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Nature Communications Oct 2023Hetero-pentameric Cys-loop receptors constitute a major type of neurotransmitter receptors that enable signal transmission and processing in the nervous system. Despite...
Hetero-pentameric Cys-loop receptors constitute a major type of neurotransmitter receptors that enable signal transmission and processing in the nervous system. Despite intense investigations into their working mechanism and pharmaceutical potentials, how neurotransmitters activate these receptors remains unclear due to the lack of high-resolution structural information in the activated open state. Here we report near-atomic resolution structures resolved in digitonin consistent with all principle functional states of the human α1β GlyR, which is a major Cys-loop receptor that mediates inhibitory neurotransmission in the central nervous system of adults. Glycine binding induces cooperative and symmetric structural rearrangements in the neurotransmitter-binding extracellular domain but asymmetrical pore dilation in the transmembrane domain. Symmetric response in the extracellular domain is consistent with electrophysiological data showing cooperative glycine activation and contribution from both α1 and β subunits. A set of functionally essential but differentially charged amino acid residues in the transmembrane domain of the α1 and β subunits explains asymmetric activation. These findings provide a foundation for understanding how the gating of the Cys-loop receptor family members diverges to accommodate specific physiological environments.
Topics: Humans; Receptors, Glycine; Ion Channel Gating; Cysteine Loop Ligand-Gated Ion Channel Receptors; Synaptic Transmission; Glycine
PubMed: 37821459
DOI: 10.1038/s41467-023-42051-6 -
Journal of Virology Sep 2023Previously, we developed an infectious hepatitis E virus (HEV) harboring the nanoKAZ gene in the hypervariable region of the open reading frame 1 (ORF1) of the HEV3b...
Previously, we developed an infectious hepatitis E virus (HEV) harboring the nanoKAZ gene in the hypervariable region of the open reading frame 1 (ORF1) of the HEV3b (JE03-1760F/P10) genome and demonstrated the usefulness for screening anti-HEV drugs that inhibit the early infection process. In the present study, we constructed another reporter HEV (HEV3b-HiBiT) by placing a minimized HiBiT tag derived from NanoLuc luciferase at the 3'-end of the viral capsid (ORF2) coding sequence. It replicated efficiently in PLC/PRF/5 cells, produced membrane-associated particles identical to those of the parental virus, and was genetically stable and infectious. The HiBiT tag was fused to both secreted ORF2s (ORF2s-HiBiT) and ORF2c capsid protein (ORF2c-HiBiT). The ORF2c-HiBiT formed membrane-associated HEV particles (eHEV3b-HiBiT). By treating these particles with digitonin, we demonstrated that the HiBiT tag was expressed on the surface of capsid and was present inside the lipid membrane. To simplify the measurement of luciferase activity and provide a more convenient screening platform, we constructed an ORF2s-defective mutant (HEV3b-HiBiT/ΔORF2s) in which the secreted ORF2s are suppressed. We used this system to evaluate the effects of introducing small interfering RNAs and treatment with an inhibitor or accelerator of exosomal release on HEV egress and demonstrated that the effects on virus release can readily be analyzed. Therefore, HEV3b-HiBiT and HEV3b-HiBiT/ΔORF2s reporters may be useful for investigating the virus life cycle and can serve as a more convenient screening platform to search for candidate drugs targeting the late stage of HEV infection such as particle formation and release. IMPORTANCE The construction of recombinant infectious viruses harboring a stable luminescence reporter gene is essential for investigations of the viral life cycle, such as viral replication and pathogenesis, and the development of novel antiviral drugs. However, it is difficult to maintain the stability of a large foreign gene inserted into the viral genome. In the present study, we successfully generated a recombinant HEV harboring the 11-amino acid HiBiT tag in the ORF2 coding region and demonstrated the infectivity, efficient virus growth, particle morphology, and genetic stability, suggesting that this recombinant HEV is useful for assays. Furthermore, this system can serve as a more convenient screening platform for anti-HEV drugs. Thus, an infectious recombinant HEV is a powerful approach not only for elucidating the molecular mechanisms of the viral life cycle but also for the screening and development of novel antiviral agents.
