Microalgae-based biotechnology is one of the most promising alternatives to conventional methods for the removal of antibiotic contaminants from diverse water matrices. However, current knowledge regarding the biochemical mechanisms and catabolic enzymes involved in microalgal biodegradation of antibiotics is scant, which limits the development of enhancement strategies to increase their engineering feasibility. In this study, we investigated the removal dynamics of amphenicols (chloramphenicol, thiamphenicol, and florfenicol), which are widely used in aquaculture, by Chlamydomonas reinhardtii under different growth modes (autotrophy, heterotrophy, and mixotrophy). We found C. reinhardtii removed >92 % chloramphenicol (CLP) in mixotrophic conditions. Intriguingly, gamma-glutamyl hydrolase (GGH) in C. reinhardtii was most significantly upregulated according to the comparative proteomics, and we demonstrated that GGH can directly bind to CLP at the Pro77 site to induce acetylation of the hydroxyl group at C3 position, which generated CLP 3-acetate. This identified role of microalgal GGH is mechanistically distinct from that of animal counterparts. Our results provide a valuable enzyme toolbox for biocatalysis and reveal a new enzymatic function of microalgal GGH. As proof of concept, we also analyzed the occurrence of these three amphenicols and their degradation intermediate worldwide, which showed a frequent distribution of the investigated chemicals at a global scale. This study describes a novel catalytic enzyme to improve the engineering feasibility of microalgae-based biotechnologies. It also raises issues regarding the different microalgal enzymatic transformations of emerging contaminants because these enzymes might function differently from their counterparts in animals.

Moonlighting proteins (MPs), characterized by their ability to perform multiple physiologically unrelated functions without alterations to their primary structures, represent a fascinating class of biomolecules with significant implications for host-pathogen interactions. This Review highlights the emerging importance of metabolic moonlighting proteins (MetMPs) in bacterial pathogenesis, focusing on their non-canonical secretion and unconventional surface anchoring mechanisms. Despite lacking typical signal peptides and anchoring motifs, MetMPs such as acetaldehyde alcohol dehydrogenase (AdhE) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are secreted and localized to the bacterial surface under stress conditions, facilitating host colonization and immune evasion. The secretion of MetMPs, often observed during conditions such as resource scarcity or infection, suggests a complex regulation akin to the overexpression of heat shock proteins in response to environmental stresses. This Review proposes two potential pathways for MetMP secretion: membrane damage-induced permeability and co-transportation with traditionally secreted proteins, highlighting a remarkable bacterial adaptability. Biophysically, surface anchoring of MetMPs is driven by electrostatic interactions, bypassing the need for conventional anchoring sequences. This mechanism is exemplified by the interaction between the bifunctional enzyme AdhE (known as Listeria adhesion protein, LAP) and the internalin B (InlB) in Listeria monocytogenes, which is mediated by charged residues facilitating adhesion to host tissues. Furthermore, MetMPs play critical roles in iron homeostasis, immune modulation, and evasion, underscoring their multifaceted roles in bacterial pathogenicity. The intricate dynamics of MetMP secretion and anchoring underline the need for further research to unravel the molecular mechanisms underpinning these processes, offering potential new targets for therapeutic intervention against bacterial infections.

To further reveal the inhibition mechanism of carbon dioxide (CO2) on Shewanella putrefaciens (S. putrefaciens), influence on metabolic function was studied by biochemical and metabolomics analysis. Accordingly, reduction of intracellular pH (pHi), depolarization of cell membrane and accumulation of reactive oxygen species (ROS) indicated that CO2 changed the membrane permeability of S. putrefaciens. Besides, adenosine triphosphate (ATP), ATPase, nicotinamide adenine dinucleotide (NAD+/NADH) and ratios of NADH/NAD+ were detected, indicating a role of CO2 in repressing respiratory pathway and electron transport. According to metabolomics results, CO2 induced differential expressions of metabolites, disordered respiratory chain and weakened energy metabolism of S. putrefaciens. Inhibition of respiratory rate-limiting enzymes also revealed that electron transfer of respiratory chain was blocked, cell respiration was weakened, and thus energy supply was insufficient under CO2 stress. These results revealed that CO2 caused disruption of metabolic function, which might be the main cause of growth inhibition for S. putrefaciens.

