FEBS Open Bio最新文献

In Thermus thermophilus, an aerobic Gram-negative eubacterium used as a model organism, more than half of the phosphorylation sites identified by proteomic analysis are located near the ligand-binding site, including the active site, of the enzyme in the three-dimensional structure. We investigated the effect of these phosphorylation events on the activity of six enzymes (three nucleoside monophosphate kinases, isocitrate kinase, malate dehydrogenase and inorganic pyrophosphatase) by introducing phosphomimetic mutations, Glu, into the phosphorylation sites. All phosphomimetic mutants showed severely reduced activity compared with the wild-type, particularly in the turnover number. The proteins analyzed in this study belong to different families and have various functions. This suggests that there is a widespread mechanism by which phosphorylation of amino acid residues near the active site reduces enzyme activity independent of the protein family and function.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) bind to RNA, regulating gene expression and splicing. HnRNP L contributes to muscle development and the pathogenesis of myotonic dystrophy. We hypothesized that hnRNP L regulates muscle expression and splicing patterns. Using nanopore long-read transcriptome sequencing and qPCR analyses, we investigated the impact of Hnrnpl knockdown on myoblasts and knockdown of the orthologous gene smooth in Drosophila. Notch signaling genes and muscle-related genes were dysregulated in both models. Several genes had altered splicing patterns, including Lamp2, Fhl1, and Dtna. The α-DB1 isoform of Dtna was downregulated, whereas the α-DB3 isoform was upregulated. Our findings indicate that hnRNP L regulates both the transcription levels and splicing patterns of genes relevant to skeletal muscle development. We demonstrate the capabilities of long-read transcriptome sequencing to study muscle development. Comparisons between nanopore long-read transcriptome sequencing and data from PCR and qPCR analyses suggest that a minimum read depth of 10 is needed on nanopore sequencing to detect splicing differences greater than 10% to 20%. Future studies could determine whether the minimum read depth that we identified in our model is valid across a broader range of genes, cell types, and conditions. There are also intriguing hints of therapeutic implications of hnRNP L regulation for muscle diseases that merit further investigation.

Cellular RNAs are not linear single-stranded stretches of nucleic acids as depicted in textbook cartoons but fold into secondary and tertiary structures through intra- and intermolecular base-pairing. They also interact with proteins to form ribonucleoproteins (RNPs), the functional units of RNA in cells. Recent methodological developments utilising high-throughput sequencing have enabled the detailed mapping of cellular RNA-protein interactions and RNA structures. While methods for the direct determination of cellular RNP structures are still lacking, the integration of high-throughput approaches and advancements with in vitro techniques such as cryogenic electron microscopy have provided insights into the functional significance of RNP structures. In this review, we will summarise the key methods used to probe cellular RNA-protein interactions and RNA structures and then provide examples of how these approaches have led to an enhanced understanding of RNP structures in gene regulation and how this has also opened new avenues for drug development.

This study investigated the reactivity of sarcosine oxidase (Sox) toward minor substrates through kinetic and structural analyses, along with mutational engineering to elucidate their reaction mechanisms. Sarcosine oxidase from Bacillus sp. (SoxB) recognizes the cyclic imino acids l-proline (l-Pro), d-proline (d-Pro), and l-thioproline (l-Tpr) as minor substrates. The reaction behavior varied depending on the substrates; notably, the absorption spectrum of l-Tpr exhibited charge transfer, which was characteristic of substrate inhibition. Crystal structures of the enzyme–substrate complexes suggested that Tyr254 causes spatial interference with cyclic imino acids at the active site. The Tyr254Ala and Tyr254Gly mutants exhibited enhanced reactivity toward cyclic imino acids by eliminating this spatial interference. Crystallographic analysis of the mutants revealed an enlarged active site, which facilitated reactions with five-membered cyclic imino acids. These mutations disrupted the electron delocalization associated with l-Tpr, thereby eliminating charge transfer and substrate inhibition. A water network was also identified near the enzyme's active site, interacting with the side chain of Tyr254. These findings provide valuable insights into substrate specificity and may facilitate the development of enzymes with broader substrate scope and enhanced catalytic activity.