PubMed: 37681960
DOI: 10.1128/jvi.00508-23 -
Plant Science : An International... Nov 2023In vascular plants, the thylakoid architecture is dominated by the highly structured multiple membrane layers known as grana. The structural diversity of the thylakoid...
In vascular plants, the thylakoid architecture is dominated by the highly structured multiple membrane layers known as grana. The structural diversity of the thylakoid system among plant species is mainly determined by the adaptation to the growth light regime, according to a paradigm stating that shade-tolerant species are featured by a high membrane extension with an enhanced number of thylakoid layers per granum. In this study, the thylakoid system was analysed in Selaginella martensii Spring, a shade-adapted rainforest species belonging to lycophytes, a diminutive plant lineage, sister clade of all other vascular plants (euphyllophytes, including ferns and seed plants). The species is characterized by giant cup-shaped chloroplasts in the upper epidermis and, quantitatively less important, disk-shaped chloroplasts in the mesophyll and lower epidermis. The study aimed at the quantitative assessment of the thylakoid appression exploiting a combination of complementary methods, including electron microscopy, selective thylakoid solubilisation, electron paramagnetic resonance, and simultaneous analysis of fast chlorophyll a fluorescence and P700 redox state. With a chlorophyll a/b ratio of 2.6 and PSI/PSII ratio of 0.31, the plant confirmed two typical hallmarks of shade-adaptation. The morphometric analysis of electron micrographs revealed a 33% fraction of non-appressed thylakoid domains. However, contrasting with the structural paradigm of thylakoid shade-adaptation in angiosperms, S. martensii privileges the increase in the granum diameter in place of the increase in the number of layers building the granum. The very wide grana diameter, 727 nm on average, largely overcame the threshold of 500 nm currently hypothesized to allow an effective diffusion of long-range electron carriers. The fraction of non-appressed membranes based on the selective solubilisation of thylakoids with digitonin was 26%, lower than the morphometric determination, indicating the presence of non-appressed domains inaccessible to the detergent, most probably because of the high three-dimensional complexity of the thylakoid system in S. martensii. Particularly, strong irregularity of grana stacks is determined by assembling thylakoid layers of variable width that tend to slide apart from each other as the number of stacked layers increases.
PubMed: 37595894
DOI: 10.1016/j.plantsci.2023.111833 -
Redox Biology Jul 2023Mitochondrial supercomplexes are observed in mammalian tissues with high energy demand and may influence metabolism and redox signaling. Nevertheless, the mechanisms...
Mitochondrial supercomplexes are observed in mammalian tissues with high energy demand and may influence metabolism and redox signaling. Nevertheless, the mechanisms that regulate supercomplex abundance remain unclear. In this study, we examined the composition of supercomplexes derived from murine cardiac mitochondria and determined how their abundance changes with substrate provision or by genetically induced changes to the cardiac glucose-fatty acid cycle. Protein complexes from digitonin-solubilized cardiac mitochondria were resolved by blue-native polyacrylamide gel electrophoresis and were identified by mass spectrometry and immunoblotting to contain constituents of Complexes I, III, IV, and V as well as accessory proteins involved in supercomplex assembly and stability, cristae architecture, carbohydrate and fat oxidation, and oxidant detoxification. Respiratory analysis of high molecular mass supercomplexes confirmed the presence of intact respirasomes, capable of transferring electrons from NADH to O. Provision of respiratory substrates to isolated mitochondria augmented supercomplex abundance, with fatty acyl substrate (octanoylcarnitine) promoting higher supercomplex abundance than carbohydrate-derived substrate (pyruvate). Mitochondria isolated from transgenic hearts that express kinase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (Glyco), which decreases glucose utilization and increases reliance on fatty acid oxidation for energy, had higher mitochondrial supercomplex abundance and activity compared with mitochondria from wild-type or phosphatase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-expressing hearts (Glyco), the latter of which encourages reliance on glucose catabolism for energy. These findings indicate that high energetic reliance on fatty acid catabolism bolsters levels of mitochondrial supercomplexes, supporting the idea that the energetic state of the heart is regulatory factor in supercomplex assembly or stability.
