The DEAD-Box RNA Helicase Ded1 Is Associated with Translating Ribosomes.

DEAD-box RNA helicases are ATP-dependent RNA binding proteins and RNA-dependent ATPases that possess weak, nonprocessive unwinding activity in vitro, but they can form long-lived complexes on RNAs when the ATPase activity is inhibited. Ded1 is a yeast DEAD-box protein, the functional ortholog of mammalian DDX3, that is considered important for the scanning efficiency of the 48S pre-initiation complex ribosomes to the AUG start codon. We used a modified PAR-CLIP technique, which we call quicktime PAR-CLIP (qtPAR-CLIP), to crosslink Ded1 to 4-thiouridine-incorporated RNAs in vivo using UV light centered at 365 nm. The irradiation conditions are largely benign to the yeast cells and to Ded1, and we are able to obtain a high efficiency of crosslinking under physiological conditions. We find that Ded1 forms crosslinks on the open reading frames of many different mRNAs, but it forms the most extensive interactions on relatively few mRNAs, and particularly on mRNAs encoding certain ribosomal proteins and translation factors. Under glucose-depletion conditions, the crosslinking pattern shifts to mRNAs encoding metabolic and stress-related proteins, which reflects the altered translation. These data are consistent with Ded1 functioning in the regulation of translation elongation, perhaps by pausing or stabilizing the ribosomes through its ATP-dependent binding.

Restoration of energy homeostasis under oxidative stress: Duo synergistic AMPK pathways regulating arginine kinases.

Rapid depletion of cellular ATP can occur by oxidative stress induced by reactive oxygen species (ROS). Maintaining energy homeostasis requires the key molecular components AMP-activated protein kinase (AMPK) and arginine kinase (AK), an invertebrate orthologue of the mammalian creatine kinase (CK). Here, we deciphered two independent and synergistic pathways of AMPK acting on AK by using the beetle Tribolium castaneum as a model system. First, AMPK acts on transcriptional factor forkhead box O (FOXO) leading to phosphorylation and nuclear translocation of the FOXO. The phospho-FOXO directly promotes the expression of AK upon oxidative stress. Concomitantly, AMPK directly phosphorylates the AK to switch the direction of enzymatic catalysis for rapid production of ATP from the phosphoarginine-arginine pool. Further in vitro assays revealed that Sf9 cells expressing phospho-deficient AK mutants displayed the lower ATP/ADP ratio and cell viability under paraquat-induced oxidative stress conditions when compared with Sf9 cells expressing wild-type AKs. Additionally, the AMPK-FOXO-CK pathway is also involved in the restoration of ATP homeostasis under oxidative stress in mammalian HEK293 cells. Overall, we provide evidence that two distinct AMPK-AK pathways, transcriptional and post-translational regulations, are coherent responders to acute oxidative stresses and distinguished from classical AMPK-mediated long-term metabolic adaptations to energy challenge.

Hsp90 mutants with distinct defects provide novel insights into cochaperone regulation of the folding cycle.

Molecular chaperones play a key role in maintaining proteostasis and cellular health. The abundant, essential, cytosolic Hsp90 (Heat shock protein, 90 kDa) facilitates the folding and activation of hundreds of newly synthesized or misfolded client proteins in an ATP-dependent folding pathway. In a simplified model, Hsp70 first helps load client onto Hsp90, ATP binding results in conformational changes in Hsp90 that result in the closed complex, and then less defined events result in nucleotide hydrolysis, client release and return to the open state. Cochaperones bind and assist Hsp90 during this process. We previously identified a series of yeast Hsp90 mutants that appear to disrupt either the 'loading', 'closing' or 'reopening' events, and showed that the mutants had differing effects on activity of some clients. Here we used those mutants to dissect Hsp90 and cochaperone interactions. Overexpression or deletion of HCH1 had dramatically opposing effects on the growth of cells expressing different mutants, with a phenotypic shift coinciding with formation of the closed conformation. Hch1 appears to destabilize Hsp90-nucleotide interaction, hindering formation of the closed conformation, whereas Cpr6 counters the effects of Hch1 by stabilizing the closed conformation. Hch1 and the homologous Aha1 share some functions, but the role of Hch1 in inhibiting progression through the early stages of the folding cycle is unique. Sensitivity to the Hsp90 inhibitor NVP-AUY922 also correlates with the conformational cycle, with mutants defective in the loading phase being most sensitive and those defective in the reopening phase being most resistant to the drug. Overall, our results indicate that the timing of transition into and out of the closed conformation is tightly regulated by cochaperones. Further analysis will help elucidate additional steps required for progression through the Hsp90 folding cycle and may lead to new strategies for modulating Hsp90 function.

Enhanced Sensing Performance of a Microchannel-Based Electrochemiluminescence Biosensor for Adenosine Triphosphate via a dsDNA Superstructure Amplification Strategy.

The sensing performance of a microchannel-based electrochemiluminescence (ECL) biosensor is related to the change ratio of charge density on the surface of microchannels caused by a target recognition reaction. In this study, adenosine triphosphate (ATP) served as a model target. The dsDNA superstructures containing a capture probe (CP, containing an ATP aptamer sequence) and alternating units of ssDNA probes of P1 and P2, CP/(P1/P2)n, were grafted onto the inner wall of microchannels first. The CP in dsDNA superstructures captured ATP molecules, causing the release of dsDNA fragments containing alternating units of P1 and P2, (P1/P2)n. The target recognition reaction significantly changed the charge density of microchannels, which altered the ECL intensity of the (1,10-phenanthroline)ruthenium(II)/tripropylamine system in the reporting interface. The ECL intensity of the constructed system had a linear relationship with the logarithm of ATP concentration ranging from 1 fM to 100 pM with a detection limit of 0.32 fM (S/N = 3). The biosensor was successfully applied to detect ATP in rat brains.

Nab3 nuclear granule accumulation is driven by respiratory capacity.

Numerous biological processes involve proteins capable of transiently assembling into subcellular compartments necessary for cellular functions. One process is the RNA polymerase II transcription cycle which involves initiation, elongation, co-transcriptional modification of nascent RNA, and termination. The essential yeast transcription termination factor Nab3 is required for termination of small non-coding RNAs and accumulates into a compact nuclear granule upon glucose removal. Nab3 nuclear granule accumulation varies in penetrance across yeast strains and a higher Nab3 granule accumulation phenotype is associated with petite strains, suggesting a possible ATP-dependent mechanism for granule disassembly. Here, we demonstrate the uncoupling of mitochondrial oxidative phosphorylation by drug treatment or deletions of nuclear-encoded ATP synthase subunit genes were sufficient to increase Nab3 granule accumulation and led to an inability to proliferate during prolonged glucose deprivation, which requires respiration. Additionally, by enriching for respiration competent cells from a petite-prone strain, we generated a low granule-accumulating strain from a relatively high one, providing another link between respiratory competency and Nab3 granules. Consistent with the resulting idea that ATP is involved in granule accumulation, the addition of extracellular ATP to semi-permeabilized cells was sufficient to reduce Nab3 granule accumulation. Deleting the SKY1 gene, which encodes a kinase that phosphorylates nuclear SR repeat-containing proteins and is involved in efficient stress granule disassembly, also resulted in increased granule accumulation. This observation implicates Sky1 in Nab3 granule biogenesis. Taken together, these findings suggest there is normally an equilibrium between termination factor granule assembly and disassembly mediated by ATP-requiring nuclear machinery.

Sepsis Encephalopathy Is Partly Mediated by miR370-3p-Induced Mitochondrial Injury but Attenuated by BAM15 in Cecal Ligation and Puncture Sepsis Male Mice.

BAM15 (a mitochondrial uncoupling agent) was tested on cecal ligation and puncture (CLP) sepsis mice with in vitro experiments. BAM15 attenuated sepsis as indicated by survival, organ histology (kidneys and livers), spleen apoptosis (activated caspase 3), brain injury (SHIRPA score, serum s100ß, serum miR370-3p, brain miR370-3p, brain TNF-α, and apoptosis), systemic inflammation (cytokines, cell-free DNA, endotoxemia, and bacteremia), and blood-brain barrier (BBB) damage (Evan's blue dye and the presence of green fluorescent E. coli in brain after an oral administration). In parallel, brain miR arrays demonstrated miR370-3p at 24 h but not 120 h post-CLP, which was correlated with metabolic pathways. Either lipopolysaccharide (LPS) or TNF-α upregulated miR370-3p in PC12 (neuron cells). An activation by sepsis factors (LPS, TNF-α, or miR370-3p transfection) damaged mitochondria (fluorescent color staining) and reduced cell ATP, possibly through profound mitochondrial activity (extracellular flux analysis) that was attenuated by BAM15. In bone-marrow-derived macrophages, LPS caused mitochondrial injury, decreased cell ATP, enhanced glycolysis activity (extracellular flux analysis), and induced pro-inflammatory macrophages (iNOS and IL-1ß) which were neutralized by BAM15. In conclusion, BAM15 attenuated sepsis through decreased mitochondrial damage, reduced neuronal miR370-3p upregulation, and induced anti-inflammatory macrophages. BAM15 is proposed to be used as an adjuvant therapy against sepsis hyperinflammation.

Optogenetic reprogramming of carbon metabolism using light-powering microbial proton pump systems.

In microbial fermentative production, ATP regeneration, while crucial for cellular processes, conflicts with efficient target chemical production because ATP regeneration exhausts essential carbon sources also required for target chemical biosynthesis. To wrestle with this dilemma, we harnessed the power of microbial rhodopsins with light-driven proton pumping activity to supplement with ATP, thereby facilitating the bioproduction of various chemicals. We first demonstrated a photo-driven ATP supply and redistribution of metabolic carbon flows to target chemical synthesis by installing already-known delta rhodopsin (dR) in Escherichia coli. In addition, we identified novel rhodopsins with higher proton pumping activities than dR, and created an engineered cell for in vivo self-supply of the rhodopsin-activator, all-trans-retinal. Our concept exploiting the light-powering ATP supplier offers a potential increase in carbon use efficiency for microbial productions through metabolic reprogramming.

Nischarin Deletion Reduces Oxidative Metabolism and Overall ATP: A Study Using a Novel NISCHΔ5-6 Knockout Mouse Model.

Nischarin (Nisch) is a cytosolic scaffolding protein that harbors tumor-suppressor-like characteristics. Previous studies have shown that Nisch functions as a scaffolding protein and regulates multiple biological activities. In the current study, we prepared a complete Nisch knockout model, for the first time, by deletion of exons 5 and 6. This knockout model was confirmed by Qrt-PCR and Western blotting with products from mouse embryonic fibroblast (MEF) cells. Embryos and adult mice of knockouts are significantly smaller than their wild-type counterparts. Deletion of Nisch enhanced cell migration, as demonstrated by wound type and transwell migration assays. Since the animals were small in size, we investigated Nisch's effect on metabolism by conducting several assays using the Seahorse analyzer system. These data indicate that Nisch null cells have lower oxygen consumption rates, lower ATP production, and lower levels of proton leak. We examined the expression of 15 genes involved in lipid and fat metabolism, as well as cell growth, and noted a significant increase in expression for many genes in Nischarin null animals. In summary, our results show that Nischarin plays an important physiological role in metabolic homeostasis.

T1121G Point Mutation in the Mitochondrial Gene COX1 Suppresses a Null Mutation in ATP23 Required for the Assembly of Yeast Mitochondrial ATP Synthase.

Nuclear-encoded Atp23 was previously shown to have dual functions, including processing the yeast Atp6 precursor and assisting the assembly of yeast mitochondrial ATP synthase. However, it remains unknown whether there are genes functionally complementary to ATP23 to rescue atp23 null mutant. In the present paper, we screen and characterize three revertants of atp23 null mutant and reveal a T1121G point mutation in the mitochondrial gene COX1 coding sequence, which leads to Val374Gly mutation in Cox1, the suppressor in the revertants. This was verified further by the partial restoration of mitochondrial ATP synthase assembly in atp23 null mutant transformed with exogenous hybrid COX1 T1121G mutant plasmid. The predicted tertiary structure of the Cox1 p.Val374Gly mutation showed no obvious difference from wild-type Cox1. By further chase labeling with isotope [35S]-methionine, we found that the stability of Atp6 of ATP synthase increased in the revertants compared with the atp23 null mutant. Taking all the data together, we revealed that the T1121G point mutation of mitochondrial gene COX1 could partially restore the unassembly of mitochondrial ATP synthase in atp23 null mutant by increasing the stability of Atp6. Therefore, this study uncovers a gene that is partially functionally complementary to ATP23 to rescue ATP23 deficiency, broadening our understanding of the relationship between yeast the cytochrome c oxidase complex and mitochondrial ATP synthase complex.

Cardiomyocytes disrupt pyrimidine biosynthesis in nonmyocytes to regulate heart repair.

Various populations of cells are recruited to the heart after cardiac injury, but little is known about whether cardiomyocytes directly regulate heart repair. Using a murine model of ischemic cardiac injury, we demonstrate that cardiomyocytes play a pivotal role in heart repair by regulating nucleotide metabolism and fates of nonmyocytes. Cardiac injury induced the expression of the ectonucleotidase ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which hydrolyzes extracellular ATP to form AMP. In response to AMP, cardiomyocytes released adenine and specific ribonucleosides that disrupted pyrimidine biosynthesis at the orotidine monophosphate (OMP) synthesis step and induced genotoxic stress and p53-mediated cell death of cycling nonmyocytes. As nonmyocytes are critical for heart repair, we showed that rescue of pyrimidine biosynthesis by administration of uridine or by genetic targeting of the ENPP1/AMP pathway enhanced repair after cardiac injury. We identified ENPP1 inhibitors using small molecule screening and showed that systemic administration of an ENPP1 inhibitor after heart injury rescued pyrimidine biosynthesis in nonmyocyte cells and augmented cardiac repair and postinfarct heart function. These observations demonstrate that the cardiac muscle cell regulates pyrimidine metabolism in nonmuscle cells by releasing adenine and specific nucleosides after heart injury and provide insight into how intercellular regulation of pyrimidine biosynthesis can be targeted and monitored for augmenting tissue repair.

What makes a histone variant a variant: Changing H2A to become H2A.Z.

Chromatin structure and underlying DNA accessibility is modulated by the incorporation of histone variants. H2A.Z, a variant of the H2A core histone family, plays a distinct and essential role in a diverse set of biological functions including gene regulation and maintenance of heterochromatin-euchromatin boundaries. Although it is currently unclear how the replacement of H2A with H2A.Z can regulate gene expression, the variance in their amino acid sequence likely contributes to their functional differences. To tease apart regions of H2A.Z that confer its unique identity, a set of plasmids expressing H2A-H2A.Z hybrids from the native H2A.Z promoter were examined for their ability to recapitulate H2A.Z function. First, we found that the H2A.Z M6 region was necessary and sufficient for interaction with the SWR1-C chromatin remodeler. Remarkably, the combination of only 9 amino acid changes, the H2A.Z M6 region, K79 and L81 (two amino acids in the α2-helix), were sufficient to fully rescue growth phenotypes of the htz1Δ mutant. Furthermore, combining three unique H2A.Z regions (K79 and L81, M6, C-terminal tail) was sufficient for expression of H2A.Z-dependent heterochromatin-proximal genes and GAL1 derepression. Surprisingly, hybrid constructs that restored the transcription of H2A.Z-dependent genes, did not fully recapitulate patterns of H2A.Z-specific enrichment at the tested loci. This suggested that H2A.Z function in transcription regulation may be at least partially independent of its specific localization in chromatin. Together, this work has identified three regions that can confer specific H2A.Z-identity to replicative H2A, furthering our understanding of what makes a histone variant a variant.

Reverting the mode of action of the mitochondrial FOF1-ATPase by Legionella pneumophila preserves its replication niche.

Legionella pneumophila, the causative agent of Legionnaires' disease, a severe pneumonia, injects via a type 4 secretion system (T4SS) more than 300 proteins into macrophages, its main host cell in humans. Certain of these proteins are implicated in reprogramming the metabolism of infected cells by reducing mitochondrial oxidative phosphorylation (OXPHOS) early after infection. Here. we show that despite reduced OXPHOS, the mitochondrial membrane potential (Δψm) is maintained during infection of primary human monocyte-derived macrophages (hMDMs). We reveal that L. pneumophila reverses the ATP-synthase activity of the mitochondrial FOF1-ATPase to ATP-hydrolase activity in a T4SS-dependent manner, which leads to a conservation of the Δψm, preserves mitochondrial polarization, and prevents macrophage cell death. Analyses of T4SS effectors known to target mitochondrial functions revealed that LpSpl is partially involved in conserving the Δψm, but not LncP and MitF. The inhibition of the L. pneumophila-induced 'reverse mode' of the FOF1-ATPase collapsed the Δψm and caused cell death in infected cells. Single-cell analyses suggested that bacterial replication occurs preferentially in hMDMs that conserved the Δψm and showed delayed cell death. This direct manipulation of the mode of activity of the FOF1-ATPase is a newly identified feature of L. pneumophila allowing to delay host cell death and thereby to preserve the bacterial replication niche during infection.

Overexpression of Neuroglobin Promotes Energy Metabolism and Autophagy Induction in Human Neuroblastoma SH-SY5Y Cells.

Neuroglobin (NGB) is an O2-binding globin mainly expressed in the central and peripheral nervous systems and cerebrospinal fluid. Previously, it was demonstrated that NGB overexpression protects cells from hypoxia-induced death. To investigate processes promoted by NGB overexpression, we used a cellular model of neuroblastoma stably overexpressing an NGB-FLAG construct. We used a proteomic approach to identify the specific profile following NGB overexpression. To evaluate the role of NGB overexpression in increasing energetic metabolism, we measured oxygen consumption rate (OCR) and the extracellular acidification rate through Seahorse XF technology. The effect on autophagy induction was evaluated by analyzing SQSTM1/p62 and LC3-II expression. Proteomic analysis revealed several differentially regulated proteins, involved in oxidative phosphorylation and integral mitochondrial proteins linked to energy metabolism. The analysis of mitochondrial metabolism demonstrated that NGB overexpression increases mitochondrial ATP production. Indeed, NGB overexpression enhances bioenergetic metabolism, increasing OCR and oxygen consumption. Analysis of autophagy induction revealed an increase of LC3-II together with a significant decrease of SQSTM1/p62, and NGB-LC3-II association during autophagosome formation. These results highlight the active participation of NGB in several cellular processes that can be upregulated in response to NGB overexpression, playing a role in the adaptive response to stress in neuroblastoma cells.

Biological synthesis of nicotinamide mononucleotide.

Nicotinamide mononucleotide (NMN) or Nicotinamide-1-ium-1-ß-D-ribofuranoside 5'-phosphate is a nucleotide that can be converted into nicotinamide adenine dinucleotide (NAD) in human cells. NMN has recently attracted great attention because of its potential as an anti-aging drug, leading to great efforts for its effective manufacture. The chemical synthesis of NMN is a challenging task since it is an isomeric compound with a complicated structure. The majority of biological synthetic routes for NMN is through the intermediate phosphoribosyl diphosphate (PRPP), which is further converted to NMN by nicotinamide phosphoribosyltransferase (Nampt). There are various routes for the synthesis of PRPP from simple starting materials such as ribose, adenosine, and xylose, but all of these require the expensive phosphate donor adenosine triphosphate (ATP). Thus, an ATP regeneration system can be included, leading to diminished ATP consumption during the catalytic process. The regulations of enzymes that are not directly involved in the synthesis of NMN are also critical for the production of NMN. The aim of this review is to present an overview of the biological production of NMN with respect to the critical enzymes, reaction conditions, and productivity.

