Protein dynamics are often invoked in explanations of enzyme catalysis, but their design has proven elusive. Here we track the role of dynamics in evolution, starting from the evolvable and thermostable ancestral protein AncHLD-RLuc which catalyses both dehalogenase and luciferase reactions. Insertion-deletion (InDel) backbone mutagenesis of AncHLD-RLuc challenged the scaffold dynamics. Screening for both activities reveals InDel mutations localized in three distinct regions that lead to altered protein dynamics (based on crystallographic B-factors, hydrogen exchange, and molecular dynamics simulations). An anisotropic network model highlights the importance of the conformational flexibility of a loop-helix fragment of Renilla luciferases for ligand binding. Transplantation of this dynamic fragment leads to lower product inhibition and highly stable glow-type bioluminescence. The success of our approach suggests that a strategy comprising (i) constructing a stable and evolvable template, (ii) mapping functional regions by backbone mutagenesis, and (iii) transplantation of dynamic features, can lead to functionally innovative proteins.

• MeSH
• buňky NIH 3T3 MeSH
• katalýza MeSH
• kinetika MeSH
• konformace proteinů MeSH
• luciferasy renil chemie   genetika   metabolismus   MeSH
• luciferasy chemie   genetika   metabolismus   MeSH
• mutace MeSH
• mutageneze MeSH
• myši MeSH
• proteinové inženýrství * MeSH
• savci MeSH
• simulace molekulární dynamiky * MeSH
• stabilita enzymů MeSH
• teplota MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Hepatocyte tumor cell lines lack the expression or induction properties of major cytochrome P450 (CYP) enzymes compared to primary human hepatocytes. The Ras/Raf/MEK/ERK signaling cascade contributes to hepatocarcinogenesis, dedifferentiation and loss of hepatocyte drug metabolism in hepatocyte tumors. In the present study, we examined whether MEK1/2 inhibitors can restore the expression of CYP genes in hepatocarcinoma HepG2 cells. We found that U0126, a prototype dual MEK1/2 inhibitor, is a potent inducer of CYP3A4, CYP3A5 and CYP3A7 mRNA expression (>100-fold) in HepG2 cells and CYP3A4 mRNA expression in primary human hepatocytes. This U0126-mediated induction is sensitive to the transcriptional inhibitor actinomycin D and was not detected for CYP2B6 or MDR1 mRNA expression. In gene reporter assays, U0126 activates a CYP3A4 promoter luciferase reporter construct containing PXR response elements (PXREs), but not a construct containing mutated PXREs. Based on a ligand binding assay and the examination of a PXR mutant expressing an obstructed ligand binding pocket, we found that U0126 is a ligand of PXR. We also found that U0126 up-regulates the mRNA expression of the nuclear receptors HNF4α, CAR, VDR and PXR but abolishes small heterodimer partner (SHP) corepressor expression in HepG2 cells. The MEK1/2 inhibitors PD0325901 and PD184352, as well as dominant-negative MEK1 expression, also down-regulate SHP mRNA expression. In contrast, dominant-negative MEK1 expression does not significantly induce CYP3A4 gene in HepG2 cells. In conclusion, we found that U0126 is an atypical PXR ligand that via direct (binding and activation of PXR) and indirect (SHP dowregulation) mechanisms selectively restores CYP3A genes in HepG2 cells.

• MeSH
• buňky Hep G2 MeSH
• butadieny farmakologie   MeSH
• cytochrom P-450 CYP3A genetika   MeSH
• hepatocyty účinky léků   enzymologie   MeSH
• inhibitory enzymů farmakologie   MeSH
• izoenzymy MeSH
• lidé MeSH
• ligandy MeSH
• luciferasy renil genetika   MeSH
• MAP kinasa-kinasa 1 antagonisté a inhibitory   MeSH
• MAP kinasa-kinasa 2 antagonisté a inhibitory   MeSH
• MAP kinasový signální systém účinky léků   MeSH
• nitrily farmakologie   MeSH
• plazmidy MeSH
• primární buněčná kultura MeSH
• promotorové oblasti (genetika) MeSH
• steroidní receptory metabolismus   MeSH
• transfekce MeSH
• upregulace MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH