Project description:In this study, three synthetic zinc-chelating peptides (ZCPs) derived from sea cucumber hydrolysates with limited or none of the common metal-chelating amino-acid residues were analyzed by flame atomic absorption spectroscopy, circular dichroism spectroscopy, size exclusion chromatography, zeta-potential, Fourier transform infrared spectroscopy, Raman spectroscopy and nuclear magnetic resonance spectroscopy. The amount of zinc bound to the ZCPs reached maximum values with ZCP:zinc at 1:1, and it was not further increased by additional zinc presence. The secondary structures of ZCPs were slightly altered, whereas no formation of multimers was observed. Furthermore, zinc increased the zeta-potential value by neutralizing the negatively charged residues. Only free carboxyl in C-terminus of ZCPs was identified as the primary binding site of zinc. These results provide the theoretical foundation to understand the mechanism of zinc chelation by peptides.
Project description:Ethyl pyruvate (EP), a simple aliphatic ester of pyruvic acid, has been shown to have antiinflammatory effects and to confer protective effects in various pathological conditions. Recently, a number of studies have reported EP inhibits high mobility group box 1 (HMGB1) secretion and suggest this might contribute to its antiinflammatory effect. Since EP is used in a calcium-containing balanced salt solution (Ringer solution), we wondered if EP directly chelates Ca(2+) and if it is related to the EP-mediated suppression of HMGB1 release. Calcium imaging assays revealed that EP significantly and dose-dependently suppressed high K(+)-induced transient [Ca(2+)]i surges in primary cortical neurons and, similarly, fluorometric assays showed that EP directly scavenges Ca(2+) as the peak of fluorescence emission intensities of Mag-Fura-2 (a low-affinity Ca(2+) indicator) was shifted in the presence of EP at concentrations of ≥7 mmol/L. Furthermore, EP markedly suppressed the A23187-induced intracellular Ca(2+) surge in BV2 cells and, under this condition, A23187-induced activations of Ca(2+)-mediated kinases (protein kinase Cα and calcium/calmodulin-dependent protein kinase IV), HMGB1 phosphorylation and subsequent secretion of HMGB1 also were suppressed. (A23187 is a calcium ionophore and BV2 cells are a microglia cell line.) Moreover, the above-mentioned EP-mediated effects were obtained independent of cell death or survival, which suggests that they are direct effects of EP. Together, these results indicate that EP directly chelates Ca(2+), and that it is, at least in part, responsible for the suppression of HMGB1 release by EP.
Project description:Breast cancer is the most common type of cancer in women globally. The overexpressed proteins, including EGFR, PI3K, AKT1, and CDK4, have a role in the growth of breast cancer cells. The 3D peptide structure of sea cucumber Cucumaria frondosa was modeled and then docked with EGFR, PI3K, AKT1, and CDK4 proteins using AutoDock Vina software. The docking result, which has the best binding affinity value, is continued with molecular dynamics simulation. The docking results showed that all peptides bind to the active sites of the four proteins. WPPNYQW and YDWRF peptides bind to proteins with lower binding affinity values than positive controls. The four proteins were in a stable state when complexed with the WPPNYQW peptide, which was seen from the RMSD and RMSF value. PI3K-YDWRF and AKT1-YDWRF complexes are stable, characterized by high RMSD values and increased volatility in several amino acids. WPPNYQW peptide has high potential as an antibreast cancer agent because it binds to the active sites of the four proteins with low binding affinity values and stable interactions. Meanwhile, the YDWRF peptide interacts with the four proteins with low binding affinity values, but the interaction is only stable on PI3K and AKT1 proteins.
Project description:We presented a genome-wide characterization for H3K9 acetylation (H3K9ac) binding regions in normal temperature and heat-stress conditions via ChIP-seq. The results revealed H3K9ac was an extensive epigenetic modulation in A. japonicus. We further identified differentially acetylated regions (DARs) under heat stress.
