment of Chol + AβO + fructose + LPS increased mitochondrial Chol content. Microglia treated with combinations containing Chol + AβO had lower apolipoprotein E (ApoE) secretion, with the combination of Chol + AβO + fructose + LPS having the strongest effect. Combination treatment with Chol + AβO + fructose + LPS also induced APOE and TNF-α expression, reduced ATP production, increased reactive oxygen species (ROS) concentration, and reduced phagocytosis events. These findings suggest that HMC3 microglia treated with the combination of Chol + AβO + fructose + LPS may be a useful high-throughput screening model amenable to testing on 96-well plates to test potential therapeutics to improve microglial function in the context of AD.

Primary mitochondrial diseases are a group of genetically and clinically heterogeneous disorders resulting from oxidative phosphorylation (OXPHOS) defects. COX11 encodes a copper chaperone that participates in the assembly of complex IV and has not been previously linked to human disease. In a previous study, we identified that COX11 knockdown decreased cellular adenosine triphosphate (ATP) derived from respiration, and that ATP levels could be restored with coenzyme Q10 (CoQ10 ) supplementation. This finding is surprising since COX11 has no known role in CoQ10 biosynthesis. Here, we report a novel gene-disease association by identifying biallelic pathogenic variants in COX11 associated with infantile-onset mitochondrial encephalopathies in two unrelated families using trio genome and exome sequencing. Functional studies showed that mutant COX11 fibroblasts had decreased ATP levels which could be rescued by CoQ10 . These results not only suggest that COX11 variants cause defects in energy production but reveal a potential metabolic therapeutic strategy for patients with COX11 variants.

The marine cyanobacterium Prochlorococcus can utilize glucose as a source of carbon. However, the relative importance of inorganic and organic carbon assimilation and the timing of glucose assimilation are still poorly understood in these numerically dominant cyanobacteria. Here, we investigated whole microbial community and group-specific primary production and glucose assimilation using incubations with radioisotopes combined with flow cytometry cell sorting. We also studied changes in the microbial community structure in response to glucose enrichments and analyzed the transcription of Prochlorocccus genes involved in carbon metabolism and photosynthesis. Our results showed a diel variation for glucose assimilation in Prochlorococcus, with maximum assimilation at midday and minimum at midnight (~2-fold change), which was different from that of the total microbial community. This suggests that the timing in glucose assimilation in Prochlorococcus is coupled to photosynthetic light reactions producing energy, it being more convenient for Prochlorococcus to show maximum glucose uptake precisely when the rest of microbial populations have their minimum glucose uptake. Many transcriptional responses to glucose enrichment occurred after 12- and 24-h periods, but community composition did not change. High-light Prochlorococcus strains were the most impacted by glucose addition, with transcript-level increases observed for genes in pathways for glucose metabolism, such as the pentose phosphate pathway, the Entner-Doudoroff pathway, glycolysis, respiration, and glucose transport. While Prochlorococcus C assimilation from glucose represented less than 0.1% of the bacteriums photosynthetic C fixation, increased assimilation during the day and glcH gene upregulation upon glucose enrichment indicate an important role of mixotrophic C assimilation by natural populations of Prochlorococcus. IMPORTANCE Several studies have demonstrated that Prochlorococcus, the most abundant photosynthetic organism on Earth, can assimilate organic molecules, such as amino acids, amino sugars, ATP, phosphonates, and dimethylsulfoniopropionate. This autotroph can also assimilate small amounts of glucose, supporting the hypothesis that Prochlorococcus is mixotrophic. Our results show, for the first time, a diel variability in glucose assimilation by natural populations of Prochlorococcus with maximum assimilation during midday. Based on our previous results, this indicates that Prochlorococcus could maximize glucose uptake by using ATP made during the light reactions of photosynthesis. Furthermore, Prochlorococcus showed a different timing of glucose assimilation from the total population, which may offer considerable fitness advantages over competitors temporal niches. Finally, we observed transcriptional changes in some of the genes involved in carbon metabolism, suggesting that Prochlorococcus can use both pathways previously proposed in cyanobacteria to metabolize glucose.

Eukaryotic DNA mismatch repair (MMR) depends on recruitment of the Mlh1-Pms1 endonuclease (human MLH1-PMS2) to mispaired DNA. Both Mlh1 and Pms1 contain a long unstructured linker that connects the N- and carboxyl-terminal domains. Here, we demonstrated the Mlh1 linker contains a conserved motif (Saccharomyces cerevisiae residues 391-415) required for MMR. The Mlh1-R401A,D403A-Pms1 linker motif mutant protein was defective for MMR and endonuclease activity in vitro, even though the conserved motif could be >750 Å from the carboxyl-terminal endonuclease active site or the N-terminal adenosine triphosphate (ATP)-binding site. Peptides encoding this motif inhibited wild-type Mlh1-Pms1 endonuclease activity. The motif functioned in vivo at different sites within the Mlh1 linker and within the Pms1 linker. Motif mutations in human cancers caused a loss-of-function phenotype when modeled in S. cerevisiae. These results suggest that the Mlh1 motif promotes the PCNA-activated endonuclease activity of Mlh1-Pms1 via interactions with DNA, PCNA, RFC, or other domains of the Mlh1-Pms1 complex.

