NAT10, an essential enzyme catalyzing RNA ac⁴C modification, is recognized as a critical regulator of tumorigenesis and progression. This study investigates the role and underlying molecular mechanisms of NAT10 in breast cancer. We found that NAT10 is significantly overexpressed in breast cancer tissues compared to adjacent normal tissues, exhibiting high diagnostic accuracy (AUC = 0.9702, p < 0.001). Consistently, NAT10 expression was also elevated in breast cancer cell lines. Knockdown of NAT10 potently inhibited cell viability, glycolysis (as indicated by reduced glucose uptake, lactate production, and ECAR), and metastatic potential (manifested as suppressed migration and invasion) in breast cancer cells. Mechanistically, NAT10 regulated TRAF6 expression and stability through ac⁴C modification; NAT10 knockdown led to reduced ac⁴C enrichment on TRAF6 mRNA and accelerated its degradation. Rescue experiments confirmed that TRAF6 overexpression partially reversed the inhibitory effects of NAT10 knockdown on glycolysis and metastasis. In vivo, NAT10 knockdown significantly suppressed tumor growth in nude mice, which was associated with reduced expression of Ki67 and TRAF6 in tumor tissues. Collectively, our findings highlight NAT10 as a key regulator of breast cancer progression via ac⁴C-mediated TRAF6 modulation, suggesting it as a promising therapeutic target for breast cancer therapy.

Phosphonate natural products have proven value to society as antibiotics and herbicides. They inhibit a range of enzyme targets usually by mimicking the enzyme substrates. In this study, we investigate a family of phosphonate biosynthetic gene clusters (BGCs) found in Burkholderia. Heterologous expression in Escherichia coli resulted in production of an antimicrobial compound. Spectroscopic characterization and chemical synthesis assigned its structure as 2,4-dioxopentylphosphonic acid. One of the biosynthetic enzymes is a member of the domain of unknown function (DUF) 849 family with homology to β-keto acid cleavage enzymes (BKACEs). In vitro characterization shows that this enzyme catalyzes chemistry that is divergent from previously characterized BKACEs. The observed catalytic activity is explained by a series of cocrystal structures with substrates and intermediates. The BGC also contains a gene encoding lumazine synthase (LS), an essential enzyme in flavin biosynthesis. Biochemical experiments revealed that 2,4-dioxopentylphosphonic acid inhibits LS. In addition, expression of the LS encoded in the BGC, or LS orthologs from a range of organisms, in E. coli conferred resistance to the new phosphonate, which we therefore name flavophos.

Pyridoxal 5'-phosphate (PLP) is an essential enzyme cofactor in hundreds of metabolic reactions. While the classical PLP salvage pathway involves phosphorylation and oxidation steps, the reaction catalysed by pyridoxal reductase represents a pivotal and yet incompletely understood player. Identified across bacteria, plants and animals, this enzyme converts pyridoxal to pyridoxine, shaping PLP recycling and preventing toxic intermediate accumulation. In humans, AKR1C isozymes exhibit dual reductase and dehydrogenase activities, potentially linking vitamin B6 homeostasis with steroid metabolism. These findings suggest a regulatory network integrating biosynthesis, recycling and degradation, although mechanistic details and physiological roles remain unresolved and require further investigation.

Tuberculosis remains one of the world's most devastating infectious diseases. Targeting the essential cell wall enzyme, DprE1 has emerged as a promising therapeutic strategy. In this study, we report the discovery of a novel selenium-containing scaffold, benzoselenazinones (BSZs), which was designed through selenium-for-sulfur replacement in benzothiazinones (BTZs) to combine DprE1 inhibition with the pharmacological benefits of organoselenium compounds. Most BSZ derivatives exhibited exceptional anti-TB activity (MICs < 0.03 μM), low cytotoxicity (Vero IC50s > 100 μM), and potent DprE1 inhibition (IC50s < 0.1 μM). Moreover, compound 11c demonstrated superior oral bioavailability (F % = 31.9%) compared to the representative PBTZ169 of BTZs in CD-1 mice, positioning it as a promising preclinical candidate. This study highlights the potential of selenium-based bioisosteres in anti-TB drug discovery and offers a novel benzoselenazinone scaffold with enhanced efficacy and safety for further development.

Pol32 is a subunit shared by DNA polymerases δ and ζ, yet its role in maintaining genome integrity remains incompletely defined. Here, we employed whole-genome sequencing of mutation-accumulation lines to systematically characterize the genome-wide effects of a POL32 deletion in diploid Saccharomyces cerevisiae. Loss of Pol32 led to substantially (>5-fold) elevated rates of loss of heterozygosity (LOH), chromosome rearrangements, and aneuploidy, but resulted in substantially less genome instability than observed in strains with low levels of DNA polymerase δ. In particular, there was only a small (<2-fold) effect of the pol32 mutation on mutation rates. Notably, a prominent hotspot for chromosome rearrangements located near the end of chromosome VII was observed in pol32 strains. Although deletion of REV3 (encoding the catalytic subunit of Pol ζ) had no significant effect on genome integrity in a wild-type background, pol32 rev3 double mutants had reduced rates of most types of chromosome alterations compared to the pol32 single mutant, implicating Pol ζ in driving the genome instability induced by the Pol32 deficiency. Together, these findings provide new insights into how a shared structural subunit of several DNA polymerases contributes to the regulation of genome stability.IMPORTANCEPol32 is a subunit of DNA polymerases δ (an essential replicative enzyme) and ζ (an error-prone DNA polymerase required for DNA repair). We show that yeast strains that lack this protein have elevated rates of mitotic recombination, large deletions/duplications, translocations, and other types of genomic alterations. The high level of genomic alterations in pol32 mutants is substantially suppressed in strains that lack DNA polymerase ζ, suggesting that this error-prone polymerase may stimulate DNA breaks in conditions of DNA replication stress. Our studies are likely to have wide relevance since sequence variants of POLD3 (the human homolog of Pol32) are associated with certain types of human tumors.

The HypA and HypB metallochaperones from Methanococcus maripaludis have unique metal-binding properties and a distinct nickel transfer mechanism.

The essential bacterial enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) is absent in humans, making the enzyme an attractive antimicrobial target. Its product DXP sits at a metabolic branchpoint and is required for biosynthesis of pyridoxal phosphate (PLP), thiamin diphosphate (ThDP), and isoprenoids. DXP is formed via decarboxylation of pyruvate and subsequent carboligation with d-glyceraldehyde-3-phosphate (d-GAP) in a ThDP-dependent manner. In the current mechanistic model, DXPS follows a ligand-gated mechanism. Pyruvate reacts with ThDP to form C2α-lactylThDP (LThDP), which coincides with a shift to a closed conformation. The flexible "spoon" and "fork" motifs become ordered and the catalytic residue H299 is placed within the active site, supporting LThDP formation and persistence in the closed conformation of the E-LThDP complex until binding of d-GAP. We aim to understand the molecular basis for stabilization of the E-LThDP complex in its closed conformation. We propose that the conserved aromatic residues Y288, F298, and F304 in theE. coli DXPS spoon and fork motifs form a cluster upon transition from the open to closed form, positioning H299 within the active site where it has roles in LThDP formation and persistence. We conducted mutagenesis studies to elucidate the roles of Y288, F298, and F304 in conformational cycling and catalysis. Variants (1) adopted open conformations, (2) displayed significantly reduced kcat, (3) promoted LThDP decarboxylation, and (4) exhibited decreased affinity for the postdecarboxylation intermediate. Our results support a model in which conserved aromatic residues within the mobile, sequence-diverse spoon/fork motifs promote the closed conformation, supporting catalysis and LThDP persistence.

The objective of this study was to identify potential natural inhibitors of peptide deformylase (PDF), an essential bacterial enzyme involved in leptospiral survival. By targeting PDF, the study aims to explore alternative therapeutic options for leptospirosis using phytochemicals derived from Cardiospermum halicacabum. A total of 22 phytochemicals reported from C. halicacabum were selected for this in silico study. Drug-likeness, pharmacokinetic properties, and toxicity profiles were evaluated using SwissADME and ProTox-II. Based on Lipinski's rule of five, 19 compounds were shortlisted and subjected to molecular docking using PyRx software to examine their binding interactions with peptide deformylase. ; Results: Docking results showed that stigmasterol (-9.3 kcal/mol), β-sitosterol (-8.7 kcal/mol), and chrysoeriol (-8.5 kcal/mol) had stronger binding affinities toward PDF than the reference drug penicillin (-7.3 kcal/mol). Interaction analysis revealed that these compounds formed stable hydrogen bonds and hydrophobic interactions with key active-site residues of the enzyme. The observed binding patterns suggest that the selected phytochemicals may effectively interfere with PDF activity. The stronger binding affinity compared to the standard drug highlights their potential as natural anti-leptospiral agents. This study also demonstrates the value of computational approaches in identifying promising lead compounds at an early stage of drug discovery. Overall, the findings indicate that phytochemicals from Cardiospermum halicacabum, particularly stigmasterol, β-sitosterol, and chrysoeriol, show promising inhibitory potential against peptide deformylase. Further experimental studies are needed to confirm these results and assess their potential application in the treatment of leptospirosis.

QM/MM study of the catalytic reaction of potato epoxide hydrolase.

Viral proteases are essential enzymes in many viral strains, playing a crucial role in the viral replication cycle. They are key targets for antiviral drug development and have significant implications for viral pathogenesis. To address the issue of Flavivirus protease substrate promiscuity, Yellow Fever virus protease (YFP), West Nile Virus Protease (WNP), Zika virus protease (ZVP), Usutu Virus Protease (UVP), and Rocio Virus Protease (RVP) were recombinantly expressed in E. coli BL21-(DE3) and purified. Mass spectrometric Proteomic Identification of protease Cleavage Sites (PICSs) was performed using peptide libraries derived from a murine cell line lysate. A surprisingly high promiscuity in protease substrate specificity was detected for all five viral proteases, with a recurrence of arginine in the P1 position. Using homology modeling, specific subsites could be identified. However, the promiscuity of peptide binding was difficult to elucidate using these models. For these reasons, the ProtTrans protein language model (pLM) was used and fine-tuned with the obtained peptide sequences. The ProtTrans T5-Encoder model, originally trained to predict same protein-chain amino acids using a huge size of protein sequence data, when fine-tuned with target peptides from the PICS experiments and decoy peptides, could classify each of these groups with up to 76% test-set accuracy. Dimensionality reduction indicated that the T5 embeddings could indeed contain similar information, which was useful for recognizing protein-peptide interactions. These results confirm the usefulness of pLMs for the prediction of protein-protein interactions and thus have important implications for antiviral drug design.

Suoquan Yishen formula attenuates ectopic lipid deposition in diabetic kidney disease by inhibiting UBC9-mediated SUMO1 modification of DRP1.

Single intravitreal injection of lipid nanoparticles delivering circular mRNA of nicotinamide phosphoribosyltransferase protects against dry AMD.

Emerging importance of ALDH2 in liver diseases and its potential therapeutic role.

Overexpression of human heme oxygenase-1 confers hypersensitivity to oxyfluorfen-induced photodynamic stress in transgenic rice (Oryza sativa L.).

RNA polymerase II (Pol II) is an essential eukaryotic enzyme that transcribes protein‑coding genes and various non‑coding RNAs. RNA polymerase II, I and III subunit L (POLR2L) is a highly conserved component shared by RNA polymerase subunits I, II, and III, which contributes to transcriptional regulation, enzymatic structural integrity, key cellular processes such as proliferation, differentiation, and stress responses. Recent research has shown that POLR2L is not merely a Pol II structural subunit but also plays key roles in disease progression, particularly cancer, where POLR2L dysregulation contributes to tumor growth, metastasis, and resistance to chemotherapy. Additionally, POLR2L is closely linked to major signaling pathways including the PI3K‑Akt, Wnt/β‑catenin, and TGF‑β pathways, highlighting the diverse roles played by POLR2L in cellular signaling. This review summarizes current knowledge on the structural and functional properties of POLR2L, its involvement in various diseases, and its potential as a therapeutic target. By outlining the diagnostic and therapeutic relevance of POLR2L, this review aims to provide a framework for understanding how POLR2L related research may inform transcriptional regulation and its impact on human health and disease.

