scholarly journals Dimethylaminomicheliolide (DMAMCL) Inhibits Cell Proliferation and Increases Apoptosis and Efficacy of Gemcitabine via Annexin A2 in Pancreatic Adenocarcinoma

2021 ◽  
Author(s):  
Kaihui Liu ◽  
Jianshuang Guo ◽  
Ning Liu ◽  
Jiyan Wang ◽  
Mukuo Wang ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Molecular Docking ◽  
Pancreatic Adenocarcinoma ◽  
Pancreatic Cancer Cell ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Chemical Proteomics ◽  
Pancreatic Cells ◽  
Proteomics Approach ◽  
Direct Binding

Abstract Background: Pancreatic adenocarcinoma is one of the highest malignant tumors in digestive tract with extremely poor survival rate. Dimethylaminomicheliolide (DMAMCL) is a clinical developing anti-cancer agent, however, little is known regarding its effects in pancreatic cancer, and the mechanisms of DMAMCL are still not fully understood.Methods: This study evaluated DMAMCL on three pancreatic cancer cell lines by cell viability assay, colony formation assay, and apoptosis assay. To identify the direct binding target of DMAMCL in pancreatic cells, a chemical proteomics approach, molecular docking and site-directed mutagenesis were performed.Results: DMAMCL inhibits proliferation and promotes apoptosis of pancreatic cancer cells. Interestingly, using a chemical proteomics approach, we identify ANXA2 as a direct binding target of DMAMCL in pancreatic cells. Molecular docking and site-directed mutagenesis confirm that Cys132 (C132) of ANXA2 is the binding site for DMAMCL. The knockdown of ANXA2 largely decreases the inhibition activity of DMAMCL, indicating that the effect of DMAMCL is mainly mediated by ANXA2 in pancreatic cancer. In addition, the combination regimen of gemcitabine and DMAMCL exhibits synergistic effect on pancreatic cancer cell lines at both proliferation and pro-apoptosis level. Conclusions: Thus, our findings elucidate the mechanisms of DMAMCL and may provide a potential strategy to enhancing the efficacy of gemcitabine in pancreatic adenocarcinoma.

scholarly journals Conformational communication mediates the reset step in t6A biosynthesis

2019 ◽  
Vol 47 (12) ◽  
pp. 6551-6567 ◽  
Author(s):  
Amit Luthra ◽  
Naduni Paranagama ◽  
William Swinehart ◽  
Susan Bayooz ◽  
Phuc Phan ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Molecular Docking ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Translational Fidelity ◽  
Substrate Analog ◽  
Binding Cavity ◽  
Saxs Analysis

Abstract The universally conserved N6-threonylcarbamoyladenosine (t6A) modification of tRNA is essential for translational fidelity. In bacteria, t6A biosynthesis starts with the TsaC/TsaC2-catalyzed synthesis of the intermediate threonylcarbamoyl adenylate (TC–AMP), followed by transfer of the threonylcarbamoyl (TC) moiety to adenine-37 of tRNA by the TC-transfer complex comprised of TsaB, TsaD and TsaE subunits and possessing an ATPase activity required for multi-turnover of the t6A cycle. We report a 2.5-Å crystal structure of the T. maritima TC-transfer complex (TmTsaB2D2E2) bound to Mg2+-ATP in the ATPase site, and substrate analog carboxy-AMP in the TC-transfer site. Site directed mutagenesis results show that residues in the conserved Switch I and Switch II motifs of TsaE mediate the ATP hydrolysis-driven reactivation/reset step of the t6A cycle. Further, SAXS analysis of the TmTsaB2D2-tRNA complex in solution reveals bound tRNA lodged in the TsaE binding cavity, confirming our previous biochemical data. Based on the crystal structure and molecular docking of TC–AMP and adenine-37 in the TC-transfer site, we propose a model for the mechanism of TC transfer by this universal biosynthetic system.

scholarly journals Characterization of iron-binding motifs in Candida albicans high-affinity iron permease CaFtr1p by site-directed mutagenesis

2002 ◽  
Vol 368 (2) ◽  
pp. 641-647 ◽  
Author(s):  
Hao-Ming FANG ◽  
Yue WANG
Keyword(s):  
Amino Acids ◽  
Candida Albicans ◽  
Iron Uptake ◽  
Iron Transport ◽  
Site Directed Mutagenesis ◽  
Detectable Effect ◽  
Directed Mutagenesis ◽  
Binding Motif ◽  
Iron Binding ◽  
Direct Binding

