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*

Proteases are widely found in organisms participating in the decomposition of proteins to maintain the organisms' normal life activities. Protease inhibitors regulate the activities of target proteases by binding to their active sites, thereby affecting protein metabolism. The key amino acid mutations in proteases and protease inhibitors can affect their physiological functions, stability, catalytic activity, and inhibition specificity. More active, stable, specific, environmentally friendly and cheap proteases and protease inhibitors might be obtained by excavating various natural mutants of proteases and protease inhibitors, analyzing their key active sites by using protein engineering methods. Here, we review the studies on proteases' key active sites and protease inhibitors to deepen the understanding of the active mechanism of proteases and their inhibitors.
Binding Sites ; Catalytic Domain ; Endopeptidases ; Peptide Hydrolases/genetics* ; Protease Inhibitors ; Proteins

To explore whether the downregulation of protein phosphatase 2A catalytic subunit(PP2Ac)involved in the pathogenesis of mitochondria fission/fusion dynamics and functional imbalance induced by human tau accumulation. After cotransfection with mito-dsRed plasmids and pIRES-eGFP-tau40 plasmids 48 hours,the rat primary hippocampal neurons were observed with a laser scanning confocal microscope for their changes in shape and distribution of mitochondria.The expressions of mitochondria fission/fusion protein and PP2Ac and PP2Ab were detected by Western blotting.Furthermore,the shape and distribution of mitochondria of rat primary hippocampal neuron and wild type 293wt cells were assayed 48 hours after co-transfection with siPP2Ac-EGFP plasmids and mito-DsRed plasmids,and the fission/fusion dynamics of 293wt cells was captured with live cell time-lapse imaging after co-transfection with siPP2Ac plasmids and mito-Dendra2 plasmids.After transfection with siPP2Ac plasmids,the relative level of mitochondria fission/fusion protein of 293wt cells was assayed by Western blotting,and mitochondria membrane potential was detected by JC-1 staining,and the cellular viability was measured by CCK8 assay.Finally,the shape and distribution and membrane potential of mitochondria of HEK293 cells with stable transfection of htau40(293htau)were detected after co-transfection with PP2Ac and mito-dsRed plasmids. Human tau40 expression decreased distribution of mitochondria and significantly lowered PP2Ac level in primary hippocampal neuron(=4.814, =0.0086).Down-regulation of PP2Ac caused mitochondria elongation and perinuclear accumulation in primary hippocampal neuron and 293wt cells;in addition,down-regulation of PP2Ac in 293wt cells significantly increased mitochondria fusion rate(=2.857, =0.0074)and the levels of mitochondria fusion protein mitofusin(MFN)1(=6.768, =0.0025),MFN2(=3.121, =0.0035),and optic atrophy 1(=3.775, =0.0199);however,the levels of dynamin-like protein-1 and Fis1 remained unchanged.The down-regulation of PP2Ac in 293wt cells led to the significant decrease in mitochondria membrane potential(=2.300, =0.0270)and cell viability(=6.249, <0.0001).Finally,up-regulation of PP2Ac attenuated the abnormalities in the shape,distribution and function of mitochondria in the 293htau cells. Down-regulation of PP2Ac is involved in the abnormal shape and distribution of mitochondria and its dysfunction induced by human tau40 in rat primary hippocampal neurons and HEK293 cells.
Animals ; Catalytic Domain ; Down-Regulation ; HEK293 Cells ; Humans ; Mitochondria ; Protein Phosphatase 2 ; Rats ; tau Proteins

Pectin methylesterase (PME) is an important pectinase that hydrolyzes methyl esters in pectin to release methanol and reduce the degree of methylation of pectin. At present, it has broad application prospects in food processing, tea beverage, paper making and other production processes. With the in-depth study of PME, the crystal structures with different sources have been reported. Analysis of these resolved crystal structures reveals that PME belongs to the right-hand parallel β-helix structure, and its catalytic residues are two aspartic acids and a glutamine, which play the role of general acid-base, nucleophile and stable intermediate, in the catalytic process. At the same time, the substrate specificity is analyzed to understand the recognition mechanism of the substrate and active sites. This paper systematically reviews these related aspects.
Carboxylic Ester Hydrolases ; chemistry ; metabolism ; Catalytic Domain ; Crystallography ; Pectins ; metabolism ; Protein Structure, Tertiary ; Substrate Specificity