Tafenoquine (TF), a quinoline-derived antimalarial compound, had been utilized both as a chemoprophylactic agent and in combination therapies such as with artesunate. Despite its potent efficacy against Plasmodium falciparum, its clinical application had been restricted due to reports of neurotoxicity and adverse neuropsychiatric reactions. Previous computational investigations indicated that TF functioned as a dual cholinesterase inhibitor with a high affinity for protein targets, findings that were subsequently corroborated through in vitro enzymatic inhibition studies. Malaria continued to represent a significant global health challenge, particularly within tropical and subtropical regions, owing to the emergence of Plasmodium falciparum and Plasmodium vivax strains resistant to conventional antimalarial drugs. Targeting essential parasitic enzymes, including aspartic proteases, had been recognized as a promising strategy for discovering novel chemotherapeutic candidates. In the present study, an in silico molecular docking approach was employed to examine a series of Tafenoquine analogues as potential inhibitors of critical Plasmodium proteins. Among the fourteen designed derivatives, TF4A, TF8A, TF3A, and TF1A exhibited stronger binding affinities than the parent compound Tafenoquine, with docking energies of −8.1, −8.5, −8.0, and −8.2 kcal/mol, respectively. Additionally, ADMET evaluation and drug-likeness analyses demonstrated that these analogues possessed acceptable pharmacokinetic characteristics and conformed to Lipinski’s rule of five, suggesting good oral bioavailability and favorable physicochemical behavior. Collectively, the computational findings indicated that halogen-substituted Tafenoquine analogues, particularly TF8A and TF1A, established stable interactions within the catalytic pockets of target proteins and exhibited enhanced binding energies. Therefore, these derivatives could be considered as promising lead scaffolds for future antimalarial drug development. Nevertheless, further in vitro and in vivo investigations would be necessary to validate their efficacy, metabolic stability, and safety profiles.

Malassezia spp. are lipophilic yeasts that colonize human and animal skin, being implicated in several dermatological and systemic disorders, including seborrheic dermatitis, pityriasis versicolor, and opportunistic fungemia in immunocompromised individuals. The increasing diffusion of resistant strains and the limited efficacy of conventional azole-based antifungal therapies have prompted the search for alternative treatment strategies. This review explores recent approaches to fighting Malassezia, focusing on both natural and synthetic compounds targeting key molecular pathways. Among the most innovative approaches are Malassezia carbonic anhydrases, essential enzymes involved in pH regulation and cellular metabolism, which represent promising and selective antifungal targets. The antifungal potential of plant-derived extracts rich in polyphenols, terpenoids, and flavonoids is also discussed, highlighting their ability to interfere with yeast growth and adhesion. Silver nanoparticles are examined as synergistic agents due to their antimicrobial properties and potential as carriers for natural bioactive compounds. Additional perspectives include the use of probiotics/postbiotics to restore skin microbiota balance, enzymes such as chitinase and chitosanase, and the inhibition of lipases, key enzymes in Malassezia lipid metabolism. Finally, the growing interest in antimicrobial peptides, both natural and synthetic, opens new avenues for targeted topical formulations. Overall, the integration of natural and synthetic approaches, supported by a deeper understanding of Malassezia's molecular mechanisms, may promote the development of more effective and safer multifactorial therapies.