Eukaryotes have evolved multiple ATP-dependent chromatin remodelers to shape the nucleosome landscape. We recently uncovered an evolutionarily conserved SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeler complex in plants reminiscent of the mammalian BAF subclass, which specifically incorporates the MINUSCULE (MINU) catalytic subunits and the TRIPLE PHD FINGERS (TPF) signature subunits. Here we report experimental evidence that establishes the functional relevance of TPF proteins for the complex activity. Our results show that depletion of TPF triggers similar pleiotropic phenotypes and molecular defects to those found in minu mutants. Moreover, we report the genomic location of MINU2 and TPF proteins as representative members of this SWI/SNF complex and their impact on nucleosome positioning and transcription. These analyses unravel the binding of the complex to thousands of genes where it modulates the position of the +1 nucleosome. These targets tend to produce 5-shifted transcripts in the tpf and minu mutants pointing to the participation of the complex in alternative transcription start site usage. Interestingly, there is a remarkable correlation between +1 nucleosome shift and 5 transcript length change suggesting their functional connection. In summary, this study unravels the function of a plant SWI/SNF complex involved in +1 nucleosome positioning and transcription start site determination.

PurposeWe constructed a 13C/31P surface coil at 3 T for studying cancer metabolism and bioenergetics. In a single scan session, hyperpolarized 13C-pyruvate MRS and 31P MRS was carried out for a healthy rat brain.MethodsAll experiments were carried out at 3 Tesla. The multinuclear surface coil was designed as two coplanar loops each tuned to either the 13C or 31P operating frequency with an LCC trap on the 13C loop. A commercial volume proton coil was used for anatomical localization and B0 shimming. Single tuned coils operating at either the 13C or 31P frequency were built to evaluate the relative performance of the multinuclear coil. Coil performance metrics consisted of measuring Q factor ratio, calculating system input power using a single-pulse acquisition, and acquiring SNR and flip angle maps using 2D CSI sequences. To observe in vivo spectra, a bolus of hyperpolarized [1-13C] pyruvate was administered via tail vein. In vivo13C and endogenous 31P spectra were obtained in a single scan session using 1D slice selective acquisitions.ResultsWhen compared with single tuned surface coils, the multinuclear coil performance showed a decrease in Q factor ratio, SNR, and transmit efficiency. Flip angle maps showed adequate flip angles within the phantom when the transmit voltage was set using an external phantom. Results show good detection of 13C labeled lactate, alanine, and bicarbonate in addition to ATP from 31P MRS.ConclusionsThe coil enables obtaining complementary information within a scan session, thus reducing the number of trials and minimizing biological variability for studies of metabolism and bioenergetics.

During translation initiation, the underlying mechanism by which the eukaryotic initiation factor (eIF) 4E, eIF4A, and eIF4G components of eIF4F coordinate their binding activities to regulate eIF4F binding to mRNA is poorly defined. Here, we used fluorescence anisotropy to generate thermodynamic and kinetic frameworks for the interaction of uncapped RNA with human eIF4F. We demonstrate that eIF4E binding to an autoinhibitory domain in eIF4G generates a high-affinity binding conformation of the eIF4F complex for RNA. In addition, we show that the nucleotide-bound state of the eIF4A component further regulates uncapped RNA binding by eIF4F, with a four-fold decrease in the equilibrium dissociation constant observed in the presence versus the absence of ATP. Monitoring uncapped RNA dissociation in real time reveals that ATP reduces the dissociation rate constant of RNA for eIF4F by ∼4-orders of magnitude. Thus, release of ATP from eIF4A places eIF4F in a dynamic state that has very fast association and dissociation rates from RNA. Monitoring the kinetic framework for eIF4A binding to eIF4G revealed two different rate constants that likely reflect two conformational states of the eIF4F complex. Furthermore, we determined that the eIF4G autoinhibitory domain promotes a more stable, less dynamic, eIF4A-binding state, which is overcome by eIF4E binding. Overall, our data support a model whereby eIF4E binding to eIF4G/4A stabilizes a high-affinity RNA-binding state of eIF4F and enables eIF4A to adopt a more dynamic interaction with eIF4G. This dynamic conformation may contribute to the ability of eIF4F to rapidly bind and release mRNA during scanning.