The development of next generation sequencing (NGS) has led to marked advancement of our understanding of genetic events mediating the initiation and progression of thyroid cancers. The NGS studies have confirmed the previously reported high frequency of mutually-exclusive oncogenic alterations affecting BRAF and RAS proto-oncogenes in all stages of thyroid cancer. Initially identified by traditional sequencing approaches, the NGS studies also confirmed the acquisition of alterations that inactivate tumor protein p53 (TP53) and activate phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) in advanced thyroid cancers. Novel alterations, such as those in telomerase reverse transcriptase (TERT) promoter and mating-type switching/sucrose non-fermenting (SWI/SNF) complex, are also likely to promote progression of the BRAF(V600E)-driven thyroid cancers. A number of genetically engineered mouse models (GEMM) of BRAF(V600E)-driven thyroid cancer have been developed to investigate thyroid tumorigenesis mediated by oncogenic BRAF and to explore the role of genetic alterations identified in the genomic analyses of advanced thyroid cancer to promote tumor progression. This review will discuss the various GEMMs that have been developed to investigate oncogenic BRAF(V600E)-driven thyroid cancers.
Animals ; Carcinogenesis ; Catalytic Domain ; Mice ; Mice, Transgenic ; Negotiating ; Proto-Oncogene Proteins B-raf ; Proto-Oncogenes ; Telomerase ; Thyroid Gland ; Thyroid Neoplasms

OBJECTIVE: To study the effect of hyperoxic exposure on the dynamic expression of heme oxygenase-1 (HO-1) and glutamate-L-cysteine ligase catalytic subunit (GCLC) in the lung tissue of preterm neonatal rats. METHODS: Cesarean section was performed for rats on day 21 of gestation to obtain 80 preterm rats, which were randomly divided into air group and hyperoxia group after one day of feeding. The rats in the air group were housed in room air under atmospheric pressure, and those in the hyperoxia group were placed in an atmospheric oxygen tank (oxygen concentration 85%-95%) in the same room. Eight rats each were selected from each group on days 1, 4, 7, 10, and 14, and lung tissue samples were collected. Hematoxylin and eosin staining was used to observe the pathological changes of lung tissue at different time points after air or hyperoxic exposure. Western blot and RT-qPCR were used to measure the protein and mRNA expression of HO-1 and GCLC in the lung tissue of preterm rats at different time points after air or hyperoxic exposure. RESULTS: Compared with the air group, the hyperoxia group had a significant reduction in the body weight (P<0.05). Compared with the air group, the hyperoxia group had structural disorder, widening of alveolar septa, a reduction in the number of alveoli, and simplification of the alveoli on the pathological section of lung tissue. Compared with the air group, the hyperoxia group had significantly lower relative mRNA expression of HO-1 in the lung tissue on day 7 and significantly higher expression on days 10 and 14 (P<0.05). Compared with the air group, the hyperoxia group had significantly lower mRNA expression of GCLC in the lung tissue on days 1, 4, and 7 and significantly higher expression on day 10 (P<0.05). Compared with the air group, the hyperoxia group had significantly higher protein expression of HO-1 in the lung tissue on all days, and the protein expression of GCLC had same results as HO-1, except on day 1 (P<0.05). CONCLUSIONS Hyperoxia exposure may lead to growth retardation and lung developmental retardation in preterm rats. Changes in the protein and mRNA expression of HO-1 and GCLC in the lung tissue of preterm rats may be associated with the pathogenesis of hyperoxia-induced lung injury in preterm rats.
Animals ; Animals, Newborn ; Catalytic Domain ; Cesarean Section ; Cysteine ; Female ; Glutamates ; Heme Oxygenase-1 ; Humans ; Hyperoxia ; Infant, Newborn ; Lung ; Pregnancy ; Rats ; Rats, Sprague-Dawley

