Gene regulation relies on the precise binding of transcription factors (TFs) at regulatory elements, but simultaneously detecting hundreds of TFs on chromatin is challenging. We developed cFOOT-seq, a cytosine deaminase-based TF footprinting assay, for high-resolution, quantitative genome-wide assessment of TF binding in both open and closed chromatin regions, even with small cell numbers. By utilizing the dsDNA deaminase SsdAtox, cFOOT-seq converts accessible cytosines to uracil while preserving genomic integrity, making it compatible with techniques like ATAC-seq for sensitive and cost-effective detection of TF occupancy at the single-molecule and single-cell level. Our approach enables the delineation of TF footprints, quantification of occupancy, and examination of chromatin influences on TF binding. Notably, cFOOT-seq, combined with FootTrack analysis, enables de novo prediction of TF binding sites and tracking of TF occupancy dynamics. We demonstrate its application in capturing cell type-specific TFs, analyzing TF dynamics during reprogramming, and revealing TF dependencies on chromatin remodelers. Overall, cFOOT-seq represents a robust approach for investigating the genome-wide dynamics of TF occupancy and elucidating the cis-regulatory architecture underlying gene regulation.
Transcription Factors/genetics* ; Humans ; Chromatin/genetics* ; Animals ; Binding Sites ; Mice ; DNA Footprinting/methods*

As important biocatalysts, nitrilases can efficiently convert nitrile groups into acids and ammonia in a mild and eco-friendly manner, being widely used in the synthesis of important pharmaceutical intermediates. Early studies reported that nitrilases only had the hydrolysis activity of catalyzing the formation of corresponding carboxylic acid products from nitriles, showing catalytic specificity. However, recent studies have shown that some nitrilases exhibit the hydration activity for catalyzing the formation of amides from nitriles, showing catalytic promiscuity. The catalytic promiscuity of nitrilases has dual effects. On the one hand, the presence of amide by-products increases the difficulties and costs of subsequent separation and purification of carboxylic acid products. On the other hand, however, if the catalytic reaction pathways of nitrilases can be precisely regulated to reshape enzyme functions, the reactions catalyzed by nitrilases can be broadened to provide new ideas for the biosynthesis of high-value amides, which is crucial for the development of artificial enzymes and biocatalysis. This review summarized the research progress in the catalytic promiscuity of nitrilases and discussed the key regulatory factors that may affect the catalytic promiscuity of nitrilases from the evolutionary origin, catalytic domains, and catalytic mechanisms, hoping to provide reference and inspiration for the application of nitrilases in biocatalysis.
Aminohydrolases/chemistry* ; Biocatalysis ; Nitriles/chemistry* ; Substrate Specificity ; Catalytic Domain ; Catalysis

This study aimed to explore the roles of microRNAs (miRNAs) in the post-transcriptional regulation of cocaine- and amphetamine-regulated transcript (CART) peptide in the bovine hypothalamus and to screen key regulatory miRNAs. Targetscan was used to predict the potential miRNAs binding to CART 3' untranslated regions (3'UTR). Bioinformatics analysis predicted 7 miRNA binding sites in the bovine CART 3'UTR, which were bta-miR-377, bta-miR-331-3p, bta-miR-491, bta-miR-493, bta-miR-758, bta-miR-877, and bta-miR-381, respectively. Reverse transcription-PCR (RT-PCR) was carried out to determine the endogenous expression of CART and target miRNAs in the bovine hypothalamus. All the 7 target miRNAs and CART were endogenously expressed in the bovine hypothalamus. The dual-luciferase reporter gene assay was employed to detect the targeted binding relationship between CART 3'UTR and target miRNAs obtained from bioinformatics analysis. The dual-luciferase reporter gene assay confirmed that the 3'UTR of CART had a targeted binding relationship with the 7 target miRNAs. Cell experiments were conducted to examine the effects of target miRNAs on the messenger RNA (mRNA) and protein levels of exogenous CART and screen for key regulatory miRNAs. The results of cell experiments showed that the 7 miRNAs downregulated the mRNA level of CART, with bta-miR-491 demonstrating the strongest downregulating effect. Bta-miR-377, bta-miR-331-3p, bta-miR-491, bta-miR-493, and bta-miR-381 downregulated the protein level of CART, with bta-miR-381 exerting the strongest downregulating effect. Animal experiments were conducted to explore the effects of key regulatory miRNAs on the mRNA and protein levels of CART in the hypothalamus and the CART concentration in the serum. The results from animal experiments showed that miR-491 and miR-381 regulated the endogenous expression of CART in the hypothalamus and the concentration in the serum by binding to the CART 3'UTR. These results suggest that miR-491 and miR-381 are the main miRNAs regulating CART expression in the bovine hypothalamus, which can affect serum CART concentration by modulating endogenous CART expression.
Animals ; MicroRNAs/metabolism* ; Cattle ; Hypothalamus/metabolism* ; 3' Untranslated Regions/genetics* ; Nerve Tissue Proteins/metabolism* ; Gene Expression Regulation ; Binding Sites ; Base Sequence ; Computational Biology/methods* ; Cocaine- and Amphetamine-Regulated Transcript Protein

