In silico characterization of fructosyl peptide oxidase properties from Eupenicillium terrenum.

Fructosyl peptide oxidase (FPOX) enzyme from Eupenicillium terrenum has a high potential to be applied as a diagnostic enzyme. The aim of the present study is the characterization of FPOX from E. terrenum using different bioinformatics tools. The computational prediction of the RNA and protein secondary structures of FPOX, solubility profile in Escherichia coli, stability, domains, and functional properties were performed. In the FPOX protein, six motifs were detected. The d-amino acid oxidase motif was found as the most important motif that is a FAD-dependent oxidoreductase. The cysteines including 97, 154, 234, 280, and 360 showed a lower score than -10 that have a low possibility for participitation in the formation of the SS bond. The 56.52% of FPOX amino acids are nonpolar. Random coils are dominant in the FPOX sequence, followed by alpha-helix and extended strand. The fpox gene is capable of generating a stable RNA secondary structure (-423.90 kcal/mol) in E. coli. FPOX has a large number of hydrophobic amino acids. FPOX showed a low solubility in E. coli which has several aggregation-prone sites in its 3-D structure. According to the scores, the best mutation candidate for increasing solubility was the conversion of methionine 302 to arginine. The melting temperature of FPOX based on its amino acid sequence was 55°C to 65°C. The amounts of thermodynamic parameters for the FPOX enzyme were -137.4 kcal/mol, -3.59 kcal/(mol K), and -6.8 kcal/mol for standard folding enthalpy, heat capacity, and folding free energy, respectively. In conclusion, the in silico study of proteins can provide a valuable method for better understanding the protein properties and functions for use in our purposes.

The interplay of aryl hydrocarbon receptor/WNT/CTNNB1/Notch signaling pathways regulate amyloid beta precursor mRNA/protein expression and effected the learning and memory of mice.

The amyloid beta precursor protein (APP) plays a pathophysiological role in the development of Alzheimer's disease as well as a physiological role in neuronal growth and synaptogenesis. The aryl hydrocarbon receptor (AhR)/WNT/Catenin Beta 1 (CTNNB1)/Notch signaling pathways stamp in many functions, including development and growth of neurons. However, the regulatory role of AhR-/WNT-/CTNNB1-/Notch-induced APP expression and its influence on hippocampal-dependent learning and memory deficits is not clear. Male BALB/C mice received 6-formylindolo[3,2-b]carbazole (an AhR agonist), CH223191(an AhR antagonist), DAPT (an inhibitor of Notch signaling), and XAV-939 (a WNT pathway inhibitor) at a single dose of 100 µg/kg, 1, 5 , and 5 mg/kg of body weight, respectively, via intraperitoneal injection alone or in combination. Gene expression analyses and protein assay were performed on the 7th and 29th days. To assess the hippocampal-dependent memory, all six mice also underwent contextual fear conditioning on the 28th day after treatments. Our results showed that endogenous ligand of AhR has a regulatory effect on APP gene. Also, the interaction of AhR/WNT/CTNNB1 has a positive regulatory effect, but Notch has a negative regulatory effect on the mRNA and protein expression of APP, which have a correlation with mice's learning skills and memory.

6-Formylindolo[3,2-b]carbazole (FICZ) Enhances The Expression of Tumor Suppressor miRNAs, miR-22, miR-515-5p, and miR-124-3p in MCF-7 Cells.

OBJECTIVE: microRNAs (miRNAs) play bifunctional roles in the initiation and progression of cancer, and recent evidence has confirmed that unusual expression of miRNAs is required for the progress of breast cancer. The regulatory role of aryl hydrocarbon receptor (AhR) and its endogenous ligand, 6-formylindolo[3,2-b]carbazole (FICZ) on the expression of tumor suppressor miRNAs, miR-22, miR-515-5p and miR-124-3p, as well as their association with the estrogen receptor alpha (ERα) were the aims of this study. MATERIALS AND METHODS: In this experimental study, the expression levels of miR-22, miR-515-5p, miR-124-3p and miR-382-5p in MCF-7 cells were determined using the quantificational real time polymerase chain reaction (qRT-PCR) assay. RESULTS: Our results revealed that miR-22, miR-515-5p, and miR-124-3p expressions were significantly increased in cells transfected with ERα siRNA. Our data also showed that miR-22, miR 515-5p, and miR-124-3p expression levels were significantly increased following FICZ treatment. Here, we found that AhR/ERα cross-talk plays a critical role in the expression of miR-22, miR-515-5p and miR-124-3p in MCF-7 cells. CONCLUSION: Overall, our data demonstrated that FICZ, as an AhR agonist could induce the expression of tumor suppressor miRNAs, miR-22, miR-515-5p, and miR-124-3p; thus, FICZ might be regarded as a potential therapeutic agent for breast cancer treatment.

Aryl hydrocarbon-estrogen alpha receptor-dependent expression of miR-206, miR-27b, and miR-133a suppress cell proliferation and migration in MCF-7 cells.

The underlying functions of miR-206, miR-133a, miR-27b, and miR-21, and their link to the estrogen receptor alpha (ERα) and aryl hydrocarbon receptor (AhR) signaling pathways remain largely unexplored. In this study, we detect the expression of miR-206, miR-133a, miR-27b, and miR-21 in MCF-7 through quantificational real-time polymerase chain reaction assay along with the activation/inhibition of ERα and AhR receptors. Aside from this, cell proliferation and migration as well as AhR-dependent CYP1A1 enzyme activity were measured. Here, we found that the forced increased expression of miR-206, miR-133a, and miR-27b were closely associated with the suppression of MCF-7 cell proliferation and migration. The anti-proliferative-metastatic effect of miR-206, miR-133a, and miR-27b was probably mediated by targeting the ERα and AhR signaling pathways. Considered together, our study indicated that the overexpression of miR-206, miR-133a, and miR-27b might be potential biomarkers for prognosis and therapeutic strategies in breast cancer.

Paradoxical effect of methimazole on liver mitochondria: In vitro and in vivo.

Methimazole is the most frequently prescribed antithyroid agent. On the other hand, several cases of liver injury are attributed to this drug. The mechanism of methimazole-induced liver injury is obscure. Hepatocytes mitochondria seem to be a target for methimazole cytotoxicity. Current investigation aimed to evaluate the effects of methimazole on the hepatocytes mitochondria in different experimental models. In the in vivo model, methimazole (100, 200 and 400mg/kg, i.p) was administered to mice and liver mitochondria were isolated and assessed. In the in vitro experiments, intact isolated liver mitochondria were incubated with increasing methimazole concentrations (10µM-100mM). It was found that methimazole decreased liver mitochondrial ATP and glutathione, increased mitochondrial swelling, lipid peroxidation and reactive oxygen species (ROS), and collapsed mitochondrial membrane potential when administered to mice. Paradoxically, methimazole not only caused no significant injury toward isolated liver mitochondria in vitro but improved mitochondrial function and protected this organelle. The differences between two investigated models in the current study might be associated with drug bioactivation and reactive metabolites formation. These findings suggest mitochondrial dysfunction as a mechanism for methimazole-induced liver injury. Moreover, methimazole seems to be a novel mitochondrial protecting agent in vitro.