SIRT3 mediates mitofusin 2 ubiquitination and degradation to suppress ischemia reperfusion-induced acute kidney injury.

Ischemia reperfusion-induced acute kidney injury (IR-induced AKI) is a life-threatening disease with many complications. Mitofusin 2 (Mfn2) ubiquitination is related to AKI. But the underlying molecular mechanisms remain unknown. This study aimed to probe the mechanism of Mfn2 ubiquitination in IR-induced AKI development. In IR-induced AKI mouse models, orbital blood and urine were collected for assessing kidney function. The kidney injury, ultrastructure of mitochondria, and histopathology in mice were evaluated after injection of G5, an ubiquitination inhibitor. Oxygen glucose deprivation/reoxygenation (OGD/R) models were established in HK-2 cells, and the mitochondria were extracted. Cell viability, apoptosis, oxidative stress, inflammatory reaction, mitochondrial membrane potential, and ATP production were measured. Mfn2 ubiquitination in mouse and cell models was evaluated. si-SIRT3 and pcDNA3.1-SIRT3 were transfected into cell models. Consequently, kidney function in mice was impaired by IR-induced AKI. Mfn2 ubiquitination and degradation promoted IR-induced AKI. OGD/R induced renal tubular epithelial cell injury and disrupted mitochondrial dynamics and functions through promoting Mfn2 ubiquitination. SIRT3 knockdown led to Mfn2 ubiquitination by binding to UBC; while its overexpression alleviated tubular epithelial cell injury. Briefly, SIRT3 mediates Mfn2 ubiquitination to relieve IR-induced AKI. This investigation may offer new insights for the treatment of IR-induced AKI injury.

Visual Detection of Adenosine Triphosphate by Taylor Rising: A Simple Point-of-Care Testing Method Based on Rolling Circle Amplification.

Rapid and sensitive point-of-care testing (POCT) is an extremely critical mission in practical applications, especially for rigorous military medicine, home health care, and in the third world. Here, we report a visual POCT method for adenosine triphosphate (ATP) detection based on Taylor rising in the corner of quadratic geometries between two rod surfaces. We discuss the principle of Taylor rising, demonstrating that it is significantly influenced by contact angle, surface tension, and density of the sample, which are controlled by ATP-dependent rolling circle amplification (RCA). In the presence of ATP, RCA reaction effectively suppresses Taylor-rising behavior, due to the increased contact angle, density, and decreased surface tension. Without addition of ATP, untriggered RCA reaction is favorable for Taylor rising, resulting in a significant height. With this proposed method, visual sensitive detection of ATP without the aid of other instruments is realized with only a 5 µL droplet, which has good selectivity and a low detection limit (17 nM). Importantly, this visual method provides a promising POCT tool for user-friendly molecular diagnostics.

Activation of Aldehyde Dehydrogenase 2 Ameliorates Glucolipotoxicity of Pancreatic Beta Cells.

Chronic hyperglycemia and hyperlipidemia hamper beta cell function, leading to glucolipotoxicity. Mitochondrial aldehyde dehydrogenase 2 (ALDH2) detoxifies reactive aldehydes, such as methylglyoxal (MG) and 4-hydroxynonenal (4-HNE), derived from glucose and lipids, respectively. We aimed to investigate whether ALDH2 activators ameliorated beta cell dysfunction and apoptosis induced by glucolipotoxicity, and its potential mechanisms of action. Glucose-stimulated insulin secretion (GSIS) in MIN6 cells and insulin secretion from isolated islets in perifusion experiments were measured. The intracellular ATP concentrations and oxygen consumption rates of MIN6 cells were assessed. Furthermore, the cell viability, apoptosis, and mitochondrial and intracellular reactive oxygen species (ROS) levels were determined. Additionally, the pro-apoptotic, apoptotic, and anti-apoptotic signaling pathways were investigated. We found that Alda-1 enhanced GSIS by improving the mitochondrial function of pancreatic beta cells. Alda-1 rescued MIN6 cells from MG- and 4-HNE-induced beta cell death, apoptosis, mitochondrial dysfunction, and ROS production. However, the above effects of Alda-1 were abolished in Aldh2 knockdown MIN6 cells. In conclusion, we reported that the activator of ALDH2 not only enhanced GSIS, but also ameliorated the glucolipotoxicity of beta cells by reducing both the mitochondrial and intracellular ROS levels, thereby improving mitochondrial function, restoring beta cell function, and protecting beta cells from apoptosis and death.

