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Intrahepatic cholangiocarcinoma (CHOL) remains a rare malignancy, ranking as the leading lethal primary liver cancer worldwide. However, the biological functions of integrator complex subunit 8 (INTS8) in CHOL remain unknown. Thus, this research aimed to explore the potential role of INTS8 as a novel diagnostic or therapeutic target in CHOL. Differentially expressed genes (DEGs) in two Gene Expression Omnibus (GEO) datasets were obtained by the "RRA" package in R software. The "maftools" package was used to visualize the CHOL mutation data from The Cancer Genome Atlas (TCGA) database. The expression of INTS8 was detected by performing quantitative reverse transcription-PCR (qRT-PCR) and immunohistochemistry in cell lines and human samples. The association between subtypes of tumour-infiltrating immune cells (TIICs) and INTS8 expression in CHOL was determined by using CIBERSORT tools. We evaluated the correlations between INTS8 expression and mismatch repair (MMR) genes and DNA methyltransferases (DNMTs) in pan-cancer analysis. Finally, the pan-cancer prognostic signature of INTS8 was identified by univariate analysis. We obtained the mutation landscapes of an RRA gene set in CHOL. The expression of INTS8 was upregulated in CHOL cell lines and human CHOL samples. Furthermore, INTS8 expression was closely associated with a distinct landscape of TIICs, MMR genes, and DNMTs in CHOL. In addition, the high INTS8 expression group presented significantly poor outcomes, including overall survival (OS), disease-specific survival (DSS) and disease-free interval (DFI) (p < 0.05) in pan-cancer. INTS8 contributes to the tumorigenesis and progression of CHOL. Our study highlights the significant role of INTS8 in CHOL and pan-cancers, providing a valuable molecular target for cancer research., (© 2021. The Author(s).)

Similar to humans, the horse relies predominantly on the evaporation of sweat from the skin surface to dissipate excess body heat. Loss of the sweat response or anhidrosis can result in life-threatening hyperthermia. Anhidrosis occurs more frequently in some breeds as well as occurs at an increased frequency among individuals with a family history, suggesting a heritable component to the pathology. Given the natural occurrence and indications of genetic components in the etiology, we utilized genomics to better understand the molecular mechanisms involved in sweat response. We performed a case-control (n = 200) GWAS targeting cases of chronic idiopathic anhidrosis in a controlled genetic background to discover the contributing regions and interrogated gene function for roles in the sweating mechanism. A region containing the KCNE4 gene, which encodes the β-subunit of a potassium channel protein with a possible function in sweat gland outflow, was associated (P = 1.13 × 10 -07 ) with chronic idiopathic anhidrosis through GWAS. A candidate mutation (NC&#95;009149.3:g.11813731A > G, rs68643109) disrupting the KCNE4 protein structure could explain the disease but requires further investigation in larger populations. We show the potential role of ion channels and cellular damage in sweat response, correlating anhidrosis as a possible effect of congenital channelopathy., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The glycoprotein hormones (GPH) are heterodimers composed of a common α subunit and a specific β subunit. They act by activating specific leucine-rich repeat G protein-coupled receptors. However, individual subunits have been shown to elicit responses in cells devoid of the receptor for the dimeric hormones. The α subunit is involved in prolactin production from different tissues. The human chorionic gonadotropin β subunit (βhCG) plays determinant roles in placentation and in cancer development and metastasis. A truncated form of the thyrotropin (TSH) β subunit is also reported to have biological effects. The GPH α- and β subunits are derived from precursor genes ( gpa and gpb , respectively), which are expressed in most invertebrate species and are still represented in vertebrates as GPH subunit paralogs ( gpa2 and gpb5 , respectively). No specific receptor has been found for the vertebrate GPA2 and GPB5 even if their heterodimeric form is able to activate the TSH receptor in mammals. Interestingly, GPA and GPB are phylogenetically and structurally related to cysteine-knot growth factors (CKGF) and particularly to a group of antagonists that act independently on any receptor. This review article summarizes the observed actions of individual GPH subunits and presents the current hypotheses of how these actions might be induced. New approaches are also proposed in light of the evolutionary relatedness with antagonists of the CKGF family of proteins., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Querat.)

GABA A receptors are vital for controlling neuronal excitability and can display significant levels of constitutive activity that contributes to tonic inhibition. However, the mechanisms underlying spontaneity are poorly understood. Here we demonstrate a strict requirement for β3 subunit incorporation into receptors for spontaneous gating, facilitated by α4, α6 and δ subunits. The crucial molecular determinant involves four amino acids (GKER) in the β3 subunit's extracellular domain, which interacts with adjacent receptor subunits to promote transition to activated, open channel conformations. Spontaneous activity is further regulated by β3 subunit phosphorylation and by allosteric modulators including neurosteroids and benzodiazepines. Promoting spontaneous activity reduced neuronal excitability, indicating that spontaneous currents will alter neural network activity. This study demonstrates how regional diversity in GABA A receptor isoform, protein kinase activity, and neurosteroid levels, can impact on tonic inhibition through the modulation of spontaneous GABA A receptor gating., (© 2021. The Author(s).)

The chemical threat agent tetramethylenedisulfotetramine (TETS) is a γ-aminobutyric acid type A receptor (GABA A R) antagonist that causes life threatening seizures. Currently, there is no specific antidote for TETS intoxication. TETS-induced seizures are typically treated with benzodiazepines, which function as nonselective positive allosteric modulators (PAMs) of synaptic GABA A Rs. The major target of TETS was recently identified as the GABA A R α2β3γ2 subtype in electrophysiological studies using recombinantly expressed receptor combinations. Here, we tested whether these in vitro findings translate in vivo by comparing the efficacy of GABA A R subunit-selective PAMs in reducing TETS-induced seizure behavior in larval zebrafish. We tested PAMs targeting α1, α2, α2/3/5, α6, ß2/3, ß1/2/3, and δ subunits and compared their efficacy to the benzodiazepine midazolam (MDZ). The data demonstrate that α2- and α6-selective PAMs (SL-651,498 and SB-205384, respectively) were effective at mitigating TETS-induced seizure-like behavior. Combinations of SB-205384 and MDZ or SL-651,498 and 2-261 (ß2/3-selective) mitigated TETS-induced seizure-like behavior at concentrations that did not elicit sedating effects in a photomotor behavioral assay, whereas MDZ alone caused sedation at the concentration required to stop seizure behavior. Isobologram analyses suggested that SB-205384 and MDZ interacted in an antagonistic fashion, while the effects of SL-651,498 and 2-261 were additive. These results further elucidate the molecular mechanism by which TETS induces seizures and provide mechanistic insight regarding specific countermeasures against this chemical convulsant., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

In nature, plants are exposed to several environmental stresses that can be continuous or recurring. Continuous stress can be lethal, but stress after priming can increase the tolerance of a plant to better prepare for future stresses. Reports have suggested that transcription factors are involved in stress memory after recurrent stress; however, less is known about the factors that regulate the resetting of stress memory. Here, we uncovered a role for Constitutive Photomorphogenesis 5A (CSN5A) in the regulation of stress memory for resetting transcriptional memory genes ( APX2 and HSP22 ) and H3K4me3 following recurrent heat stress. Furthermore, CSN5A is also required for the deposition of H3K4me3 following recurrent heat stress. Thus, CSN5A plays an important role in the regulation of histone methylation and transcriptional stress memory after recurrent heat stress.

The ubiquitin-proteasome system is the major pathway for the maintenance of protein homeostasis. Its inhibition causes accumulation of ubiquitinated proteins; this accumulation has been associated with several of the most common neurodegenerative diseases. Several genetic factors have been identified for most neurodegenerative diseases, however, most cases are considered idiopathic, thus making the study of the mechanisms of protein accumulation a relevant field of research. It is often mentioned that the biggest risk factor for neurodegenerative diseases is aging, and several groups have reported an age-related alteration of the expression of some of the 26S proteasome subunits and a reduction of its activity. Proteasome subunits interact with proteins that are known to accumulate in neurodegenerative diseases such as α-synuclein in Parkinson's, tau in Alzheimer's, and huntingtin in Huntington's diseases. These interactions have been explored for several years, but only until recently, we are beginning to understand them. In this review, we discuss the known interactions, the underlying patterns, and the phenotypes associated with the 26S proteasome subunits in the etiology and progression of neurodegenerative diseases where there is evidence of proteasome involvement. Special emphasis is made in reviewing proteasome subunits that interact with proteins known to have an age-related altered expression or to be involved in neurodegenerative diseases to explore key effectors that may trigger or augment their progression. Interestingly, while the causes of age-related reduction of some of the proteasome subunits are not known, there are specific relationships between the observed neurodegenerative disease and the affected proteasome subunits., (Copyright © 2020 IMSS. Published by Elsevier Inc. All rights reserved.)