Diacylglycerol (DAG) is an intermediate product in lipid metabolism and plays an important physiological role in human body. It is mainly prepared by hydrolyzing lipid with lipase. However, research on the detection method of 1, 2-diacylglycerol (1, 2-DAG) and 1, 3-diacylglycerol (1, 3-DAG) and catalytic specificity of lipase was not enough, which limits its wide application. To address these challenges, an efficient quantitative detection method was first established for 1, 2-DAG (0.025-0.200 g/L) and 1, 3-DAG (0.025-0.150 g/L) by combining supercritical fluid chromatography with evaporative light scattering detector and optimizing the detection and analysis parameters. Based on the molecular docking between Thermomyces lanuginosus lipase (TLL) and triolein, five potential substrate binding sites were selected for site-specific saturation mutation to construct a mutation library for enzyme activity and position specificity screening. The specificity of sn-1, 3 of the I202V mutant was the highest in the library, which was 11.7% higher than the specificity of the wild type TLL. In summary, the position specificity of TLL was modified based on a semi-rational design, and an efficient separation and detection method of DAG isomers was also established, which provided a reference for the study of the catalytic specificity of lipase.
Humans ; Diglycerides ; Molecular Docking Simulation ; Binding Sites ; Catalysis ; Lipase/genetics*

Bacillus subtilis is recognized as a generally-regarded-as-safe strain, and has been widely used in the biosynthesis of high value-added products, including N-acetylneuraminic acid (NeuAc) which is widely used as a nutraceutical and a pharmaceutical intermediate. Biosensors responding to target products are widely used in dynamic regulation and high-throughput screening in metabolic engineering to improve the efficiency of biosynthesis. However, B. subtilis lacks biosensors that can efficiently respond to NeuAc. This study first tested and optimized the transport capacity of NeuAc transporters, and obtained a series of strains with different transport capacities for testing NeuAc-responsive biosensors. Subsequently, the binding site sequence of Bbr_NanR responding to NeuAc was inserted into different sites of the constitutive promoter of B. subtilis, and active hybrid promoters were obtained. Next, by introducing and optimizing the expression of Bbr_NanR in B. subtilis with NeuAc transport capacity, we obtained an NeuAc-responsive biosensor with wide dynamic range and higher activation fold. Among them, P535-N2 can sensitively respond to changes in intracellular NeuAc concentration, with the largest dynamic range (180-20 245) AU/OD. P566-N2 shows a 122-fold of activation, which is 2 times of the reported NeuAc-responsive biosensor in B. subtilis. The NeuAc-responsive biosensor developed in this study can be used to screen enzyme mutants and B. subtilis strains with high NeuAc production efficiency, providing an efficient and sensitive analysis and regulation tool for biosynthesis of NeuAc in B. subtilis.
N-Acetylneuraminic Acid/metabolism* ; Bacillus subtilis/metabolism* ; Promoter Regions, Genetic/genetics* ; Binding Sites ; Biosensing Techniques

Objective: The docking and superantigen activity sites of staphylococcal enterotoxin-like W (SElW) and T cell receptor (TCR) were predicted, and its SElW was cloned, expressed and purified. Methods: AlphaFold was used to predict the 3D structure of SElW protein monomers, and the protein models were evaluated with the help of the SAVES online server from ERRAT, Ramachandran plot, and Verify_3D. The ZDOCK server simulates the docking conformation of SElW and TCR, and the amino acid sequences of SElW and other serotype enterotoxins were aligned. The primers were designed to amplify selw, and the fragment was recombined into the pMD18-T vector and sequenced. Then recombinant plasmid pMD18-T was digested with BamHⅠand Hind Ⅲ. The target fragment was recombined into the expression plasmid pET-28a(+). After identification of the recombinant plasmid, the protein expression was induced by isopropyl-beta-D- thiogalactopyranoside. The SElW expressed in the supernatant was purified by affinity chromatography and quantified by the BCA method. Results: The predicted three-dimensional structure showed that the SElW protein was composed of two domains, the amino-terminal and the carboxy-terminal. The amino-terminal domain was composed of 3 α-helices and 6 β-sheets, and the carboxy-terminal domain included 2 α-helices and 7 antiparallel β-sheets composition. The overall quality factor score of the SElW protein model was 98.08, with 93.24% of the amino acids having a Verify_3D score ≥0.2 and no amino acids located in disallowed regions. The docking conformation with the highest score (1 521.328) was selected as the analysis object, and the 19 hydrogen bonds between the corresponding amino acid residues of SElW and TCR were analyzed by PyMOL. Combined with sequence alignment and the published data, this study predicted and found five important superantigen active sites, namely Y18, N19, W55, C88, and C98. The highly purified soluble recombinant protein SElW was obtained with cloning, expression, and protein purification. Conclusions: The study found five superantigen active sites in SElW protein that need special attention and successfully constructed and expressed the SElW protein, which laid the foundation for further exploration of the immune recognition mechanism of SElW.
Humans ; Enterotoxins/genetics* ; Superantigens/genetics* ; Catalytic Domain ; Selenoprotein W/metabolism* ; Receptors, Antigen, T-Cell

Covalently anchoring of a ligand/metal via polar amino acid side chain(s) is often observed in metalloenzyme, while the substitutability of metal-binding sites remains elusive. In this study, we utilized a zinc-dependent alcohol dehydrogenase from Thermoanaerobacter brockii (TbSADH) as a model enzyme, analyzed the sequence conservation of the three residues Cys37, His59, and Asp150 that bind the zinc ion, and constructed the mutant library. After experimental validation, three out of 224 clones, which showed comparative conversion and ee values as the wild-type enzyme in the asymmetric reduction of the model substrate tetrahydrofuran-3-one, were screened out. The results reveal that the metal-binding sites in TbSADH are substitutable without tradeoff in activity and stereoselectivity, which lay a foundation for designing ADH-catalyzed new reactions via metal ion replacement.
Alcohol Dehydrogenase/metabolism* ; Catalytic Domain ; Ligands ; Protein Domains ; Zinc/metabolism*

Objective: To investigate the effects of glucose-6-phosphatase catalytic subunit (G6PC) recombinant adenovirus on proliferation and cell cycle regulation of liver cancer cells. Methods: Recombinant adenovirus AdG6PC was constructed. Huh7 cells and SK-Hep1 cells were set as Mock, AdGFP and AdG6PC group. Cell proliferation and clone formation assay were used to observe the proliferation of liver cancer cells. Transwell and scratch assay were used to observe the invasion and migration of liver cancer cells. Cell cycle flow cytometry assay was used to analyze the effect of G6PC overexpression on the proliferation cycle of liver cancer cells. Western blot was used to detect the effect of G6PC overexpression on the cell-cycle protein expression in liver cancer cells. Results: The recombinant adenovirus AdG6PC was successfully constructed. Huh7 and SK-Hep1 cells proliferation assay showed that the number of proliferating cells in the AdG6PC group was significantly lower than the other two groups (P < 0.05). Clone formation assay showed that the number of clones was significantly lower in AdG6PC than the other two groups (P < 0.05), suggesting that G6PC overexpression could significantly inhibit the proliferation of liver cancer cells. Transwell assay showed that the number of cell migration was significantly lower in AdG6PC than the other two groups (P < 0.05). Scratch repair rate was significantly lower in AdG6PC than the other two groups (P < 0.05), suggesting that G6PC overexpression can significantly inhibit the invasion and migration of liver cancer cells. Cell cycle flow cytometry showed that G6PC overexpression had significantly inhibited the Huh7 cells G(1)/S phase transition. Western blot result showed that G6PC overexpression had down-regulated the proliferation in cell-cycle related proteins expression. Conclusion: G6PC inhibits the proliferation, cell-cycle related expression, and migration of liver cancer cells by inhibiting the G(1)/S phase transition.
Catalytic Domain ; Cell Cycle Checkpoints ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Glucose-6-Phosphatase/metabolism* ; Humans ; Liver Neoplasms/genetics*

A new coronavirus (SARS-CoV-2) has been identified as the etiologic agent for the COVID-19 outbreak. Currently, effective treatment options remain very limited for this disease; therefore, there is an urgent need to identify new anti-COVID-19 agents. In this study, we screened over 6,000 compounds that included approved drugs, drug candidates in clinical trials, and pharmacologically active compounds to identify leads that target the SARS-CoV-2 papain-like protease (PLpro). Together with main protease (M
Antiviral Agents/therapeutic use* ; Binding Sites ; COVID-19/virology* ; Coronavirus Papain-Like Proteases/metabolism* ; Crystallography, X-Ray ; Drug Evaluation, Preclinical ; Drug Repositioning ; High-Throughput Screening Assays/methods* ; Humans ; Imidazoles/therapeutic use* ; Inhibitory Concentration 50 ; Molecular Dynamics Simulation ; Mutagenesis, Site-Directed ; Naphthoquinones/therapeutic use* ; Protease Inhibitors/therapeutic use* ; Protein Structure, Tertiary ; Recombinant Proteins/isolation & purification* ; SARS-CoV-2/isolation & purification*

Animals ; Binding Sites ; Cochlea ; Homeodomain Proteins/chemistry* ; Mice ; Transcription Factors ; Tumor Suppressor Proteins/chemistry*