Topics: Mice; Animals; Phosphofructokinase-2; Heart; Mitochondria, Heart; Glucose; Fatty Acids; Mammals
PubMed: 37210780
DOI: 10.1016/j.redox.2023.102740 -
The Journal of Biological Chemistry Jun 2023Phosphatidylserine (PS) synthase from Candida albicans, encoded by the CHO1 gene, has been identified as a potential drug target for new antifungals against systemic...
Phosphatidylserine (PS) synthase from Candida albicans, encoded by the CHO1 gene, has been identified as a potential drug target for new antifungals against systemic candidiasis. Rational drug design or small molecule screening are effective ways to identify specific inhibitors of Cho1, but both will be facilitated by protein purification. Due to the transmembrane nature of Cho1, methods were needed to solubilize and purify the native form of Cho1. Here, we used six non-ionic detergents and three styrene maleic acids (SMAs) to solubilize an HA-tagged Cho1 protein from the total microsomal fractions. Blue native PAGE and immunoblot analysis revealed a single band corresponding to Cho1 in all detergent-solubilized fractions, while two bands were present in the SMA2000-solubilized fraction. Our enzymatic assay suggests that digitonin- or DDM-solubilized enzyme has the most PS synthase activity. Pull-downs of HA-tagged Cho1 from the digitonin-solubilized fraction reveal an apparent MW of Cho1 consistent with a hexamer. Furthermore, negative-staining electron microscopy analysis and AlphaFold2 structure prediction modeling suggest the hexamer is composed of a trimer of dimers. We purified Cho1 protein to near-homogeneity as a hexamer using affinity chromatography and TEV protease treatment, and optimized Cho1 enzyme activity for manganese and detergent concentrations, temperature (24 °C), and pH (8.0). The purified Cho1 has a K for its substrate CDP-diacylglycerol of 72.20 μM with a V of 0.079 nmol/(μg∗min) while exhibiting a sigmoidal kinetic curve for its other substrate serine, indicating cooperative binding. Purified hexameric Cho1 can potentially be used in downstream structure determination and small drug screening.
Topics: Candida albicans; CDPdiacylglycerol-Serine O-Phosphatidyltransferase; Detergents; Digitonin
PubMed: 37116705
DOI: 10.1016/j.jbc.2023.104756 -
Journal of Immunology (Baltimore, Md. :... May 2023Hepatic innate immune function plays an important role in the pathogenesis of many diseases. Importantly, a growing body of literature has firmly established the spatial...
Hepatic innate immune function plays an important role in the pathogenesis of many diseases. Importantly, a growing body of literature has firmly established the spatial heterogeneity of hepatocyte metabolic function; however, whether innate immune function is zonated remains unknown. To test this question, we exposed adult C57BL/6 mice to endotoxemia, and hepatic tissue was assessed for the acute phase response (APR). The zone-specific APR was evaluated in periportal and pericentral/centrilobular hepatocytes isolated using digitonin perfusion and on hepatic tissue using RNAscope and immunohistochemistry. Western blot, EMSA, chromatin immunoprecipitation, and immunohistochemistry were used to determine the role of the transcription factor NF-κB in mediating hepatic C-reactive protein (CRP) expression. Finally, the ability of mice lacking the NF-κB subunit p50 (p50-/-) to raise a hepatic APR was evaluated. We found that endotoxemia induces a hepatocyte transcriptional APR in both male and female mice, with Crp, Apcs, Fga, Hp, and Lbp expression being enriched in pericentral/centrilobular hepatocytes. Focusing our work on CRP expression, we determined that NF-κB transcription factor subunit p50 binds to consensus sequence elements present in the murine CRP promoter. Furthermore, pericentral/centrilobular hepatocyte p50 nuclear translocation is temporally associated with zone-specific APR during endotoxemia. Lastly, the APR and CRP expression is blunted in endotoxemic p50-/- mice. These results demonstrate that the murine hepatocyte innate immune response to endotoxemia includes zone-specific activation of transcription factors and target gene expression. These results support further study of zone-specific hepatocyte innate immunity and its role in the development of various disease states.
