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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