Bivalve metamorphosis is a developmental transition from a free-living larva to a benthic juvenile (spat), regulated by a complex interaction of neurotransmitters and neurohormones such as L-DOPA and epinephrine (catecholamine). We recently suggested an N-Methyl-D-aspartate (NMDA) receptor pathway as an additional and previously unknown regulator of bivalve metamorphosis. To explore this theory further, we successfully induced metamorphosis in the Pacific oyster, Crassostrea gigas, by exposing competent larvae to L-DOPA, epinephrine, MK-801 and ifenprodil. Subsequently, we cloned three NMDA receptor subunits CgNR1, CgNR2A and CgNR2B, with sequence analysis suggesting successful assembly of functional NMDA receptor complexes and binding to natural occurring agonists and the channel blocker MK-801. NMDA receptor subunits are expressed in competent larvae, during metamorphosis and in spat, but this expression is neither self-regulated nor regulated by catecholamines. In-situ hybridisation of CgNR1 in competent larvae identified NMDA receptor presence in the apical organ/cerebral ganglia area with a potential sensory function, and in the nervous network of the foot indicating an additional putative muscle regulatory function. Furthermore, phylogenetic analyses identified molluscan-specific gene expansions of key enzymes involved in catecholamine biosynthesis. However, exposure to MK-801 did not alter the expression of selected key enzymes, suggesting that NMDA receptors do not regulate the biosynthesis of catecholamines via gene expression., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

c-Myc modulator 1 (MM1), also known as PFDN5, is the fifth subunit of prefoldin. It was previously reported that MM1-based prefoldin promotes folding of actin during assembly of cytoskeleton, which plays key roles in cell migration. However, no evidence supports that MM1 affects cell migration. In the present study, we found that MM1 promotes cell migration in multiple cell lines. Further study revealed that MM1 promotes polymerization of β-actin into filamentous form and increases both density and length of filopodia. Effects of MM1 on filopodia formation and cell migration depend on its prefoldin activity. Though c-Myc is repressed by MM1, simultaneous knock-down of c-Myc fails to rescue migration inhibition induced by MM1 ablation. Taken together, we here, for the first time, report that prefoldin subunit MM1 is involved in cell migration; this involvement of MM1 in cell migration is due to its prefoldin activity to boost polymerization of β-actin during filopodia formation. Our findings may be helpful to elucidate the mechanism of cell migration and cancer metastasis., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The bacterial RNA polymerase (RNAP) is a multi-subunit protein complex (α2ββ'ω σ) containing the smallest subunit, ω. Although identified early in RNAP research, its function remained ambiguous and shrouded with controversy for a considerable period. It was shown before that the protein has a structural role in maintaining the conformation of the largest subunit, β', and its recruitment in the enzyme assembly. Despite evolutionary conservation of ω and its role in the assembly of RNAP, E. coli mutants lacking rpoZ (codes for ω) are viable due to the association of the global chaperone protein GroEL with RNAP. To get a better insight into the structure and functional role of ω during transcription, several dominant lethal mutants of ω were isolated. The mutants showed higher binding affinity compared to that of native ω to the α2ββ' subassembly. We observed that the interaction between α2ββ' and these lethal mutants is driven by mostly favorable enthalpy and a small but unfavorable negative entropy term. However, during the isolation of these mutants we isolated a silent mutant serendipitously, which showed a lethal phenotype. Silent mutant of a given protein is defined as a protein having the same sequence of amino acids as that of wild type but having mutation in the gene with alteration in base sequence from more frequent code to less frequent one due to codon degeneracy. Eventually, many silent mutants were generated to understand the role of rare codons at various positions in rpoZ . We observed that the dominant lethal mutants of ω having either point mutation or silent in nature are more structured in comparison to the native ω. However, the silent code's position in the reading frame of rpoZ plays a role in the structural alteration of the translated protein. This structural alteration in ω makes it more rigid, which affects the plasticity of the interacting domain formed by ω and α2ββ'. Here, we attempted to describe how the conformational flexibility of the ω helps in maintaining the plasticity of the active site of RNA polymerase. The dominant lethal mutant of ω has a suppressor mapped near the catalytic center of the β' subunit, and it is the same for both types of mutants.

Homocysteine (HCY) molecule combines distinct pharmacological properties as an agonist of N -methyl-d-aspartate receptors (NMDARs) and a reducing agent. Whereas NMDAR activation by HCY was elucidated, whether the redox modulation contributes to its action is unclear. Here, using patch-clamp recording and imaging of intracellular Ca 2+ , we study dithiothreitol (DTT) effects on currents and Ca 2+ responses activated by HCY through native NMDARs and recombinant diheteromeric GluN1/2A, GluN1/2B, and GluN1/2C receptors. Within a wide range (1-800 μM) of [HCY]s, the concentration-activation relationships for recombinant NMDARs revealed a biphasicness. The high-affinity component obtained between 1 and 100 µM [HCY]s corresponding to the NMDAR activation was not affected by 1 mM DTT. The low-affinity phase observed at [HCY]s above 200 μM probably originated from thiol-dependent redox modulation of NMDARs. The reduction of NMDAR disulfide bonds by either 1 mM DTT or 1 mM HCY decreased GluN1/2A currents activated by HCY. In contrast, HCY-elicited GluN1/2B currents were enhanced due to the remarkable weakening of GluN1/2B desensitization. In fact, cleaving NMDAR disulfide bonds in neurons reversed the HCY-induced Ca 2+ accumulation, making it dependent on GluN2B- rather than GluN2A-containing NMDARs. Thus, estimated concentrations for the HCY redox effects exceed those in the plasma during intermediate hyperhomocysteinemia but may occur during severe hyperhomocysteinemia.

Acid-sensing ion channel (ASIC) subunits 1a and 3 are highly expressed in central and peripheral sensory neurons, respectively. Endogenous biomolecule zinc plays a critical role in physiological and pathophysiological conditions. Here, we found that currents recorded from heterologously expressed ASIC1a/3 channels using the whole-cell patch-clamp technique were regulated by zinc with dual effects. Co-application of zinc dose-dependently potentiated both peak amplitude and the sustained component of heteromeric ASIC1a/3 currents; pretreatment with zinc between 3 to 100 µM exerted the same potentiation as co-application. However, pretreatment with zinc induced a significant inhibition of heteromeric ASIC1a/3 channels when zinc concentrations were over 250 µM. The potentiation of heteromeric ASIC1a/3 channels by zinc was pH dependent, as zinc shifted the pH dependence of ASIC1a/3 currents from a pH 50 of 6.54 to 6.77; whereas the inhibition of ASIC1a/3 currents by zinc was also pH dependent. Furthermore, we systematically mutated histidine residues in the extracellular domain of ASIC1a or ASIC3 and found that histidine residues 72 and 73 in both ASIC1a and ASIC3, and histidine residue 83 in the ASIC3 were responsible for bidirectional effects on heteromeric ASIC1a/3 channels by zinc. These findings suggest that histidine residues in the extracellular domain of heteromeric ASIC1a/3 channels are critical for zinc-mediated effects.

Background: Glycosylphosphatidylinositol (GPI) addition is one of the several post-translational modifications to proteins that increase their affinity for membranes. In eukaryotes, the GPI transamidase complex (GPI-T) catalyzes the attachment of pre-assembled GPI anchors to GPI-anchored proteins (GAPs) through a transamidation reaction. A mutation in AtGPI8 (gpi8-2), the putative catalytic subunit of GPI-T in Arabidopsis, is transmitted normally through the female gametophyte (FG), indicating the FG tolerates loss of GPI transamidation. In contrast, gpi8-2 almost completely abolishes male gametophyte (MG) function. Still, the unexpected finding that gpi8-2 FGs function normally requires further investigation. Additionally, specific developmental defects in the MG caused by loss of GPI transamidation remain poorly characterized., Results: Here we investigated the effect of loss of AtPIG-S, another GPI-T subunit, in both gametophytes. Like gpi8-2, we showed that a mutation in AtPIG-S (pigs-1) disrupted synergid localization of LORELEI (LRE), a putative GAP critical for pollen tube reception by the FG. Still, pigs-1 is transmitted normally through the FG. Conversely, pigs-1 severely impaired male gametophyte (MG) function during pollen tube emergence and growth in the pistil. A pPIGS:GFP-PIGS transgene complemented these MG defects and enabled generation of pigs-1/pigs-1 seedlings. However, the pPIGS:GFP-PIGS transgene seemingly failed to rescue the function of AtPIG-S in the sporophyte, as pigs-1/pigs-1, pPIGS:GFP-PIGS seedlings died soon after germination., Conclusions: Characterization of pigs-1 provided further evidence that the FG tolerates loss of GPI transamidation more than the MG and that the MG compared to the FG may be a better haploid system to study the role of GPI-anchoring. Pigs-1 pollen develops normally and thus represent a tool in which GPI anchor biosynthesis and transamidation of GAPs have been uncoupled, offering a potential way to study free GPI in plant development. While previously reported male fertility defects of GPI biosynthesis mutants could have been due either to loss of GPI or GAPs lacking the GPI anchor, our results clarified that the loss of mature GAPs underlie male fertility defects of GPI-deficient pollen grains, as pigs-1 is defective only in the downstream transamidation step.

DNA-directed RNA polymerase II (pol II) is composed of ten core and two dissociable subunits. The dissociable subcomplex is a heterodimer of Rpb4/Polr2d and Rpb7/Polr2g, which are encoded by RPB4/polr2d and RPB7/polr2g genes, respectively. Functional studies of Rpb4/Polr2d in yeast have revealed that Rpb4 plays a role primarily in pol II-mediated RNA synthesis and partly in various mRNA regulations including pre-mRNA splicing, nuclear export of mRNAs and decay of mRNAs. Although Rpb4 is evolutionally highly conserved from yeast to human, it is dispensable for survival in budding yeast S. cerevisiae, whereas it was indispensable for survival in fission yeast S. pombe, slime molds and fruit fly. To elucidate whether Rpb4/Polr2d is necessary for development and survival of vertebrate animals, we generated polr2d-deficient zebrafish. The polr2d mutant embryos exhibited progressive delay of somitogenesis at the onset of 11 h postfertilization (hpf). Mutant embryos then showed increased cell death at 15 hpf, displayed hypoplasia such as small eye and cardiac edema by 48 hpf and prematurely died by 60 hpf. In accordance with these developmental defects, our RT-qPCR revealed that expression of housekeeping and zygotic genes was diminished in mutants. Collectively, we conclude that Rpb4/Polr2d is indispensable for vertebrate development.

Mucociliary clearance, the continuous removal of mucus-trapped particles by cilia-driven directed transport of the airway lining fluid, is the primary innate defense mechanism of the airways. It is potently activated by acetylcholine (ACh) addressing muscarinic receptors with a currently less defined role of nicotinic ACh receptors (nAChR). We here set out to determine their contribution in driving ciliary activity in an explanted mouse trachea preparation utilizing selected agonists and antagonists and nAChR-subunit deficient mice. Nicotine (100 µM) induced an increase in ciliary beat frequency, accompanied by a sharp, but not long lasting increase in particle transport speed (PTS) on the mucosal surface showing marked desensitization within the next 30 min. Nicotine-induced PTS acceleration was sensitive to the general nAChR inhibitors mecamylamine and d-tubocurarine as well as to the α3β4-nAChR antagonist α-conotoxin AulB, but not to other antagonists primarily addressing α3β2-nAChR or α4-, α7- and α9-containing nAChR. Agonists at α3β*-nAChR (epibatidine, cytisine), but not cotinine mimicked the effect. Tracheas from mice with genetic deletion of nAChR subunits α5, α7, α9, α10, α9/10, and β2 retained full PTS response to nicotine, whereas this was entirely lost in tracheas from mice lacking the β4-subunit. Collectively, our data show that nicotinic stimulation of α3β4-nAChR acutely increases PTS to the same extent as the established strong activator ATP. In view of the marked desensitization observed in the present setting, the physiological relevance of these receptors in adapting mucociliary clearance to rapidly changing endogenous or environmental stimuli remains open., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Glioblastoma is the most common primary brain tumor. Glioblastoma cells secrete a significant amount of glutamate, which serve as a potential growth factor in glioma pathobiology through their specific receptor subtypes including α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR). Glioblastoma express AMPAR subunits; however, its regulation and activation with downstream intracellular signaling are not well-understood. Phosphorylated-extracellular signaling-regulated kinase (ERK)1/2 is known to regulate the ionotropic glutamate receptors in cortical neurons. The mitogen-activated protein kinase cascade is frequently activated in several tumors, including glioma. Nonetheless, the association of ERK signaling with AMPAR subunits in glioblastoma is undetermined. Here, we demonstrated potential role of AMPAR in invasion, and the modulation of AMPAR subunits at transcript level by ERK signaling in glioblastoma cells. Inhibition of ERK signaling specifically downregulated the expression of calcium-permeable AMPAR subunits, GluA1 and GluA4, and upregulated calcium-impermeable AMPAR subunit GluA2 implying differential regulation of the expression of calcium-permeable AMPAR subunits of glioblastoma. Concomitantly, it significantly decreased the invasion of U87MG cells. Taken together, these findings suggest that the AMPAR enhances invasion of glioblastoma, and ERK signaling modulates the differential expression of calcium-permeable AMPAR phenotype that might play a crucial role in the invasive propensity of glioblastoma cells., (© 2019 International Federation for Cell Biology.)

Whole-cell patch-clamp technique was employed to record chloride ionic current I GABA evoked by fast (600 msec) application of GABA to hippocampal pyramidal neurons and cerebellar Purkinje cells isolated from rat brain. GABA solution in the application pipette was either neutral (pH 7.4) or acidic (pH 7.0 or 6.0). Application of protons to neurons causes a rapid, reversible, and dose-dependent decrease in the amplitude of I GABA ; the effect was more pronounced on hippocampal neurons (carrying both synaptic and extrasynaptic GABA A receptors) than in cerebellar Purkinje cells (predominantly equipped with synaptic GABA A receptors). In hippocampal neurons, pharmacological isolation of extrasynaptic component from total I GABA was performed with GABA A receptor antagonist gabazine (50 nM). The extrasynaptic component of I GABA was stronger blocked by protons than total I GABA . It was concluded that acidic medium produced more potent blocking effect on extrasynaptic GABA A receptors than on synaptic ones.

Large conductance Ca 2+ -activated K + (BK Ca ) channels are ubiquitously expressed in plasma membrane of both excitable and non-excitable cells and possess significant physiological functions. A tetrameric complex of α subunit (BKα) forms a functional pore of BK Ca channel. The properties of BK Ca channel, such as voltage-dependence, Ca 2+ sensitivity and pharmacological responses, are extensively modulated by co-expressing accessory β subunits (BKβ), which can associate with BKα in one to one manner. Although the functional significance of newly identified γ subunits (BKγ) has been revealed, the stoichiometry between BKα and BKγ1 remains unclear. In the present study, we utilized a single molecule fluorescence imaging with a total internal reflection fluorescence (TIRF) microscope to directly count the number of green fluorescent protein (GFP)-tagged BKγ1 (BKγ1-GFP) within a single BK Ca channel complex in HEK293 cell expression system. BKγ1-GFP significantly enhanced the BK channel activity even when the intracellular Ca 2+ concentration was kept lower, i.e., 10 nM, than the physiological resting level. BKγ1-GFP stably formed molecular complexes with BKα-mCherry in the plasma membrane. Counting of GFP bleaching steps revealed that a BK Ca channel can contain up to four BKγ1 per channel at the maximum. These results suggest that BKγ1 forms a BK Ca channel complex with BKα in a 1 : 1 stoichiometry in a human cell line.

The COP9 (constitutive photomorphogenesis 9) signalosome (CSN) is an evolutionarily conserved protein complex which regulates various growth and developmental processes. However, the role of CSN during environmental stress is largely unknown. Using Arabidopsis as model organism, we used CSN hypomorphic mutants to study the role of the CSN in plant responses to environmental stress and found that heat stress specifically enhanced the growth of csn5a-1 but not the growth of other hypomorphic photomorphogenesis mutants tested. Following heat stress, csn5a-1 exhibits an increase in cell size, ploidy, photosynthetic activity, and number of lateral roots and an upregulation of genes connected to the auxin response. Immunoblot analysis revealed an increase in deneddylation of CUL1 but not CUL3 following heat stress in csn5a-1, implicating improved CUL1 activity as a basis for the improved growth of csn5a-1 following heat stress. Studies using DR5::N7-VENUS and DII-VENUS reporter constructs confirm that the heat-induced growth is due to an increase in auxin signaling. Our results indicate that CSN5A has a specific role in deneddylation of CUL1 and that CSN5A is required for the recovery of AUX/IAA repressor levels following recurrent heat stress to regulate auxin homeostasis in Arabidopsis., Competing Interests: There is no conflict of interest.

Epilepsy is a common primary neurological disorder characterized by the chronic tendency of a patient to experience epileptic seizures, which are abnormal body movements or cognitive states that result from excessive, hypersynchronous brain activity. Epilepsy has been found to have numerous etiologies and, although about two-thirds of epilepsies were classically considered idiopathic, the majority of those are now believed to be of genetic origin. Mutations in genes involved in gamma-aminobutyric acid (GABA)-mediated inhibitory neurotransmission have been associated with a broad range of epilepsy syndromes. Mutations in the GABA-A receptor gamma 2 subunit gene ( GABRG2 ), for example, have been associated with absence epilepsy and febrile seizures in humans. Several rodent models of GABRG2 loss of function depict clinical features of the disease; however, alternative genetic models more amenable for the study of ictogenesis and for high-throughput screening purposes are still needed. In this context, we generated a gabrg2 knockout (KO) zebrafish model (which we called R23X) that displayed light/dark-induced reflex seizures. Through high-resolution in vivo calcium imaging of the brain, we showed that this phenotype is associated with widespread increases in neuronal activity that can be effectively alleviated by the anti-epileptic drug valproic acid. Moreover, these seizures only occur at the larval stages but disappear after 1 week of age. Interestingly, our whole-transcriptome analysis showed that gabrg2 KO does not alter the expression of genes in the larval brain. As a result, the gabrg2 -/- zebrafish is a novel in vivo genetic model of early epilepsies that opens new doors to investigate ictogenesis and for further drug-screening assays., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

N-methyl-d-aspartate receptors (NMDARs) play an essential role in excitatory neurotransmission within the mammalian central nervous system (CNS). NMDARs are heteromultimers containing GluN1, GluN2, and/or GluN3 subunits, thus giving rise to a wide variety of subunit combinations, each with unique functional and pharmacological properties. Importantly, GluN1/GluN3A and GluN1/GluN3B receptors form glycine-gated receptors. Here, we combined electrophysiology with rapid solution exchange in order to determine whether the presence of specific N-glycans and/or interactions with specific lectins regulates the functional properties of GluN1/GluN3A and GluN1/GluN3B receptors expressed in human embryonic kidney 293 (HEK293) cells. We found that removing putative N-glycosylation sites alters the functional properties of GluN1/GluN3B receptors, but has no effect on GluN1/GluN3A receptors. Moreover, we found that the functional properties of both GluN1/GluN3A and GluN1/GluN3B receptors are modulated by a variety of lectins, including Concanavalin A (ConA), Wheat Germ Agglutinin (WGA), and Aleuria Aurantia Lectin (AAL), and this effect is likely mediated by a reduction in GluN1 subunit-mediated desensitization. We also found that AAL has the most profound effect on GluN1/GluN3 receptors, and this effect is mediated partly by a single N-glycosylation site on the GluN3 subunit (specifically, N565 on GluN3A and N465 on GluN3B). Finally, we found that lectins mediate their effect only when applied to non-activated receptors and have no effect when applied in the continuous presence of glycine. These findings provide further evidence to distinguish GluN1/GluN3 receptors from the canonical GluN1/GluN2 receptors and offer insight into how GluN1/GluN3 receptors may be regulated in the mammalian CNS., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The vacuolar H + -adenosine triphosphatase (ATPase) subunit V0C (ATP6V0C), a proton-conducting, pore-forming subunit of vacuolar ATPase, maintains pH homeostasis and induces organelle acidification. The intracellular and extracellular pH of cancer cells affects their growth; however, the role of ATP6V0C in highly invasive esophageal cancer cells (ECCs) remains unclear. In this study, we examined the role of ATP6V0C in glucose metabolism in ECCs. The ATP6V0C depletion attenuated ECC proliferation, invasion, and suppressed glucose metabolism, as indicated by reduced glucose uptake and decreased lactate and adenosine triphosphate (ATP) production in cells. Consistent with this, expression of glycolytic enzyme and the extracellular acidification rate (ECAR) were also decreased by ATP6V0C knockdown. Mechanistically, ATP6V0C interacted with pyruvate kinase isoform M2 (PKM2), a key regulator of glycolysis in ECCs. The ATP6V0C depletion reduced PKM2 phosphorylation at tyrosine residue 105 (Tyr 105 ), leading to inhibition of nuclear translocation of PKM2. In addition, ATP6V0C was recruited at hypoxia response element (HRE) sites in the lactate dehydrogenase A ( LDHA ) gene for glycolysis. Thus, our data suggest that ATP6V0C enhances aerobic glycolysis and motility in ECCs.

Glycogen phosphorylase kinase β-subunit (PHKB) is a regulatory subunit of phosphorylase kinase (PHK), involving in the activation of glycogen phosphorylase (GP) and the regulation of glycogen breakdown. Emerging evidence suggests that PHKB plays a role in tumor progression. However, the function of PHKB in HCC progression remains elusive. Here, our study revealed that the expression of PHKB significantly decreased in HCC tissues, and the low expression of PHKB could serve as an independent indicator for predicting poor prognosis in HCC. Functional experiments showed that PHKB knockdown significantly promoted cell proliferation both in vitro and in vivo , whereas PHKB overexpression resulted in opposing effects. Additionally, in vitro assays revealed that the over (or high) expression of PHKB greatly hindered HCC cell invasion and increased apoptosis rates. Also, we found that the over (or high) expression of PHKB effectively suppressed the epithelial-mesenchymal transition, which was further confirmed by our clinical data. Intriguingly, the biological function of PHKB in HCC was independent of glycogen metabolism. Mechanically, PHKB could inhibit AKT and STAT3 signaling pathway activation in HCC. Collectively, our data demonstrate that PHKB acts as a novel prognostic indicator for HCC, which exerts its suppression function via inactivating AKT and STAT3. Our data might provide novel insights into progression and facilitate the development of a new therapeutic strategy for HCC., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Excessive activation of the NLRP3 inflammasome is a key component contributing to the pathogenesis of various inflammatory diseases. However, the molecular mechanisms underlying its activation and regulation remain poorly defined. The objective of this study was to explore the possible function of the K + channel pore-forming subunit Kir6.1 in regulating NLRP3 inflammasome activation and insulin resistance. Here, we demonstrate that Kir6.1 depletion markedly activates the NLRP3 inflammasome, whereas enhanced Kir6.1 expression produces opposing effects both in mice in vivo and in primary cells in vitro. We also demonstrate that Kir6.1 controls insulin resistance by inhibiting NLRP3 inflammasome activation in mice. We further show that Kir6.1 physically associates with NLRP3 and thus inhibits the interactions between the NLRP3 inflammasome subunits. Our results reveal a previously unrecognized function of Kir6.1 as a negative regulator of the NLRP3 inflammasome and insulin resistance, which is mediated by virtue of its ability to inhibit NLRP3 inflammasome assembly. These data provide novel insights into the regulatory mechanism of NLRP3 inflammasome activation and suggest that Kir6.1 is a promising therapeutic target for inflammasome-mediated inflammatory diseases.

During neuronal development, AMPA receptors (AMPARs) and NMDA receptors (NMDARs) are important for neuronal differentiation. Kainate receptors (KARs) are closely related to AMPARs and involved in the regulation of cortical network activity. However, their role for neurite growth and differentiation of cortical neurons is unclear. Here, we used KAR agonists and overexpression of selected KAR subunits and their auxiliary neuropilin and tolloid-like proteins, NETOs, to investigate their influence on dendritic growth and network activity in organotypic cultures of rat visual cortex. Kainate at 500 nM enhanced network activity and promoted development of dendrites in layer II/III pyramidal cells, but not interneurons. GluK2 overexpression promoted dendritic growth in pyramidal cells and interneurons. GluK2 transfectants were highly active and acted as drivers for network activity. GluK1 and NETO1 specifically promoted dendritic growth of interneurons. Our study provides new insights for the roles of KARs and NETOs in the morphological and physiological development of the visual cortex.

Copy number variation (CNV) at chromosomal region 15q11.2 is linked to increased risk of neurodevelopmental disorders including autism and schizophrenia. A significant gene at this locus is cytoplasmic fragile X mental retardation protein (FMRP) interacting protein 1 ( CYFIP1 ). CYFIP1 protein interacts with FMRP, whose monogenic absence causes fragile X syndrome (FXS). Fmrp knock-out has been shown to reduce tonic GABAergic inhibition by interacting with the δ-subunit of the GABA A receptor (GABA A R). Using in situ hybridization (ISH), qPCR, Western blotting techniques, and patch clamp electrophysiology in brain slices from a Cyfip1 haploinsufficient mouse, we examined δ-subunit mediated tonic inhibition in the dentate gyrus (DG). In wild-type (WT) mice, DG granule cells (DGGCs) responded to the δ-subunit-selective agonist THIP with significantly increased tonic currents. In heterozygous mice, no significant difference was observed in THIP-evoked currents in DGGCs. Phasic GABAergic inhibition in DGGC was also unaltered with no difference in properties of spontaneous IPSCs (sIPSCs). Additionally, we demonstrate that DG granule cell layer (GCL) parvalbumin-positive interneurons (PV + -INs) have functional δ-subunit-mediated tonic GABAergic currents which, unlike DGGC, are also modulated by the α 1 -selective drug zolpidem. Similar to DGGC, both IPSCs and THIP-evoked currents in PV + -INs were not different between Cyfip1 heterozygous and WT mice. Supporting our electrophysiological data, we found no significant change in hippocampal δ-subunit mRNA expression or protein level and no change in α 1 /α 4 -subunit mRNA expression. Thus, Cyfip1 haploinsufficiency, mimicking human 15q11.2 microdeletion syndrome, does not alter hippocampal phasic or tonic GABAergic inhibition, substantially differing from the Fmrp knock-out mouse model., (Copyright © 2019 Trent et al.)

RNA polymerase II is a conserved multi-subunit enzyme made up of twelve different subunits. Two of these subunits, Rpb4 and Rpb7, have been shown to perform functions in both transcription as well as outside of transcription in Saccharomyces cerevisiae. However, our knowledge about the roles of these subunits in Schizosaccharomyces pombe and higher eukaryotes is still limited. Moreover, both Rpb4 and Rpb7 are indispensable for viability of S. pombe and higher eukaryotes, in comparison to S. cerevisiae where deletion of only Rpb7 results in lethality. Therefore in this study, we used S. pombe strains expressing reduced levels of these subunits to determine their impact on the S. pombe proteome employing i-TRAQ based proteomics approach. Furthermore, proteomic profiling was carried out at two different time points to gain a temporal insight into the processes regulated by Rpb4 and Rpb7. The results showed that reduced levels of either Rpb4 or Rpb7 affected the expression of proteins involved in metabolism and ribosome biogenesis at both the time points. Our polysomal profiling experiments further revealed a role of these subunits in translation. Taken together, our results suggest a key role of Rpb4 and Rpb7 subunits in ribosome biogenesis and protein translation in S. pombe. SIGNIFICANCE: Rpb4 and Rpb7 subunits of RNA polymerase II are known for their diverse roles in regulating transcription, mRNA export, mRNA decay, stress response and translation in S. cerevisiae. However, their roles in other organisms are yet to be characterized in detail. Different lines of evidence also suggest that these subunits may function independently as well as a complex in budding yeast. Therefore, in the present study we employed a genome-wide quantitative proteomics-based approach to gain deeper insights into their cellular roles, and to examine if they regulate similar or different biological pathways in fission yeast. Our results provide evidence that they are both involved in primarily regulating metabolic pathways and ribosome biogenesis and also, play a role in protein translation in S. pombe., (Copyright © 2019. Published by Elsevier B.V.)

Background: Previous studies have investigated α1GABA A and α5GABA A receptor mechanisms in the behavioral effects of ethanol (EtOH) in monkeys. However, genetic studies in humans and preclinical studies with mutant mice suggest a role for α2GABA A and/or α3GABA A receptors in the effects of EtOH. The development of novel positive allosteric modulators (PAMs) with functional selectivity (i.e., selective efficacy) at α2GABA A and α3GABA A receptors allows for probing of these subtypes in preclinical models of the discriminative stimulus and reinforcing effects of EtOH in rhesus macaques., Methods: In discrimination studies, subjects were trained to discriminate EtOH (2 g/kg, intragastrically) from water under a fixed-ratio (FR) schedule of food delivery. In oral self-administration studies, subjects were trained to self-administer EtOH (2% w/v) or sucrose (0.3 to 1% w/v) under an FR schedule of solution availability., Results: In discrimination studies, functionally selective PAMs at α2GABA A and α3GABA A (HZ-166) or α3GABA A (YT-III-31) receptors substituted fully (maximum percentage of EtOH-lever responding ≥80%) for the discriminative stimulus effects of EtOH without altering response rates. Full substitution for EtOH also was engendered by a nonselective PAM (triazolam), an α5GABA A -preferring PAM (QH-ii-066) and a PAM at α2GABA A , α3GABA A , and α5GABA A receptors (L-838417). A partial (MRK-696) or an α1GABA A -preferring (zolpidem) PAM only engendered partial substitution (i.e., ~50 to 60% EtOH-lever responding). In self-administration studies, pretreatments with the functionally selective PAMs at α2GABA A and α3GABA A (XHe-II-053 and HZ-166) or α3GABA A (YT-III-31 and YT-III-271) receptors increased EtOH, but not sucrose, drinking at doses that had few, or no, observable sedative-motor effects., Conclusions: Our results confirm prior findings regarding the respective roles of α1GABA A and α5GABA A receptors in the discriminative stimulus effects of EtOH and, further, suggest a key facilitatory role for α3GABA A and potentially α2GABA A receptors in several abuse-related effects of EtOH in monkeys. Moreover, they reveal a potential role for these latter subtypes in EtOH's sedative effects., (© 2019 by the Research Society on Alcoholism.)

Objectives: Small ribosomal protein subunit 7 (RPS7) is an important structural components of the ribosome involved in protein synthesis, previous studies demonstrated that RPS7 was associated with several malignancies, but the role of RPS7 in prostate cancer (PCa) remains unclear. To decipher such a puzzle, in the current study, we deciphered the role and mechanism of RPS7 during the progression of PCa., Material and Methods: In this study, the expression of mRNA was performed by quantitative real-time PCR. The protein level was identified by Western blotting. Kaplan-Meier survival analysis was demonstrated the relation between the abnormal expression of RPS7 mRNA and the overall survival. Cell proliferation was assessed by MTT assay and cell counting, meanwhile, cell migration was checked by transwell assay., Results: RPS7 is higher expressed in PCa (p < 0.001), and the overexpression of RPS7 is closely associated with poor outcome of PCa patients after radical prostatectomy (p < 0.001). Inhibition the expression of RPS7 with a specific RPS7 siRNA could markedly attenuate prostate tumor growth and migration (p < 0.05). Mechanistic data reveals that inhibition of RPS7 could up-regulate the epithelial protein marker, E-cadherin (p < 0.05), and down-regulate the mesenchymal protein markers, such as N-cadherin and Snail (p < 0.001)., Conclusions: RPS7 is a newly verified tumor promoter in PCa, and promotes cell migration by targeting epithelial-to-mesenchymal transitionpathway. Thus, inhibition of RPS7-epithelial to-mesenchymal transition signaling might represent a prospective approach toward limiting prostate tumor progression., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The aminoacylation of tRNAs by aminoacyl-tRNA synthetases (ARSs) is a central reaction in biology. Multiple regulatory pathways use the aminoacylation status of cytosolic tRNAs to monitor and regulate metabolism. The existence of equivalent regulatory networks within the mitochondria is unknown. Here, we describe a functional network that couples protein synthesis to DNA replication in animal mitochondria. We show that a duplication of the gene coding for mitochondrial seryl-tRNA synthetase (SerRS2) generated in arthropods a paralog protein (SLIMP) that forms a heterodimeric complex with a SerRS2 monomer. This seryl-tRNA synthetase variant is essential for protein synthesis and mitochondrial respiration. In addition, SLIMP interacts with the substrate binding domain of the mitochondrial protease LON, thus stimulating proteolysis of the DNA-binding protein TFAM and preventing mitochondrial DNA (mtDNA) accumulation. Thus, mitochondrial translation is directly coupled to mtDNA levels by a network based upon a profound structural modification of an animal ARS., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Regulation of cellular volume is an essential process to balance volume changes during cell proliferation and migration or when intracellular osmolality increases due to transepithelial transport. We previously characterized the key role of volume-regulated anion channels (VRAC) in the modulation of the volume of trabecular meshwork (TM) cells and, in turn, the aqueous humour (AH) outflow from the eye. The balance between the secretion and the drainage of AH determines the intraocular pressure (IOP) that is the major casual risk factor for glaucoma. Glaucoma is an ocular disease that causes irreversible blindness due to the degeneration of retinal ganglion cells. The recent identification of Leucine-Rich Repeat-Containing 8 (LRRC8A-E) proteins as the molecular components of VRAC opens the field to elucidate their function in the physiology of TM and glaucoma. Human TM cells derived from non-glaucomatous donors and from open-angle glaucoma patients were used to determine the expression and the functional activity of LRRC8-mediated channels. Expression levels of LRRC8A-E subunits were decreased in HTM glaucomatous cells compared to normotensive HTM cells. Consequently, the activity of VRAC currents and volume regulation of TM cells were significantly affected. Impaired cell volume regulation will likely contribute to altered aqueous outflow and intraocular pressure.

The small GTPase Arl8 is known to be involved in the periphery-directed motility of lysosomes. However, the overall importance of moving these vesicles is still poorly understood. Here we show that Drosophila Arl8 is required not only for the proper distribution of lysosomes, but also for autophagosome-lysosome fusion in starved fat cells, endosome-lysosome fusion in garland nephrocytes, and developmentally programmed secretory granule degradation (crinophagy) in salivary gland cells. Moreover, proper Arl8 localization to lysosomes depends on the shared subunits of the BLOC-1 and BORC complexes, which also promote autophagy and crinophagy. In conclusion, we demonstrate that Arl8 is responsible not only for positioning lysosomes but also acts as a general lysosomal fusion factor., (Copyright © 2018 Elsevier B.V. All rights reserved.)

N-methyl D-aspartate (NMDA) receptors play a crucial role in normal brain function, pathogenesis of neurodegenerative and psychiatric disorders. Functional tetra-heteromeric NMDA receptor contains two obligatory GluN1 subunits and two identical or different non-GluN1 subunits that evolve from six different genes including four GluN2 (A-D) and two GluN3 (A-B) subunits. Since NMDA receptors confer varied physiological properties and spatiotemporal distributions in the brain, pharmacological agents that target NMDA receptors with specific GluN2 subunits have significant potential for therapeutic applications. In the present work, by using electrophysiology techniques, we have studied the role of ligand binding domain (LBD) interactions in determining the effect of well-characterized pharmacological agents including agonists, competitive antagonists, channel blockers and an allosteric modulator. Remarkably, point mutations at the distal end (site-II&III) of GluN1 LBD interface increased memantine potency up to sevenfold when co-expressed with wild type GluN2A receptors but exhibit no effect on Mg 2+ activity. Conversely, mutations at the proximal end (site-I) of the LBD interface did not affect the memantine but altered Zn 2+ and Mg 2+ potency towards opposite directions. These results indicate that GluN1/2A LBD interface interactions play a key role in determining channel function. Further, subtle changes in LBD interaction can be readily translated to the downstream extracellular vestibule of channel pore to adopt a conformation that may affect memantine, Zn 2+ and Mg 2+ binding. Further studies on NMDA receptor LBD to transmembrane domain signal propagation mechanisms will help develop GluN2 subunit selective biomolecules that can be used for the treatment of neurological and psychiatric disorders., (Copyright © 2018 Elsevier B.V. All rights reserved.)

p97 is an essential hexameric AAA+ ATPase involved in a wide range of cellular processes. Mutations in the enzyme are implicated in the etiology of an autosomal dominant neurological disease in which patients are heterozygous with respect to p97 alleles, containing one copy each of WT and disease-causing mutant genes, so that, in vivo, p97 molecules can be heterogeneous in subunit composition. Studies of p97 have, however, focused on homohexameric constructs, where protomers are either entirely WT or contain a disease-causing mutation, showing that for WT p97, the N-terminal domain (NTD) of each subunit can exist in either a down (ADP) or up (ATP) conformation. NMR studies establish that, in the ADP-bound state, the up/down NTD equilibrium shifts progressively toward the up conformation as a function of disease mutant severity. To understand NTD functional dynamics in biologically relevant p97 heterohexamers comprising both WT and disease-causing mutant subunits, we performed a methyl-transverse relaxation optimized spectroscopy (TROSY) NMR study on a series of constructs in which only one of the protomer types is NMR-labeled. Our results show positive cooperativity of NTD up/down equilibria between neighboring protomers, allowing us to define interprotomer pathways that mediate the allosteric communication between subunits. Notably, the perturbed up/down NTD equilibrium in mutant subunits is partially restored by neighboring WT protomers, as is the two-pronged binding of the UBXD1 adaptor that is affected in disease. This work highlights the plasticity of p97 and how subtle perturbations to its free-energy landscape lead to significant changes in NTD conformation and adaptor binding., Competing Interests: The authors declare no conflict of interest.

The serine/threonine phosphatase PP2A regulates a vast portion of the phosphoproteome including pathways involved in apoptosis, proliferation and DNA damage response and PP2A inactivation is a vital step in malignant transformation. Many groups have explored the therapeutic venue of combining PP2A reactivation with kinase inhibition to counteract the very changes in tumor suppressors and oncogenes that lead to cancer development. Conversely, inhibition of PP2A to complement chemotherapy and radiation-induced cancer cell death is also an area of active investigation. Here we review the studies that utilize PP2A targeted agents as combination therapy in cancer. A potential role for PP2A in tumor immunity is also highlighted., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: AMP-activated protein kinase (AMPK), a heterotrimer with α1 or α2 catalytic subunits, acts as an energy sensor and regulates cellular homeostasis. Whereas AMPKα1 is necessary for myogenesis in skeletal muscle, the role of AMPKα2 in myogenic differentiation and energy metabolism-related gene expressions has remained unclear. We here examined the specific roles of AMPKα1 and AMPKα2 in the myogenic differentiation and mitochondria and energy metabolism-related gene expressions in C2C12 cells., Materials and Methods: Stable C2C12 cell lines expressing a scramble short hairpin RNA (shRNA) or shRNAs specific for AMPKα1 (shAMPKα1), AMPKα2 (shAMPKα2), or both AMPKα1 and AMPKα2 (shPanAMPK) were generated by lentivirus infection. Lentiviruses encoding wild-type AMPKα2 (WT-AMPKα2) or AMPKα2 with a mutated nuclear localization signal (ΔNLS-AMPKα2) were also constructed for introduction into myoblasts. Myogenesis was induced by culture of C2C12 myoblasts for 6 days in differentiation medium., Results: The amount of AMPKα2 increased progressively, whereas that of AMPKα1 remained constant, during the differentiation of myoblasts into myotubes. Expression of shPanAMPK or shAMPKα1, but not that of shAMPKα2, attenuated the proliferation of myoblasts as well as the phosphorylation of both acetyl-CoA carboxylase and the autophagy-initiating kinase ULK1 in myotubes. Up-regulation of myogenin mRNA, a marker for the middle stage of myogenesis, was attenuated in differentiating myotubes expressing shPanAMPK or shAMPKα1. In contrast, up-regulation of gene expression for muscle creatine kinase (MCK), a late-stage differentiation marker, as well as for genes related to mitochondrial biogenesis including the transcriptional coactivator peroxisome proliferator-activated receptor-γ coactivator-1α1 and α4 (PGC-1α1 and PGC-1α4) and mitochondria-specific genes such as cytochrome c were attenuated in myotubes expressing shAMPKα2 or shPanAMPK. The diameter of myotubes expressing shPanAMPK or shAMPKα2 was reduced, whereas that of those expressing shAMPKα1 was increased, compared with myotubes expressing scramble shRNA. A portion of AMPKα2 became localized to the nucleus during myogenic differentiation. The AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) and 2-deoxyglucose (2DG) each induced the nuclear translocation of WT-AMPKα2, but not that of ΔNLS-AMPKα2. Finally, expression of WT-AMPKα2 increased the mRNA abundance of PGC-1α1 and MCK mRNAs as well as cell diameter and tended to increase that of PGC-1α4, whereas that of ΔNLS-AMPKα2 increased only the abundance of MCK mRNA, in myotubes depleted of endogenous AMPKα2., Conclusion: TAMPKα1 and AMPKα2 have distinct roles in myogenic differentiation of C2C12 cells, with AMPKα1 contributing to the middle stage of myogenesis and AMPKα2 to the late stage. AMPKα2 regulates gene expressions including MCK, PGC-1α1 and PGC-1α4 and mitochondria-specific genes such as cytochrome c during the late stage of differentiation. Furthermore, the nuclear translocation of AMPKα2 is necessary for maintenance of PGC-1α1 mRNA during myogenesis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Protein Phosphatase 2A (PP2A) encompasses a large family of Ser/Thr phosphatases, consisting of a catalytic C subunit and a structural A subunit that are, in most cases, further bound to a regulatory B-type subunit. The B-type subunits determine function and regulation of PP2A trimers, but despite their importance in PP2A biology, their roles in controlling dephosphorylation of a given substrate in a given cell or tissue remain poorly defined, particularly in the context of a complete organism. Besides twenty PP2A subunit encoding genes, some of which are tissue-specifically expressed, five additional genes encode major regulators of active PP2A trimer assembly, and at least seven genes encode cellular PP2A inhibitors, further adding to the complexity of the mammalian PP2A system. In this review, we summarize current knowledge on physiologic functions of PP2A in germ cell maturation, embryonic development, metabolic regulation, tumor suppression, and homeostasis of adult brain, heart, liver, immune system, lung, intestine, kidney, skin, bone and eye, all retrieved from in vivo studies using PP2A transgenic, knockout or knockin mice. Data from 63 mouse models, generated between 1998 and now, reveal the essentiality of PP2A in vivo, and shed light on tissue-specific functions of particular PP2A subunits on the one hand, and functional redundancies on the other hand. In future, it remains of utmost importance to further characterize the existing models, as well as to generate novel models, with the aim of deepening our insights in PP2A (patho)physiology and, particularly, in the therapeutic potential of PP2A targeting in human disease., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Voltage-gated calcium (Ca V ) channels are associated with β and α 2 δ auxiliary subunits. This review will concentrate on the function of the α 2 δ protein family, which has four members. The canonical role for α 2 δ subunits is to convey a variety of properties on the Ca V 1 and Ca V 2 channels, increasing the density of these channels in the plasma membrane and also enhancing their function. More recently, a diverse spectrum of non-canonical interactions for α 2 δ proteins has been proposed, some of which involve competition with calcium channels for α 2 δ or increase α 2 δ trafficking and others which mediate roles completely unrelated to their calcium channel function. The novel roles for α 2 δ proteins which will be discussed here include association with low-density lipoprotein receptor-related protein 1 (LRP1), thrombospondins, α-neurexins, prion proteins, large conductance (big) potassium (BK) channels, and N -methyl-d-aspartate (NMDA) receptors., Competing Interests: No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.

Reconsolidation, a process by which long-term memories are rendered malleable following retrieval, has been shown to occur across many different species and types of memory. However, there are conditions under which memories do not reconsolidate, and the reasons for this are poorly understood. One emerging theory is that these boundary conditions are mediated by a form of metaplasticity: cellular changes through which experience can affect future synaptic plasticity. We review evidence that N-methyl-D-aspartate receptors (NMDARs) might contribute to this phenomenon, and hypothesize that resistance to memory destabilization may be mediated by the ratio of GluN2A/GluN2B subunits that make up these receptors. Qualities such as memory strength and the age of the memory may increase the GluN2A/GluN2B ratio, reducing the ability of reactivation cues to induce destabilization, thereby preventing reconsolidation. Other examples of experience-dependent learning and evolutionary perspectives of reconsolidation are also discussed., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In this paper, we report an example of the engineered expression of tetrameric β-galactosidase (β-gal) containing varying numbers of active monomers. Specifically, by combining wild-type and single-nucleotide polymorphism plasmids at varying ratios, tetrameric β-gal was expressed in vitro with one to four active monomers. The kinetics of individual enzyme molecules revealed four distinct populations, corresponding to the number of active monomers in the enzyme. Using single-molecule-level enzyme kinetics, we were able to measure an accurate in vitro mistranslation frequency (5.8 × 10 -4 per base). In addition, we studied the kinetics of the mistranslated β-gal at the single-molecule level., Competing Interests: The authors declare no conflict of interest.

GABA A receptors play a dominant role in mediating inhibition in the mature mammalian brain, and defects of GABAergic neurotransmission contribute to the pathogenesis of a variety of neurological and psychiatric disorders. Two types of GABAergic inhibition have been described: αβγ receptors mediate phasic inhibition in response to transient high-concentrations of synaptic GABA release, and αβδ receptors produce tonic inhibitory currents activated by low-concentration extrasynaptic GABA. Both αβδ and αβγ receptors are important targets for general anesthetics, which induce apparently different changes both in GABA-dependent receptor activation and in desensitization in currents mediated by αβγ vs. αβδ receptors. Many of these differences are explained by correcting for the high agonist efficacy of GABA at most αβγ receptors vs. much lower efficacy at αβδ receptors. The stoichiometry and subunit arrangement of recombinant αβγ receptors are well established as β-α-γ-β-α, while those of αβδ receptors remain controversial. Importantly, some potent general anesthetics selectively bind in transmembrane inter-subunit pockets of αβγ receptors: etomidate acts at β + /α - interfaces, and the barbiturate R-5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (R-mTFD-MPAB) acts at α + /β - and γ + /β - interfaces. Thus, these drugs are useful as structural probes in αβδ receptors formed from free subunits or concatenated subunit assemblies designed to constrain subunit arrangement. Although a definite conclusion cannot be drawn, studies using etomidate and R-mTFD-MPAB support the idea that recombinant α1β3δ receptors may share stoichiometry and subunit arrangement with α1β3γ2 receptors., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Molecular investigations were performed in order to determine the major characteristics of voltage-gated Na + channel β-subunits in mouse vas deferens. The use of real-time quantitative PCR showed that the expression of Scn1b was significantly higher than that of other β-subunit genes (Scn2b - Scn4b). Immunoreactivity of Scn1b proteins was also detected in the inner circular and outer longitudinal smooth muscle of mouse vas deferens. In whole-cell recordings, the actions of 4,9-anhydroTTX on voltage-gated Na + current peak amplitude in myocytes (i.e., native I Na ) were compared with its inhibitory potency on recombinant Na V 1.6 channels (expressed in HEK293 cells). A depolarizing rectangular voltage-pulse elicited a fast and transient inward native I Na and recombinant Na V 1.6 expressed in HEK293 cells (i.e., recombinant I Na ). The current decay of native I Na was similar to the recombinant Na V 1.6 current co-expressed with β 1 -subunits. The current-voltage (I-V) relationships of native I Na were similar to those of recombinant Na V 1.6 currents co-expressed with β 1 -subunits. Application of 4,9-anhydroTTX inhibited the peak amplitude of native I Na (K i  = 510 nM), recombinant I Na (K i  = 112 nM), and recombinant I Na co-expressed with β 1 -subunits (K i  = 92 nM). The half-maximal (V half ) activation and inactivation of native I Na values were similar to those observed in recombinant I Na co-expressed with β 1 -subunits. These results suggest that β 1 -subunit proteins are likely to be expressed mainly in the smooth muscle layers of murine vas deferens and that 4,9-anhydroTTX inhibited not only native I Na but also recombinant I Na and recombinant I Na co-expressed with β 1 -subunits in a concentration-dependent manner.