Topics: Male; Female; Animals; Mice; NF-kappa B; C-Reactive Protein; Endotoxemia; Mice, Inbred C57BL; Liver; NF-kappa B p50 Subunit; Immunity, Innate
PubMed: 36946778
DOI: 10.4049/jimmunol.2200900 -
Science Signaling Feb 2023Synaptotagmin-11 (Syt11) is a vesicle-trafficking protein that is linked genetically to Parkinson's disease (PD). Likewise, the protein α-synuclein regulates vesicle...
Synaptotagmin-11 (Syt11) is a vesicle-trafficking protein that is linked genetically to Parkinson's disease (PD). Likewise, the protein α-synuclein regulates vesicle trafficking, and its abnormal aggregation in neurons is the defining cytopathology of PD. Because of their functional similarities in the same disease context, we investigated whether the two proteins were connected. We found that Syt11 was palmitoylated in mouse and human brain tissue and in cultured cortical neurons and that this modification to Syt11 disrupted α-synuclein homeostasis in neurons. Palmitoylation of two cysteines adjacent to the transmembrane domain, Cys and Cys, localized Syt11 to digitonin-insoluble portions of intracellular membranes and protected it from degradation by the endolysosomal system. In neurons, palmitoylation of Syt11 increased its abundance and enhanced the binding of α-synuclein to intracellular membranes. As a result, the abundance of the physiologic tetrameric form of α-synuclein was decreased, and that of its aggregation-prone monomeric form was increased. These effects were replicated by overexpression of wild-type Syt11 but not a palmitoylation-deficient mutant. These findings suggest that palmitoylation-mediated increases in Syt11 amounts may promote pathological α-synuclein aggregation in PD.
Topics: Mice; Animals; Humans; Synaptotagmins; Parkinson Disease; alpha-Synuclein; Lipoylation; Neurons
PubMed: 36787382
DOI: 10.1126/scisignal.add7220 -
The Journal of Biological Chemistry Mar 2023The nitric oxide synthase interacting protein (NOSIP), an E3-ubiquitin ligase, is involved in various processes like neuronal development, craniofacial development,...
The nitric oxide synthase interacting protein (NOSIP), an E3-ubiquitin ligase, is involved in various processes like neuronal development, craniofacial development, granulopoiesis, mitogenic signaling, apoptosis, and cell proliferation. The best-characterized function of NOSIP is the regulation of endothelial nitric oxide synthase activity by translocating the membrane-bound enzyme to the cytoskeleton, specifically in the G2 phase of the cell cycle. For this, NOSIP itself has to be translocated from its prominent localization, the nucleus, to the cytoplasm. Nuclear import of NOSIP was suggested to be mediated by the canonical transport receptors importin α/β. Recently, we found NOSIP in a proteomic screen as a potential importin 13 cargo. Here, we describe the nuclear shuttling characteristics of NOSIP in living cells and in vitro and show that it does not interact directly with importin α. Instead, it formed stable complexes with several importins (-β, -7, -β/7, -13, and transportin 1) and was also imported into the nucleus in digitonin-permeabilized cells by these factors. In living HeLa cells, transportin 1 seems to be the major nuclear import receptor for NOSIP. A detailed analysis of the NOSIP-transportin 1 interaction revealed a high affinity and an unusual binding mode, involving the N-terminal half of transportin 1. In contrast to nuclear import, nuclear export of NOSIP seems to occur mostly by passive diffusion. Thus, our results uncover additional layers in the larger process of endothelial nitric oxide synthase regulation.
Topics: Active Transport, Cell Nucleus; HeLa Cells; Humans; Protein Binding; Nitric Oxide Synthase Type III; Proteome; Ubiquitin-Protein Ligases; beta Karyopherins
PubMed: 36690276
DOI: 10.1016/j.jbc.2023.102932 -
Forensic Toxicology Jan 2023AMB-FUBINACA is a synthetic cannabinoid receptor agonist (SCRA) which is primarily metabolised by hepatic enzymes producing AMB-FUBINACA carboxylic acid. The...