For patients with heart failure, myocardial ATP level can be reduced to one-half of that observed in healthy controls. This marked reduction (from ≈8 mM in healthy controls to as low as 3-4 mM in heart failure) has been suggested to contribute to impaired myocardial contraction and to the decreased pump function characteristic of heart failure. However, in vitro measures of maximum myofilament force generation, maximum shortening velocity, and the actomyosin ATPase activity show effective KM values for MgATP ranging from ≈10 μM to 150 μM, well below the intracellular ATP level in heart failure. Thus, it is not clear that the fall of myocardial ATP observed in heart failure is sufficient to impair the function of the contractile proteins. Therefore, we tested the effect of low MgATP levels on myocardial contraction using demembranated cardiac muscle preparations that were exposed to MgATP levels typical of the range found in non-failing and failing hearts. Consistent with previous studies, we found that a 50% reduction in MgATP level (from 8 mM to 4 mM) did not reduce maximum force generation or maximum velocity of shortening. However, we found that a 50% reduction in MgATP level caused a 20%-25% reduction in maximal power generation (measured during muscle shortening against a load) and a 20% slowing of cross-bridge cycling kinetics. These results suggest that the decreased cellular ATP level occurring in heart failure contributes to the impaired pump function of the failing heart. Since the ATP-myosin ATPase dissociation constant is estimated to be submillimolar, these findings also suggest that MgATP concentration affects cross-bridge dynamics through a mechanism that is more complex than through the direct dependence of MgATP concentration on myosin ATPase activity. Finally, these studies suggest that therapies targeted to increase adenine nucleotide pool levels in cardiomyocytes might be beneficial for treating heart failure.

The tumour microenvironment (TME) imposes a major obstacle to infiltrating T-lymphocytes and suppresses their function. Several immune checkpoint proteins that interfere with ligand/receptor interactions and impede T-cell anti-tumour responses have been identified. Immunotherapies that block immune checkpoints have revolutionized the treatment paradigm for many patients with advanced-stage tumours. However, metabolic constraints and soluble factors that exist within the TME exacerbate the functional exhaustion of tumour-infiltrating T-cells. Here we review these multifactorial constraints and mechanisms - elevated immunosuppressive metabolites and enzymes, nutrient insufficiency, hypoxia, increased acidity, immense amounts of extracellular ATP and adenosine, dysregulated bioenergetic and purinergic signalling, and ionic imbalance - that operate in the TME and collectively suppress T-cell function. We discuss how scientific advances could help overcome the complex TME obstacles for tumour-infiltrating T-lymphocytes, aiming to stimulate further research for developing new therapeutic strategies by harnessing the full potential of the immune system in combating cancer.

The AAA+ protein, Skd3 (human CLPB), solubilizes proteins in the mitochondrial intermembrane space, which is critical for human health. Skd3 variants with defective protein-disaggregase activity cause severe congenital neutropenia (SCN) and 3-methylglutaconic aciduria type 7 (MGCA7). How Skd3 disaggregates proteins remains poorly understood. Here, we report a high-resolution structure of a Skd3-substrate complex. Skd3 adopts a spiral hexameric arrangement that engages substrate via pore-loop interactions in the nucleotide-binding domain (NBD). Substrate-bound Skd3 hexamers stack head-to-head via unique, adaptable ankyrin-repeat domain (ANK)-mediated interactions to form dodecamers. Deleting the ANK linker region reduces dodecamerization and disaggregase activity. We elucidate apomorphic features of the Skd3 NBD and C-terminal domain that regulate disaggregase activity. We also define how Skd3 subunits collaborate to disaggregate proteins. Importantly, SCN-linked subunits sharply inhibit disaggregase activity, whereas MGCA7-linked subunits do not. These advances illuminate Skd3 structure and mechanism, explain SCN and MGCA7 inheritance patterns, and suggest therapeutic strategies.

The prefrontal cortex (PFC) is a hub for cognitive control, and dopamine profoundly influences its functions. In other brain regions, astrocytes sense diverse neurotransmitters and neuromodulators and, in turn, orchestrate regulation of neuroactive substances. However, basic physiology of PFC astrocytes, including which neuromodulatory signals they respond to and how they contribute to PFC function, is unclear. Here, we characterize divergent signaling signatures in mouse astrocytes of the PFC and primary sensory cortex, which show differential responsiveness to locomotion. We find that PFC astrocytes express receptors for dopamine but are unresponsive through the Gs/Gi-cAMP pathway. Instead, fast calcium signals in PFC astrocytes are time locked to dopamine release and are mediated by α1-adrenergic receptors both ex vivo and in vivo. Further, we describe dopamine-triggered regulation of extracellular ATP at PFC astrocyte territories. Thus, we identify astrocytes as active players in dopaminergic signaling in the PFC, contributing to PFC function though neuromodulator receptor crosstalk.