A peptide motif Glu-Xaa-Xaa-Glu has been implicated in direct binding of ferric iron in several proteins involved in iron transport, sensing or storage. However, it is not known whether the motif alone is sufficient for iron binding and whether functional replacement of the conserved residues by other amino acids with similar properties is possible. We previously identified a Candida albicans iron permease, CaFtr1p, which contains five Glu-Xaa-Xaa-Glu motifs [Ramanan and Wang (2000) Science 288, 1062—1065]. In this study, we investigated the role of each of these motifs in iron uptake by site-directed mutagenesis. Substitution of Ala for any one of the two Glu residues in Glu-Gly-Leu-Glu158—161 abolished iron-uptake activity, while the same substitution in any of the other four motifs had little effect, indicating that only the motif at position 158—161 is required for iron transport. We then evaluated the importance of each of the residues within and immediately adjacent to this motif in iron uptake. The permease remained active when any one of the Glu residues was replaced by Asp, while it became inactive when both were replaced. We also found that the amino acid immediately in front of Glu-Gly-Leu-Glu158—161 must be either Arg or Lys. In addition, substitution of any of the two residues in the middle with several structurally distinct amino acids had no detectable effect on iron uptake. Here we propose to extend the iron-binding motif to Arg/Lys-Glu/Asp-Xaa-Xaa-Glu or Arg/Lys-Glu-Xaa-Xaa-Glu/Asp, which may serve as a guide for the identification of potential iron-binding sites in proteins.

scholarly journals Two Key Amino Acids Variant of α-l-arabinofuranosidase from Bacillus subtilis Str. 168 with Altered Activity for Selective Conversion Ginsenoside Rc to Rd

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1733
Author(s):  
Ru Zhang ◽  
Shi Quan Tan ◽  
Bian Ling Zhang ◽  
Zi Yu Guo ◽  
Liang Yu Tian ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Catalytic Mechanism ◽  
Specific Activity ◽  
Homology Model ◽  
Good Alternative ◽  
Glycoside Hydrolases ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Efficient Expression ◽  
Ginsenoside Rc

α-l-arabinofuranosidase is a subfamily of glycosidases involved in the hydrolysis of l-arabinofuranosidic bonds, especially in those of the terminal non-reducing arabinofuranosyl residues of glycosides, from which efficient glycoside hydrolases can be screened for the transformation of ginsenosides. In this study, the ginsenoside Rc-hydrolyzing α-l-arabinofuranosidase gene, BsAbfA, was cloned from Bacilus subtilis, and its codons were optimized for efficient expression in E. coli BL21 (DE3). The recombinant protein BsAbfA fused with an N-terminal His-tag was overexpressed and purified, and then subjected to enzymatic characterization. Site-directed mutagenesis of BsAbfA was performed to verify the catalytic site, and the molecular mechanism of BsAbfA catalyzing ginsenoside Rc was analyzed by molecular docking, using the homology model of sequence alignment with other β-glycosidases. The results show that the purified BsAbfA had a specific activity of 32.6 U/mg. Under optimal conditions (pH 5, 40 °C), the kinetic parameters Km of BsAbfA for pNP-α-Araf and ginsenoside Rc were 0.6 mM and 0.4 mM, while the Kcat/Km were 181.5 s−1 mM−1 and 197.8 s−1 mM−1, respectively. More than 90% of ginsenoside Rc could be transformed by 12 U/mL purified BsAbfA at 40 °C and pH 5 in 24 h. The results of molecular docking and site-directed mutagenesis suggested that the E173 and E292 variants for BsAbfA are important in recognizing ginsenoside Rc effectively, and to make it enter the active pocket to hydrolyze the outer arabinofuranosyl moieties at C20 position. These remarkable properties and the catalytic mechanism of BsAbfA provide a good alternative for the effective biotransformation of the major ginsenoside Rc into Rd.

Structural and biochemical analyses of theStreptococcus pneumoniaeL,D-carboxypeptidase DacB

2015 ◽  
Vol 71 (2) ◽  
pp. 283-292
Author(s):  
Juan Zhang ◽  
Yi-Hu Yang ◽  
Yong-Liang Jiang ◽  
Cong-Zhao Zhou ◽  
Yuxing Chen
Keyword(s):  
Crystal Structure ◽  
Molecular Docking ◽  
Catalytic Mechanism ◽  
Substrate Recognition ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Binding Properties ◽  
Biochemical Analyses ◽  
Key Residues ◽  
Structural Insights

The L,D-carboxypeptidase DacB plays a key role in the remodelling ofStreptococcus pneumoniaepeptidoglycan during cell division. In order to decipher its substrate-binding properties and catalytic mechanism, the 1.71 Å resolution crystal structure of DacB fromS. pneumoniaeTIGR4 is reported. Structural analyses in combination with comparisons with the recently reported structures of DacB fromS. pneumoniaeD39 and R6 clearly demonstrate that DacB adopts a zinc-dependent carboxypeptidase fold and belongs to the metallopeptidase M15B subfamily. In addition, enzymatic activity assays further confirm that DacB indeed acts as an L,D-carboxypeptidase towards the tetrapeptide L-Ala-D-iGln-L-Lys-D-Ala of the peptidoglycan stem, withKmandkcatvalues of 2.84 ± 0.37 mMand 91.49 ± 0.05 s−1, respectively. Subsequent molecular docking and site-directed mutagenesis enable the assignment of the key residues that bind to the tetrapeptide. Altogether, these findings provide structural insights into substrate recognition in the metallopeptidase M15B subfamily.

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