Hyperlipidemia is a common chronic disease characterized by elevated levels of lipids in the blood. There is some evidence that suggests that berberine (BBR) might be beneficial for the treatment of hyperlipidemia. However, its low intestinal bioavailability limits its potential therapeutic action. In the present study, we explored the effect and the underlying mechanism of berberine–cinnamic acid co-crystal (BBR-CA), which is self-assembled from CA and BBR and displays a high intestinal bioavailability. In mice, BBR-CA showed the ability to decrease body weight gain and hepatic lipid accumulation in animals fed a high-fat diet. To further characterize the molecular basis of this effect, we established a hyperlipidemia cell model by treating human hepatocellular carcinoma cells (HepG2) with free fatty acids. Similarly to our in vivo experiments, lipid accumulation in free fatty acids-induced HepG2 cells was also reduced by BBR-CA. We hypothesized that BBR-CA might act through the regulation of sterol regulatory element-binding proteins-1 (SREBP-1), a key factor regulating lipid synthesis, and, indeed, SREBP-1 protein expression was inhibited by BBR-CA treatment, resulting in the decreased expression of its downstream proteins stearoyl-CoA desaturase 1 and acetyl-CoA carboxylase. Furthermore, the phosphorylation of phosphatidylinositol 3-kinase (PI3K), AKT and mammalian target of rapamycin (mTOR) was inhibited by BBR-CA, contributing to decreased active SREBP-1 in the nucleus, and was reversed and enhanced by the PI3K agonist recilisib and inhibitor LY294002, respectively. Taken together, our results suggest that BBR-CA could function by modulating the PI3K/AKT/mTOR signaling pathway, resulting in decreased nuclear expression of SREBP-1, as well as reduced expression of stearoyl-CoA desaturase 1 and acetyl-CoA carboxylase, thus alleviating hyperlipidemia. Further experimental validation is required to confirm these results.

Tight junctions (TJs) are formed where two or three cells meet and are therefore categorized, respectively, into bicellular TJs (bTJs) and tricellular TJs (tTJs). Angubindin-1 is the first tTJ modulator enhancing intestinal macromolecule permeation via binding to the key tTJ proteins, angulin-1 and angulin-3. It is a fragment (amino acids 421–664) derived from domain IV of Clostridium perfringens iota toxin. Here, we identified critical residues (L562, L598, E638, V640, Y643, K644) of angubindin-1 to be essential for binding to angulins by alanine scanning. Mutants substituting these amino acids with alanine exhibited reduced binding to angulin-expressing cells. Simultaneous substitution of all these amino acids lost binding to angulins and resulted in the loss of tTJ-modulating functions of angubindin-1. These insights highlight crucial residues for the tTJ-modulating activity of angubindin-1, which may hold promise in the design of noninvasive, targeted therapeutics using angubindin-1 as a prototype tTJ modulator to enhance the permeation of drugs.

Primordial germ cells (PGCs) are the progenitor cells of sperm and eggs. Xenotransplantation of chicken PGCs can achieve germline transmission. However, there are still challenges in obtaining many PGCs from endangered birds in vitro. In this study, at first, by incorporating 2i factors, the embryonic stem cells (ESCs) culture conditions were optimized, successfully yielding and validating pluripotent ESCs clones. Then, during induction ESCs, bFGF, activin A, and 1% KSR were added to Epiblast-like cells (EpiLCs). Quantitative real-time polymerase chain reaction (qRT-PCR) showed Pax6, Eomes, and Vimentin expression patterns similar to primary epiblast, indicating successful EpiLCs induction. During EpiLCs to Primordial germ cell-like cells (PGCLCs) transformation, we evaluated BMP4, BMP8b, EGF, LIF, and SCF combinations' impact on induction efficiency. Flow cytometry, qRT-PCR, and immunofluorescence showed high expression of Cvh, C-kit, Dazl, CVH, and DAZL in PGCLCs, suggesting successful EpiLCs differentiation. Induced PGCLCs injected into 2.5-day chick embryos migrated to gonads by day 7–7.5, demonstrating migration and colonization. This study optimized a two-step protocol for in vitro differentiation of chicken ESCs into PGCLCs. This research's results not only provide a reference for obtaining many PGCLCs in vitro but also open up a new approach for the development and application of genetic resource preservation technology in domestic chickens.