Hepatocellular carcinoma (HCC) is a prevalent form of primary liver cancer, commonly related to chronic hepatitis B virus (HBV) infection. As an essential enzyme in glycolysis, Enolase-1 (ENO1) is implicated in the progression of multiple types of cancer. The aim of this study was to explore the ENO1's function in HBV-associated HCC. The expression of ENO1 in HBV-HCC was determined with RT-qPCR, immunohistochemistry, and Western blot. ENO1 and HBx overexpression or knockdown was performed through transfection. EdU staining, TUNEL staining, wound healing assay, and Transwell assay were utilized to evaluate the malignant biological behavior of HBV-HCC cells. HBV replication in HBV-HCC cells was assessed by measuring HBV DNA, HBsAg, and HBV cccDNA levels. The interaction of HBx with ENO1 was analyzed by co-IP and Western blot analysis. This study showed the elevated ENO1 expression in HBV-HCC. ENO1 overexpression in HBV-HCC cells markedly enhanced proliferation, migration, invasion, and HBV replication, while significantly inhibiting apoptosis. Conversely, ENO1 silencing produced the opposite effects. HBx was found to upregulate ENO1 expression. By rescue assays, HBx silencing suppressed the malignant behavior of HBV-HCC cells, but was reversed by ENO1 overexpression. Additionally, the enhancement of HBx overexpression on the malignant behavior of HBV-HCC cells was counteracted by ENO1 silencing. HBx facilitates the malignant advancement of HBV-associated HCC by elevating ENO1 expression.

Osteosarcoma (OS) is a primary bone malignancy; however, its exact mechanism of development remains largely unknown. Recent studies have shown that miR-381-3p can affect the development of various cancers. However, its biological effects and mechanisms of OS progression have not yet been elucidated. This study aimed to examine the biological role of miR-381-3p in OS. We screened differentially expressed microRNAs in OS using bioinformatics tools. miR-381-3p expression was assessed by quantitative real-time-polymerase chain reaction, and the effect of miR-381-3p on OS growth was evaluated in vitro and in vivo by functional assays. The direct interaction between PFKFB3 and miR-381-3p was validated by dual luciferase reporter assay. Finally, metabolic alterations in OS cells were monitored using an XF96 Metabolic Flux Analyzer. miR-381-3p expression was significantly downregulated in OS samples and cells. miR-381-3p also participated in suppressing OS cell growth and was associated with the Warburg effect. The PFKFB3 gene, encoding an essential glycolytic enzyme, was identified as a downstream gene of miR-381-3p, and PFKFB3 overexpression partly rescued the inhibitory impact of miR-381-3p on OS growth. miR-381-3p directly targets and negatively regulates PFKFB3 expression, thereby inhibiting OS proliferation by controlling the Warburg effect. The miR-381-3p/PFKFB3 axis may be a promising therapeutic target for OS.

Astaxanthin (AST), a carotenoid pigment, has garnered significant interest due to its potent antioxidant, anti-inflammatory, and antibacterial properties, indicating that it is a valuable natural additive in the aquaculture, nutraceutical, and cosmetic industries. To date, Paracoccus spp., a known astaxanthin-producing bacteria, has emerged as a potential microbial source of substantial AST production yield and biosynthetic capabilities. This study reports the biochemical and genomic characterization of two Paracoccus isolates, GCUPA1 and GCUPA3, focusing on their potential as sources of natural carotenoids. Both strains were characterized by distinctive red-orange pigmentation and identified as P. marcusii based on 16S rRNA analysis. Spectroscopic and chromatographic analyses were performed to identify the predominant carotenoids, and the results established AST as the predominant carotenoid in both strains. The extracted pigments exhibited significant antioxidant activity in the 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay, indicating their potential to reduce oxidative stress. Genome phylogeny revealed that both strains were closely related to the carotenoid-producing strain, P. marcusii CP157, confirming their taxonomic position within the species. Notably, the complete genome sequences revealed intact carotenoid biosynthetic gene clusters (BGCs) that encode all essential enzymes (crtWZYIBE) required for astaxanthin synthesis from isoprenoid precursors, with high nucleotide identity between strains. These findings establish P. marcusii GCUPA1 and GCUPA3 as a potential cell factory for sustainable astaxanthin production and suggest significant advantages in terms of processing efficiency and production economics compared to existing microbial systems.