Mitochondria are small double-membraned organelles responsible for the generation of energy used in the body in the form of ATP. Mitochondria are unique in that they contain their own circular mitochondrial genome termed mtDNA. mtDNA codes for 37 genes, and together with the nuclear genome (nDNA), dictate mitochondrial structure and function. Not surprisingly, pathogenic variants in the mtDNA or nDNA can result in mitochondrial disease. Mitochondrial disease primarily impacts tissues with high energy demands, including the heart. Mitochondrial cardiomyopathy is characterized by the abnormal structure or function of the myocardium secondary to genetic defects in either the nDNA or mtDNA. Mitochondrial cardiomyopathy can be isolated or part of a syndromic mitochondrial disease. Common manifestations of mitochondrial cardiomyopathy are a phenocopy of hypertrophic cardiomyopathy, dilated cardiomyopathy, and cardiac conduction defects. The underlying pathophysiology of mitochondrial cardiomyopathy is complex and likely involves multiple abnormal processes in the cell, stemming from deficient oxidative phosphorylation and ATP depletion. Possible pathophysiology includes the activation of alternative metabolic pathways, the accumulation of reactive oxygen species, dysfunctional mitochondrial dynamics, abnormal calcium homeostasis, and mitochondrial iron overload. Here, we highlight the clinical assessment of mtDNA-related mitochondrial cardiomyopathy and offer a novel hypothesis of a possible integrated, multivariable pathophysiology of disease.

This article presents the biochemical intra-skeletal-muscle basis of exercise intensity domains: moderate (M), heavy (H), very heavy (VH) and severe (S). Threshold origins are mediated by a 'Pi double-threshold' mechanism of muscle fatigue, which assumes (1) additional ATP usage, underlying muscle V̇O2 and metabolite slow components, is initiated when inorganic phosphate (Pi) exceeds a critical value (Picrit); (2) exercise is terminated because of fatigue, when Pi reaches a peak value (Pipeak); and (3) the Pi increase and additional ATP usage increase mutually stimulate each other forming a positive feedback. M/H and H/VH borders are defined by Pi on-kinetics in relation to Picrit and Pipeak. The values of the ATP usage activity, proportional to power output (PO), for the M/H, H/VH and VH/S borders are lowest in untrained muscle and highest in well-trained muscle. The metabolic range between the M/H and H/VH border (or 'H space') decreases with muscle training, while the difference between the H/VH and VH/S border (or 'VH space') is only weakly dependent on training status. The absolute magnitude of the muscle V̇O2 slow-component, absent in M exercise, rises gradually with PO to a maximal value in H exercise, and then decreases with PO in VH and S exercise. Simulations of untrained, physically active and well-trained muscle demonstrate that the muscle M/H border need not be identical to the whole-body M/H border determined from pulmonary V̇O2 on-kinetics and blood lactate, while suggesting that the biochemical origins of the H/VH border reside within skeletal muscle and correspond to whole-body critical power.

The enzyme nitrogenase couples adenosine triphosphate (ATP) hydrolysis to the multielectron reduction of atmospheric dinitrogen into ammonia. Despite extensive research, the mechanistic details of ATP-dependent energy transduction and dinitrogen reduction by nitrogenase are not well understood, requiring new strategies to monitor its structural dynamics during catalytic action. Here, we report cryo-electron microscopy structures of the nitrogenase complex prepared under enzymatic turnover conditions. We observe that asymmetry governs all aspects of the nitrogenase mechanism, including ATP hydrolysis, protein-protein interactions, and catalysis. Conformational changes near the catalytic iron-molybdenum cofactor are correlated with the nucleotide-hydrolysis state of the enzyme.

This work reports a new ATP-independent bioluminescent probe (bor-DTZ) for detecting hydrogen peroxide that is compatible with the Nanoluciferase enzyme. The probe is designed with an arylboronate ester protecting group appended to a diphenylterazine core via a self-immolative phenolate linker. Reaction with hydrogen peroxide reveals diphenylterazine, which can then react with Nanoluciferase to produce a detectable bioluminescent signal. Bor-DTZ shows a dose-dependent response to hydrogen peroxide and selectivity over other biologically relevant reactive oxygen species and can be applied to detect either intra- or extracellular species. We further demonstrate the ability of this platform to monitor fluxes in extracellular hydrogen peroxide in a breast cancer cell line in response to the anticancer treatment, cisplatin.