Electrical pulse stimulation (EPS) represents a useful tool to study exercise-related adaptations of muscle cells in vitro. Here, we examine the metabolic and secretory response of primary human muscle cells from metabolically healthy individuals to the EPS protocol reflecting the episodic nature of real-life exercise training. This intermittent EPS protocol alternates high-frequency stimulation periods with low-frequency resting periods. Continuous EPS was used as a comparator. Radiometric assessment of glucose and fatty acid metabolism was complemented by examination of mitochondrial OxPHOS proteins, fiber-type markers, and the release of selected myokines and extracellular vesicles into the media. Both EPS protocols facilitated glycogen synthesis and incomplete fatty acid oxidation (intermediary metabolites accumulation), while complete glucose and fatty acid oxidation (CO₂ production) was increased only after the intermittent stimulation. Continuous stimulation elicited robust release of the contraction-regulated myokines (IL6, IL8) into the media. Both EPS protocols increased expression of oxidative fiber-type markers (MYH2, MYH7), while inducing protein expression of a putative myokine, growth differentiation factor11 (GDF11) and a release of extracellular vesicles into the media. In conclusion, intermittent electrical pulse stimulation enhanced the rate of complete glucose and fatty acid oxidation in differentiated muscle cells from metabolically healthy individuals, while it was comparable to continuous stimulation in modulating markers of oxidative fibers and a putative myokine GDF11, and less effective in stimulating the release of myokines IL6, IL8, and extracellular vesicles into the media. Intermittent EPS—a protocol mimicking the episodic nature of exercise—can be used for studying metabolism and the secretome of skeletal muscle cells in vitro.

The global accumulation of plastic waste, exceeding 360 million tonnes annually, represents a critical environmental challenge due to their widespread use and extreme recalcitrance in natural environments. Furthermore, the end-of-life processing of bioplastics, which are often marketed as eco-friendly, remains problematic, with biodegradation often requiring industrial conditions. Enzyme-based depolymerization of polyesters, such as polyethylene terephthalate (PET) and bioplastics (e.g., polylactic acid (PLA), poly(butylene adipate-co-terephthalate) (PBAT), and polyhydroxyalkanoates (PHAs)), has emerged as a promising alternative, offering a green approach to postconsumer plastic management with a reduced environmental impact and in alignment with circular economy principles. This review summarizes recent advances in enzymatic degradation of oil-derived and bio-based polyesters. Key recent developments are discussed including novel high-throughput screenings, computational workflow for improvement of PET hydrolases and de novo design of biocatalysts, microbial platforms, and enzyme-embedded self-biodegrading bioplastics. Collectively, these innovations are redefining the role of biocatalysis in tackling synthetic polymer pollution. Looking ahead, the integration of enzymatic depolymerization with upcycling pathways, standardized kinetic metrics, and one-pot bioprocesses represents a viable strategy for sustainable plastic waste valorization.

Epigallocatechin-3-gallate (EGCG), the main catechin in green tea, is associated with antidiabetic and anti-obesity effects, although its acute hepatic actions remain unclear. We investigated short-term effects of EGCG (10–500 μm) using isolated perfused rat livers and complementary assays in mitochondrial, microsomal, and cytosolic fractions. EGCG markedly inhibited gluconeogenesis from lactate (up to 52%), glycerol (33%), and alanine (47%), while it stimulated glycolysis, glycogenolysis, and oleic acid oxidation (+42% total ketone bodies). Oxygen uptake was stimulated under glycogenolytic and fatty acid oxidizing conditions but inhibited under gluconeogenic conditions. Mechanistic analyses revealed EGCG-induced mild mitochondrial uncoupling, inhibition of pyruvate carboxylase and glucose-6-phosphatase (with no effect on fructose-1,6-bisphosphatase) and stimulation of phosphoenolpyruvate carboxykinase. EGCG shifted cytosolic and mitochondrial NADH/NAD⁺ ratios toward oxidation, increased mitochondrial and plasma membrane permeability (LDH leakage from 10 μm), and altered redox-sensitive fluxes, while the total hepatic ATP content remained unchanged. In summary, EGCG's multifaceted actions suggest that suppression of gluconeogenesis may contribute to its antihyperglycemic effect and the stimulation of fatty acid oxidation to its anti-obesity action. Finally, EGCG's membrane-disruptive properties raise concerns about potential hepatotoxicity in compromised livers.