PURPOSE
AMB-FUBINACA is a synthetic cannabinoid receptor agonist (SCRA) which is primarily metabolised by hepatic enzymes producing AMB-FUBINACA carboxylic acid. The metabolising enzymes associated with this biotransformation remain unknown. This study aimed to determine if AMB-FUBINACA metabolism could be reduced in the presence of carboxylesterase (CES) inhibitors and recreational drugs commonly consumed with it. The affinity and activity of the AMB-FUBINACA acid metabolite at the cannabinoid type-1 receptor (CB) was investigated to determine the activity of the metabolite.
METHODS
The effect of CES1 and CES2 inhibitors, and delta-9-tetrahydrocannabinol (Δ-THC) on AMB-FUBINACA metabolism were determined using both human liver microsomes (HLM) and recombinant carboxylesterases. Radioligand binding and cAMP assays comparing AMB-FUBINACA and AMB-FUBINACA acid were carried out in HEK293 cells expressing human CB.
RESULTS
AMB-FUBINACA was rapidly metabolised by HLM in the presence and absence of NADPH. Additionally, CES1 and CES2 inhibitors both significantly reduced AMB-FUBINACA metabolism. Furthermore, digitonin (100 µM) significantly inhibited CES1-mediated metabolism of AMB-FUBINACA by ~ 56%, while the effects elicited by Δ-THC were not statistically significant. AMB-FUBINACA acid produced only 26% radioligand displacement consistent with low affinity binding. In cAMP assays, the potency of AMB-FUBINACA was ~ 3000-fold greater at CB as compared to the acid metabolite.
CONCLUSIONS
CES1A1 was identified as the main hepatic enzyme responsible for the metabolism of AMB-FUBINACA to its less potent carboxylic acid metabolite. This biotransformation was significantly inhibited by digitonin. Since other xenobiotics may also inhibit similar SCRA metabolic pathways, understanding these interactions may elucidate why some users experience high levels of harm following SCRA use.
Topics: Humans; Cannabinoids; Dronabinol; Digitonin; HEK293 Cells; Cannabinoid Receptor Agonists
PubMed: 36652070
DOI: 10.1007/s11419-022-00649-3 -
Biochimica Et Biophysica Acta.... Apr 2023The FF-ATP synthase uses the energy stored in the electrochemical proton gradient to synthesize ATP. This complex is found in the inner mitochondrial membrane as a...
Deletion of the ATP20 gene in Ustilago maydis produces an unstable dimer of FF-ATP synthase associated with a decrease in mitochondrial ATP synthesis and a high HO production.
The FF-ATP synthase uses the energy stored in the electrochemical proton gradient to synthesize ATP. This complex is found in the inner mitochondrial membrane as a monomer and dimer. The dimer shows higher ATPase activity than the monomer and is essential for cristae folding. The monomer-monomer interface is constituted by subunits a, i/j, e, g, and k. The role of the subunit g in a strict respiratory organism is unknown. A gene knockout was generated in Ustilago maydis to study the role of subunit g on mitochondrial metabolism and cristae architecture. Deletion of the ATP20 gene, encoding the g subunit, did not affect cell growth or glucose consumption, but biomass production was lower in the mutant strain (gΔ strain). Ultrastructure observations showed that mitochondrial size and cristae shape were similar in wild-type and gΔ strains. The mitochondrial membrane potential in both strains had a similar magnitude, but oxygen consumption was higher in the WT strain. ATP synthesis was 20 % lower in the gΔ strain. Additionally, the mutant strain expressed the alternative oxidase in the early stages of growth (exponential phase), probably as a response to ROS stress. Dimer from mutant strain was unstable to digitonin solubilization, avoiding its isolation and kinetic characterization. The isolated monomeric state activated by n-dodecyl-β-D-maltopyranoside showed similar kinetic constants to the monomer from the WT strain. A decrease in mitochondrial ATP synthesis and the presence of the AOX during the exponential growth phase suggests that deletion of the g gene induces ROS stress.
Topics: Hydrogen Peroxide; Mitochondrial Proton-Translocating ATPases; Reactive Oxygen Species; Adenosine Triphosphate
PubMed: 36509127
DOI: 10.1016/j.bbabio.2022.148950