Archives of biochemistry and biophysics最新文献

Aminoacyl-tRNA synthetases (AARSs) are essential for decoding the genetic code by accurately attaching amino acids to their corresponding tRNAs, making them attractive drug targets for treating various diseases. The natural product reveromycin A (RMA) demonstrates therapeutic potential for osteoporosis and other osteoclast-related disorders by selectively inducing osteoclast apoptosis, with human cytoplasmic isoleucyl-tRNA synthetase (HcIleRS) identified as its putative functional target. In this study, recombinant HcIleRS was expressed and characterized, and RMA was demonstrated to potently inhibit HcIleRS activity with an IC₅₀ value of 36 nM measured using an ATP consumption assay. The dissociation constant (K_d) for RMA binding to HcIleRS was measured at 429 nM, which improved to 90 nM and 28 nM in the presence of an intermediate analog and the substrate isoleucine (l-Ile), respectively. Two co-crystal structures of Saccharomyces cerevisiae IleRS (ScIleRS) complexed with RMA and l-Ile, resolved under the same crystallization conditions, revealed that l-Ile facilitates RMA binding to the tRNA^Ile CCA-end binding site in the catalytic domain by increasing hydrophobic stacking interactions between RMA and active site residues. Consequently, RMA not only directly competes with tRNA^Ile for binding to the catalytic domain but also disrupts its interactions with the editing domain by blocking necessary conformational movements. Notably, the C18 hemisuccinate chain of RMA exhibited alternative conformations in the two structures, suggesting that its interaction with the KMSKS motif is not essential for the high-affinity binding. This substrate-aided cooperative binding mechanism facilitates the potent inhibition of IleRS by RMA and offers valuable insights for developing potential combination therapies targeting AARSs.

Background: Atherosclerosis (AS) is a leading global cause of cardiovascular mortality. Gynostemma pentaphyllum (GP), a well-recognized traditional Chinese medicine with a long history of medicinal application, has emerged as a promising candidate for anti-atherosclerotic intervention due to its well-documented potential in regulating cardiovascular homeostasis.

Purpose: This study aimed to systematically elucidate the anti-atherosclerotic mechanisms of GP using metabolomics, network pharmacology, molecular docking, molecular dynamics (MD) simulations, and in vitro validation.

Methods: Leaf and root samples from five-leaf and seven-leaf gynostemma were analyzed by UHPLC-MS/MS for metabolite profiling. Active components and targets of GP were retrieved from TCMSP and PharmMapper, while AS-related targets were collected from GeneCards, OMIM, DrugBank, and DisGeNET. PPI networks were constructed using Cytoscape, and functional enrichment was analyzed via GO and KEGG. Molecular docking and MD simulations assessed binding affinities between GP components and core targets. Based on the bioinformatics analysis, PPARγ was chosen as a key therapeutic target for further in vitro experimental validation.

Results: Metabolomics identified 1898 compounds, with 208 differentially accumulated metabolites between GP varieties. Network pharmacology revealed 24 active components, 168 potential targets, and 69 AS-overlapping targets. Twelve core genes were identified, including AKT1, ALB, PPARγ, ESR1, CASP3, MMP9, EGFR, SRC, MMP2, MAPK1, MPO, and MAPK8. Enrichment analysis linked these targets to lipid metabolism, efferocytosis, and inflammation pathways. Molecular docking and MD simulations confirmed strong and stable binding of Gypenoside XL to PPARγ. Gypenoside A, one of the relatively abundant constituents among gypenosides (GPs), was also directly bound to PPARγ. In vitro, GPs reduced lipid accumulation, upregulated PPARγ and LXRα, suppressed NF-κB phosphorylation and nuclear translocation and attenuated oxidative stress. These coordinated regulations on lipid metabolism, inflammation, and oxidative stress collectively mitigate key pathological processes of AS, including foam cell formation and atherosclerotic plaque progression.

Conclusion: GP exerts anti-atherosclerotic effects through a multi-component, multi-target mechanism, with activation of the PPARγ-LXRα pathway and inhibition of NF-κB driven inflammation being central to its therapeutic action.

Studies have revealed an association between elevated neuronal cholesterol and neuronal dysfunction, in particular, mitochondrial impairment. However, the mechanism by which cholesterol disrupts neuronal mitochondrial function remains unclear, which prompts our current investigation. Using cultured HT22 mouse hippocampal neuronal cells as an in-vitro model, we found that the unmetabolized cholesterol, rather than its ester derivatives, can alter the MTT activity in cultured neuronal cells in a concentration-dependent manner, with an apparent IC₅₀ ≤1 μM. At low micromolar concentrations (≤10 μM), cholesterol selectively disrupts mitochondrial function without causing overt cell death or reducing cell density. Functional and structural analyses revealed increased mitochondrial lipid peroxidation, loss of mitochondrial membrane potential, opening of the mitochondrial permeability transition pore, disruption of mitochondrial membrane integrity and ultrastructure, reduced mitochondrial density, and decreased cellular ATP levels. Seahorse-based bioenergetic profiling further demonstrated marked reductions in basal respiration, maximal respiratory capacity, and ATP-linked respiration, indicating a broad impairment of mitochondrial oxidative metabolism. In contrast, higher cholesterol concentrations (100 μM) induced overt cytotoxicity. Furthermore, genes involved in cholesterol biosynthesis (e.g., HMGCR, HMGCS1) and transport (e.g., STARD4, ABCA1), as well as mitochondrial energy metabolism pathways, are altered in cholesterol-treated neuronal cells. These results suggest that free cholesterol at very low concentrations can induce selective mitochondrial toxicity in cultured neurons and impairs mitochondrial ATP production. These findings shed lights on the crucial role of dysregulated cholesterol homeostasis in the pathogenesis of neurodegenerative diseases and also form the basis for therapeutic interventions.

Acetyl Coenzyme A (acetyl CoA) is a core metabolite that is involved in various interlinked metabolic pathways. Its levels within distinct subcellular compartments reflect the overall metabolic energy status of the cells. Acetyl CoA serves diverse cellular roles, including as an intermediate metabolite, an allosteric regulator, providing building blocks for anabolic pathways, and facilitating protein acetylation. Since acetyl-CoA participates in multiple metabolic pathways, its dysregulation is associated with a wide range of metabolic disorders. In this study, a genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor was engineered to monitor real-time acetyl CoA levels selectively. The nanosensor, designated as SenACe, enabled the quantification of acetyl CoA dynamics in both in vitro assays and living cells. While examining its selectivity and specificity, SenACe exhibits maximum ratiometric output for acetyl CoA, thereby demonstrating its efficiency as a FRET sensor. The SenACe underwent site-directed mutagenesis to create two mutants, R614A and K880T. Among these nanosensors, the wild-type SenACe-62μ was found to be the most suitable nanosensor, binding acetyl CoA with a binding affinity (K_d) of 0.62 μM and covering a concentration range of 0.1 μM to 20 μM. This variant was further utilized for in vivo real-time analysis of acetyl CoA dynamics in cellular systems (bacteria, yeast, and mammalian cells) via confocal microscopy.