This study examined the pattern of resistance to widely applied synthetic pyrethroids, i.e., cypermethrin and deltamethrin, against larvae of Rhipicephalus microplus ticks sampled from Marathwada region in Maharashtra, India. The study also examined the role of α- and β-esterases and glutathione-S-transferase (GST) in resistance development. All eight R. microplus isolates tested were resistant to deltamethrin (RL IV), having RR50 values from 6.88 to 131.26. LPT analysis exhibited the resistance level II deltamethrin resistance in Beed and Hingoli, III in Dharashiv, and IV in Sambhajinagar, Parbhani, Latur, Jalna, and Nanded isolates. The LIT analysis showed that Dharashiv field isolates had the lowest LC50 value of 229.09 ppm against cypermethrin, while Sambhajinagar field isolates had the highest at 489.78 ppm. The RR50 ranged from 1145.45 to 2448.9. Seven isolates were level I resistant to cypermethrin while the Jalna isolate was level II resistant. In larvae treated with deltamethrin and cypermethrin, the activity of α- and β-esterase enzymes increased significantly compared to control groups. The enzyme ratios in treated larvae ranged from 0.7533 to 1.7023 for α-esterase and 0.7434 to 3.2054 for β-esterase. The Hingoli isolate treated with cypermethrin exhibited the highest α-esterase activity (903.261), whereas Sambhajinagar isolate had the highest GST enzyme ratio (2.8224) after deltamethrin exposure. When exposed to cypermethrin, the Hingoli isolate showed the highest GST enzyme ratio, 2.0832. The present study provides the current resistance status in tick populations from Marathwada region indicating deltamethrin and cypermethrin to be ineffective for tick control. The results also suggest that SP compounds should be regulated in this region and alternative control strategies should be introduced.

OBJECTIVE: To investigate the protective effects of stir-fried Semen Armeniacae Amarum (SAA) against aristolochic acid I (AAI)-induced nephrotoxicity and DNA adducts and elucidate the underlying mechanism involved for ensuring the safe use of Asari Radix et Rhizoma.
METHODS: In vitro, HEK293T cells overexpressing Flag-tagged multidrug resistance-associated protein 3 (MRP3) were constructed by Lentiviral transduction, and inhibitory effect of top 10 common pairs of medicinal herbs with Asari Radix et Rhizoma in clinic on MRP3 activity was verified using a self-constructed fluorescence screening system. The mRNA, protein expressions, and enzyme activity levels of NAD(P)H quinone dehydrogenase 1 (NQO1) and cytochrome P450 1A2 (CYP1A2) were measured in differentiated HepaRG cells. Hepatocyte toxicity after inhibition of AAI metabolite transport was detected using cell counting kit-8 assay. In vivo, C57BL/6 mice were randomly divided into 5 groups according to a random number table, including: control (1% sodium bicarbonate), AAI (10 mg/kg), stir-fried SAA (1.75 g/kg) and AAI + stir-fried SAA (1.75 and 8.75 g/kg) groups, 6 mice in each group. After 7 days of continuous gavage administration, liver and kidney damages were assessed, and the protein expressions and enzyme activity of liver metabolic enzymes NQO1 and CYP1A2 were determined simultaneously.
RESULTS: In vivo, combination of 1.75 g/kg SAA and 10 mg/kg AAI suppressed AAI-induced nephrotoxicity and reduced dA-ALI formation by 26.7%, and these detoxification effects in a dose-dependent manner (P<0.01). Mechanistically, SAA inhibited MRP3 transport in vitro, downregulated NQO1 expression in vivo, increased CYP1A2 expression and enzymatic activity in vitro and in vivo, respectively (P<0.05 or P<0.01). Notably, SAA also reduced AAI-induced hepatotoxicity throughout the detoxification process, as indicated by a 41.3% reduction in the number of liver adducts (P<0.01).
CONCLUSIONS: Stir-fried SAA is a novel drug candidate for the suppression of AAI-induced liver and kidney damages. The protective mechanism may be closely related to the regulation of transporters and metabolic enzymes.

Arachidonic acid (AA), an important polyunsaturated fatty acid in the brain, is hydrolyzed by a direct action of phospholipase A2 (PLA2) or through the combined action of phospholipase C and diacylglycerol lipase, and released into the cytoplasm. Various derivatives of AA can be synthesized mainly through the cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P450 (P450) enzyme pathways. AA and its metabolic enzymes and metabolites play important roles in a variety of neurophysiological activities. The abnormal metabolites and their catalytic enzymes in the AA cascade are related to the pathogenesis of various central nervous system (CNS) diseases, including epilepsy. Here, we systematically reviewed literatures in PubMed about the latest randomized controlled trials, animal studies and clinical studies concerning the known features of AA, its metabolic enzymes and metabolites, and their roles in epilepsy. The exclusion criteria include non-original studies and articles not in English.

Metabolic reprogramming plays critical roles in the development and progression of tumor by providing cancer cells with a sufficient supply of nutrients and other factors needed for fast-proliferating. Emerging evidence indicates that long noncoding RNAs (lncRNAs) are involved in the initiation of metastasis via regulating the metabolic reprogramming in various cancers. In this paper, we aim to summarize that lncRNAs could participate in intracellular nutrient metabolism including glucose, amino acid, lipid, and nucleotide, regardless of whether lncRNAs have tumor-promoting or tumor-suppressor function. Meanwhile, modulation of lncRNAs in glucose metabolic enzymes in glycolysis, pentose phosphate pathway and tricarboxylic acid cycle (TCA) in cancer is reviewed. We also discuss therapeutic strategies targeted at interfering with enzyme activity to decrease the utilization of glucoses, amino acid, nucleotide acid and lipid in tumor cells. This review focuses on our current understanding of lncRNAs participating in cancer cell metabolic reprogramming, paving the way for further investigation into the combination of such approaches with existing anti-cancer therapies.

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Several enzymes of intermediary metabolism have been identified to bind RNA in cells, with potential consequences for the bound RNAs and/or the enzyme. In this study, we investigate the RNA-binding activity of the mitochondrial enzyme malate dehydrogenase 2 (MDH2), which functions in the tricarboxylic acid (TCA) cycle and the malate-aspartate shuttle. We confirmed in cellulo RNA binding of MDH2 using orthogonal biochemical assays and performed enhanced cross-linking and immunoprecipitation (eCLIP) to identify the cellular RNAs associated with endogenous MDH2. Surprisingly, MDH2 preferentially binds cytosolic over mitochondrial RNAs, although the latter are abundant in the milieu of the mature protein. Subcellular fractionation followed by RNA-binding assays revealed that MDH2-RNA interactions occur predominantly outside of mitochondria. We also found that a cytosolically retained N-terminal deletion mutant of MDH2 is competent to bind RNA, indicating that mitochondrial targeting is dispensable for MDH2-RNA interactions. MDH2 RNA binding increased when cellular NAD+ levels (MDH2\'s cofactor) were pharmacologically diminished, suggesting that the metabolic state of cells affects RNA binding. Taken together, our data implicate an as yet unidentified function of MDH2-binding RNA in the cytosol.

The development of pediatric oral drugs is hampered by a lack of predictive simulation tools. These tools, in turn, require data on the physiological variables that influence oral drug absorption, including the expression of drug transporter proteins (DTPs) and drug-metabolizing enzymes (DMEs) in the intestinal tract. The expression of hepatic DTPs and DMEs shows age-related changes, but there are few data on protein levels in the intestine of children. In this study, tissue was collected from different regions of the small and large intestine from neonates (i.e., surgically removed tissue) and from pediatric patients (i.e., gastroscopic duodenal biopsies). The protein expression of clinically relevant DTPs and DMEs was determined using a targeted mass spectrometry approach. The regional distribution of DTPs and DMEs was similar to adults. Most DTPs, with the exception of MRP3, MCT1, and OCT3, and all DMEs showed the highest protein expression in the proximal small intestine. Several proteins (i.e., P-gp, ASBT, CYP3A4, CYP3A5, CYP2C9, CYP2C19, and UGT1A1) showed an increase with age. Such increase appeared to be even more pronounced for DMEs. This exploratory study highlights the developmental changes in DTPs and DMEs in the intestinal tract of the pediatric population. Additional evaluation of protein function in this population would elucidate the implications of the presented changes in protein expression on absorption of orally administered drugs in neonates and pediatric patients.