Project description:Protein hydrolysates from sea cucumber (Apostichopus japonicus) gonads are rich in active materials with remarkable angiotensin-converting enzyme (ACE) inhibitory activity. Alcalase was used to hydrolyze sea cucumber gonads, and the hydrolysate was separated by the ultrafiltration membrane to produce a low-molecular-weight peptide component (less than 3 kDa) with good ACE inhibitory activity. The peptide component (less than 3 kDa) was isolated and purified using a combination method of ACE gel affinity chromatography and reverse high-performance liquid chromatography. The purified fractions were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the resulting products were filtered using structure-based virtual screening (SBVS) to obtain 20 peptides. Of those, three noncompetitive inhibitory peptides (DDQIHIF with an IC50 value of 333.5 μmol·L-1, HDWWKER with an IC50 value of 583.6 μmol·L-1, and THDWWKER with an IC50 value of 1291.8 μmol·L-1) were further investigated based on their favorable pharmacochemical properties and ACE inhibitory activity. Molecular docking studies indicated that the three peptides were entirely enclosed within the ACE protein cavity, improving the overall stability of the complex through interaction forces with the ACE active site. The total free binding energies (ΔGtotal) for DDQIHIF, HDWWKER, and THDWWKER were -21.9 Kcal·mol-1, -71.6 Kcal·mol-1, and -69.1 Kcal·mol-1, respectively. Furthermore, a short-term assay of antihypertensive activity in spontaneously hypertensive rats (SHRs) revealed that HDWWKER could significantly decrease the systolic blood pressure (SBP) of SHRs after intravenous administration. The results showed that based on the better antihypertensive activity of the peptide in SHRs, the feasibility of targeted affinity purification and computer-aided drug discovery (CADD) for the efficient screening and preparation of ACE inhibitory peptide was verified, which provided a new idea of modern drug development method for clinical use.
Project description:Heat stress (HS) is an important factor for the survival of the marine organism Apostichopus japonicus. Lysine acetylation is a pivotal post-translational modification that modulates diverse physiological processes including heat shock response (HSR). In this study, 4028 lysine acetylation sites in 1439 proteins were identified in A. japonicus by acetylproteome sequencing. A total of 13 motifs were characterized around the acetylated lysine sites. Gene Ontology analysis showed that major acetylated protein groups were involved in "oxidation-reduction process", "ribosome", and "protein binding" terms. Compared to the control group, the acetylation quantitation of 25 and 41 lysine sites changed after 6 and 48 h HS. Notably, lysine acetyltransferase CREB-binding protein (CBP) was identified to have differential acetylation quantitation at multiple lysine sites under HS. Various chaperones, such as caseinolytic peptidase B protein homolog (CLBP), T-complex protein 1 (TCP1), and cyclophilin A (CYP1), showed differential acetylation quantitation after 48 h HS. Additionally, many translation-associated proteins, such as ribosomal proteins, translation initiation factor (IF), and elongation factors (EFs), had differential acetylation quantitation under HS. These proteins represented specific interaction networks. Collectively, our results offer novel insight into the complex HSR in A. japonicus and provide a resource for further mechanistic studies examining the regulation of protein function by lysine acetylation.
Project description:Celiac disease is an autoimmune disease triggered by oral ingestion of gluten, with certain gluten residues resistant to digestive tract enzymes. Within the duodenum, the remaining peptides incite immunogenic responses, including the generation of autoantibodies and inflammation, leading to irreversible damage. Our previous exploration unveiled a glutenase called Bga1903 derived from the Gram-negative bacterium Burkholderia gladioli. The cleavage pattern of Bga1903 indicates its moderate ability to mitigate the toxicity of pro-immunogenic peptides. The crystal structure of Bga1903, along with the identification of subsites within its active site, was determined. To improve its substrate specificity toward prevalent motifs like QPQ within gluten peptides, the active site of Bga1903 underwent site-directed mutagenesis according to structural insights and enzymatic kinetics. Among the double-site mutants, E380Q/S387L exhibits an approximately 34-fold increase in its specificity constant toward the QPQ sequence, favoring glutamines at the P1 and P3 positions compared to the wild type. The increased specificity of E380Q/S387L not only enhances its ability to break down pro-immunogenic peptides but also positions this enzyme variant as a promising candidate for oral therapy for celiac disease.