Cells are largely compartmentalized into numerous interacting organelles with dedicated functions in lipid metabolism, energy generation, or protein turnover. In the past, each organelle has been considered as an isolated unit with an individual proteome, membrane composition, and shape. However, this view is changing rapidly as organelles communicate via contact sites, fuse directly with each other, or correspond via vesicular carriers. Each of these processes disturbs the initial individual character of each organelle and they thus need to be tightly controlled and regulated., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Oligomeric proteins assemble with exceptional selectivity, even in the presence of closely related proteins, to perform their cellular roles. We show that most proteins related by gene duplication of an oligomeric ancestor have evolved to avoid hetero-oligomerization and that this correlates with their acquisition of distinct functions. We report how coassembly is avoided by two oligomeric small heat-shock protein paralogs. A hierarchy of assembly, involving intermediates that are populated only fleetingly at equilibrium, ensures selective oligomerization. Conformational flexibility at noninterfacial regions in the monomers prevents coassembly, allowing interfaces to remain largely conserved. Homomeric oligomers must overcome the entropic benefit of coassembly and, accordingly, homomeric paralogs comprise fewer subunits than homomers that have no paralogs., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Variations in the early postnatal environment of rodents produce long-term changes in responses to stress that may underlie neuropsychiatric diseases such as anxiety, depression and schizophrenia. GABA A receptors undergo marked changes in their subunit composition during this period, involving a regionally-dependent replacement of α 2 with α 1 subunits, the so-called α-subunit switch. In this study we examined the effects of early-life environment on adulthood GABA A receptor α 1 and α 2 subunit expression and the synaptic clustering of GABA A receptors. Male and female mice were exposed to either 15min daily handling sessions (EH) or no intervention (NH) over postnatal day (PND) 1-14. Adulthood behavioural differences in anxiety were assessed on the elevated plus-maze. Immunoperoxidase histochemistry was used to examine the density of the α 1 and α 2 subunit proteins. Double-labelling immunofluorescence and confocal microscopy were used to study GABA A receptor synaptic clustering. NH animals showed increased anxiety-type behaviours in the elevated plus maze relative to EH mice. NH males showed a loss of α 2 subunits from the thalamus and lower layers of the somatosensory cortex, whilst NH females showed a reduction of α 2 but increase in α 1 protein in lower layers of the primary somatosensory cortex only. The NH condition also reduced α 1 subunit expression in dentate gyrus (DG) in both males and females. Regardless of sex, NH mice showed reduced colocalisation of GABA A receptor α 2 subunits with the synaptic marker gephyrin relative to the control condition. These findings suggest that early-life environment has long-lasting effects on GABA A receptors, leading to long-term changes in adulthood behaviour, and are of relevance to neurodevelopmental explanations of stress-augmented neuropsychiatric disorders., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Voltage-gated sodium channel (VGSC) beta (β) subunits have been called the "overachieving" auxiliary ion channel subunit. Indeed, these subunits regulate the trafficking of the sodium channel complex at the plasma membrane and simultaneously tune the voltage-dependent properties of the pore-forming alpha-subunit. It is now known that VGSC β-subunits are capable of similar modulation of multiple isoforms of related voltage-gated potassium channels, suggesting that their abilities extend into the broader voltage-gated channels. The gene family for these single transmembrane immunoglobulin beta-fold proteins extends well beyond the traditional VGSC β1-β4 subunit designation, with deep roots into the cell adhesion protein family and myelin-related proteins - where inherited mutations result in a myriad of electrical signaling disorders. Yet, very little is known about how VGSC β-subunits support protein trafficking pathways, the basis for their modulation of voltage-dependent gating, and, ultimately, their role in shaping neuronal excitability. An evolutionary approach can be useful in yielding new clues to such functions as it provides an unbiased assessment of protein residues, folds, and functions. An approach is described here which indicates the greater emergence of the modern β-subunits roughly 400 million years ago in the early neurons of Bilateria and bony fish, and the unexpected presence of distant homologues in bacteriophages. Recent structural breakthroughs containing α and β eukaryotic sodium channels containing subunits suggest a novel role for a highly conserved polar contact that occurs within the transmembrane segments. Overall, a mixture of approaches will ultimately advance our understanding of the mechanism for β-subunit interactions with voltage-sensor containing ion channels and membrane proteins.

The affinity for K + of silkworm nerve Na + /K + -ATPase is markedly lower than that of mammalian Na + /K + -ATPase (Homareda 2010). In order to obtain clues on the molecular basis of the difference in K + affinities, we cloned cDNAs of silkworm (Bombyx mori) nerve Na + /K + -ATPase α and β subunits, and analyzed the deduced amino acid sequences. The molecular masses of the α and β subunits were presumed to be 111.5 kDa with ten transmembrane segments and 37.7 kDa with a single transmembrane segment, respectively. The α subunit showed 75% identity and 93% homology with the pig Na + /K + -ATPase α1 subunit. On the other hand, the amino acid identity of the β subunit with mammalian counterparts was as low as 30%. Cloned α and β cDNAs were co-expressed in cultured silkworm ovary-derived cells, BM-N cells, which lack endogenous Na + /K + -ATPase. Na + /K + -ATPase expressed in the cultured cells showed a low affinity for K + and a high affinity for Na + , characteristic of the silkworm nerve Na + /K + -ATPase. These results suggest that the β subunit is responsible for the affinity for K + of Na + /K + -ATPase.

The cotton aphid, Aphis gossypii Glover, is one of the most agriculturally important insect pests. Neonicotinoid insecticides and sulfoxaflor have generally shown excellent control of A. gossypii, but these aphids have recently developed resistance against neonicotinoid insecticides. We previously characterized a field-collected A. gossypii Kushima clone that showed higher resistance to nitro-substituted neonicotinoids, such as imidacloprid, than to cyano-substituted neonicotinoids, such as acetamiprid. This Kushima clone harbors the R81T mutation in the nicotinic acetylcholine receptor (nAChR) β1 subunit; this mutation is the source of neonicotinoid insecticide resistance. In the present study, electrophysiological analyses and molecular modeling were employed to investigate the differential effects of the R81T mutation on cyano- and nitro-substituted neonicotinoids and sulfoxaflor. We isolated full-length coding sequences of A. gossypii nAChR α1, α2, and β1 subunits. When co-expressed in Xenopus laevis oocytes with chicken β2 nAChR, A. gossypii α1 evoked inward currents in a concentration-dependent manner in response to acetylcholine (ACh) and showed sensitivity to neonicotinoid and sulfoxaflor. Additionally, the chicken β2 T77R+E79V (equivalent double mutant of R81T) mutation resulted in a lower effect to cyano-substituted neonicotinoids and sulfoxaflor than to nitro-substituted neonicotinoids. Electrophysiological data and nAChR homology modeling analysis suggested that the Kushima clone exhibited different levels of resistance to cyano- and nitro-substituted neonicotinoid insecticides., (Copyright © 2017 Elsevier Inc. All rights reserved.)