Type 2 diabetic mice enter a state of spontaneous hibernation-like suspended animation following accumulation of uric acid.

Hibernation is an example of extreme hypometabolic behavior. How mammals achieve such a state of suspended animation remains unclear. Here we show that several strains of type 2 diabetic mice spontaneously enter into hibernation-like suspended animation (HLSA) in cold temperatures. Nondiabetic mice injected with ATP mimic the severe hypothermia analogous to that observed in diabetic mice. We identified that uric acid, an ATP metabolite, is a key molecular in the entry of HLSA. Uric acid binds to the Na+ binding pocket of the Na+/H+ exchanger protein and inhibits its activity, acidifying the cytoplasm and triggering a drop in metabolic rate. The suppression of uric acid biosynthesis blocks the occurrence of HLSA, and hyperuricemic mice induced by treatment with an uricase inhibitor can spontaneously enter into HLSA similar to that observed in type 2 diabetic mice. In rats and dogs, injection of ATP induces a reversible state of HLSA similar to that seen in mice. However, ATP injection fails to induce HLSA in pigs due to the lack of their ability to accumulate uric acid. Our results raise the possibility that nonhibernating mammals could spontaneously undergo HLSA upon accumulation of ATP metabolite, uric acid.

Structural basis of the complete poxvirus transcription initiation process.

Poxviruses express their genes in the cytoplasm of infected cells using a virus-encoded multi-subunit polymerase (vRNAP) and unique transcription factors. We present cryo-EM structures that uncover the complete transcription initiation phase of the poxvirus vaccinia. In the pre-initiation complex, the heterodimeric early transcription factor VETFs/l adopts an arc-like shape spanning the polymerase cleft and anchoring upstream and downstream promoter elements. VETFI emerges as a TBP-like protein that inserts asymmetrically into the DNA major groove, triggers DNA melting, ensures promoter recognition and enforces transcription directionality. The helicase VETFs fosters promoter melting and the phospho-peptide domain (PPD) of vRNAP subunit Rpo30 enables transcription initiation. An unprecedented upstream promoter scrunching mechanism assisted by the helicase NPH-I probably fosters promoter escape and transition into elongation. Our structures shed light on unique mechanisms of poxviral gene expression and aid the understanding of thus far unexplained universal principles in transcription.

Valproate activates the Snf1 kinase in Saccharomyces cerevisiae by decreasing the cytosolic pH.

Valproate (VPA) is a widely used mood stabilizer, but its therapeutic mechanism of action is not understood. This knowledge gap hinders the development of more effective drugs with fewer side effects. Using the yeast model to elucidate the effects of VPA on cellular metabolism, we determined that the drug upregulated expression of genes normally repressed during logarithmic growth on glucose medium and increased levels of activated (phosphorylated) Snf1 kinase, the major metabolic regulator of these genes. VPA also decreased the cytosolic pH (pHc) and reduced glycolytic production of 2/3-phosphoglycerate. ATP levels and mitochondrial membrane potential were increased, and glucose-mediated extracellular acidification decreased in the presence of the drug, as indicated by a smaller glucose-induced shift in pH, suggesting that the major P-type proton pump Pma1 was inhibited. Interestingly, decreasing the pHc by omeprazole-mediated inhibition of Pma1 led to Snf1 activation. We propose a model whereby VPA lowers the pHc causing a decrease in glycolytic flux. In response, Pma1 is inhibited and Snf1 is activated, resulting in increased expression of normally repressed metabolic genes. These findings suggest a central role for pHc in regulating the metabolic program of yeast cells.