PURPOSE: Amplified mesenchymal-epithelial transition factor, MET, is a receptor tyrosine kinase (RTK) that has been considered a druggable target in non-small cell lung cancer (NSCLC). Although multiple MET tyrosine kinase inhibitors (TKIs) are being actively developed for MET-driven NSCLC, the mechanisms of acquired resistance to MET-TKIs have not been well elucidated. To understand the mechanisms of resistance and establish therapeutic strategies, we developed an in vitro model using the MET-amplified NSCLC cell line EBC-1. MATERIALS AND METHODS: We established capmatinib-resistant NSCLC cell lines and identified alternative signaling pathways using 3′ mRNA sequencing and human phospho-RTK arrays. Copy number alterations were evaluated by quantitative polymerase chain reaction and cell proliferation assay; activation of RTKs and downstream effectors were compared between the parental cell line EBC-1 and the resistant cell lines. RESULTS: We found that EBC-CR1 showed an epidermal growth factor receptor (EGFR)‒dependent growth and sensitivity to afatinib, an irreversible EGFR TKI. EBC-CR2 cells that had overexpression of EGFR-MET heterodimer dramatically responded to combined capmatinib with afatinib. In addition, EBC-CR3 cells derived from EBC-CR1 cells that activated EGFR with amplified phosphoinositide-3 kinase catalytic subunit α (PIK3CA) were sensitive to combined afatinib with BYL719, a phosphoinositide 3-kinase α (PI3Kα) inhibitor. CONCLUSION: Our in vitro studies suggested that activation of EGFR signaling and/or genetic alteration of downstream effectors like PIK3CA were alternative resistance mechanisms used by capmatinib-resistant NSCLC cell lines. In addition, combined treatments with MET, EGFR, and PI3Kα inhibitors may be effective therapeutic strategies in capmatinib-resistant NSCLC patients.
Carcinoma, Non-Small-Cell Lung ; Catalytic Domain ; Cell Line ; Cell Proliferation ; Humans ; In Vitro Techniques ; Parents ; Phosphotransferases ; Polymerase Chain Reaction ; Protein-Tyrosine Kinases ; Receptor, Epidermal Growth Factor ; RNA, Messenger

BACKGROUND: Apart from its blood pressure-lowering effect by blocking the renin-angiotensin-aldosterone system, telmisartan, an angiotensin II type 1 receptor blocker (ARB), exhibits various ancillary effects including cardiovascular protective effects in vitro. Nonetheless, the protective effects of telmisartan in cerebrocardiovascular diseases are somewhat variable in large-scale clinical trials. Dysregulation of endothelial nitric oxide (NO) synthase (eNOS)-derived NO contributes to the developments of various vascular diseases. Nevertheless, the direct effects of telmisartan on endothelial functions including NO production and vessel relaxation, and its action mechanism have not been fully elucidated. Here, we investigated the mechanism by which telmisartan regulates NO production and vessel relaxation in vitro and in vivo. METHODS: We measured nitrite levels in culture medium and mouse serum, and performed inhibitor studies and western blot analyses using bovine aortic endothelial cells (BAECs) and a hyperglycemic mouse model. To assess vessel reactivity, we performed acetylcholine (ACh)-induced vessel relaxation assay on isolated rat aortas. RESULTS: Telmisartan decreased NO production in normoglycemic and hyperglycemic BAECs, which was accompanied by reduced phosphorylation of eNOS at Ser¹¹⁷⁹ (p-eNOS-Ser¹¹⁷⁹). Telmisartan increased the expression of protein phosphatase 2A catalytic subunit (PP2Ac) and co-treatment with okadaic acid completely restored telmisartan-inhibited NO production and p-eNOS-Ser¹¹⁷⁹ levels. Of the ARBs tested (including losartan and fimasartan), only telmisartan decreased NO production and p-eNOS-Ser¹¹⁷⁹ levels, and enhanced PP2Ac expression. Co-treatment with GW9662 had no effect on telmisartan-induced changes. In line with in vitro observations, telmisartan reduced serum nitrite and p-eNOS-Ser¹¹⁷⁹ levels, and increased PP2Ac expression in high fat diet-fed mice. Furthermore, telmisartan attenuated ACh-induced rat aorta relaxation. CONCLUSION: We demonstrated that telmisartan inhibited NO production and vessel relaxation at least in part by PP2A-mediated eNOS-Ser¹¹⁷⁹ dephosphorylation in a peroxisome proliferator-activated receptor γ-independent manner. These results may provide a mechanism that explains the inconsistent cerebrocardiovascular protective effects of telmisartan.
Acetylcholine ; Animals ; Aorta ; Blotting, Western ; Catalytic Domain ; Endothelial Cells ; In Vitro Techniques ; Losartan ; Mice ; Mice, Obese ; Nitric Oxide Synthase Type III ; Nitric Oxide ; Okadaic Acid ; Peroxisomes ; Phosphorylation ; Protein Phosphatase 2 ; Rats ; Receptor, Angiotensin, Type 1 ; Relaxation ; Renin-Angiotensin System ; Vascular Diseases