Number of Hydroxyl Groups on the B‑Ring of Flavonoids Affects Their Antioxidant Activity and Interaction with Phorbol Ester Binding Site of PKCδ C1B Domain: In Vitro and in Silico Studies

Although flavonoids have been reported for their benefits and nutraceutical potential use, the importance of their structure on their beneficial effects, especially on signal transduction mechanisms, has not been well clarified. In this study, three flavonoids, pinocembrin, naringenin, and eriodictyol, were chosen to determine the effect of hydroxyl groups on the B-ring of flavonoid structure on their antioxidant activity. In vitro assays, including DPPH scavenging activity, ROS quantification by flow cytometer, and proteins immunoblotting, and in silico analysis by molecular docking between the flavonoids and C1B domain of PKCδ phorbol ester binding site were both used to complete this study. Eriodictyol (10 μM), containing two hydroxyl groups on the B-ring, exhibited significantly higher (<i>p</i> < 0.05) antioxidant activity than pinocembrin and naringenin. The IC₅₀ values of eriodictyol, naringenin, and pinocembrin were 17.4 ± 0.40, 30.2 ± 0.61, and 44.9 ± 0.57 μM, respectively. In addition, eriodictyol at 10 μM remarkably inhibited the phosphorylation of PKCδ at 63.4% compared with PMA-activated RAW264.7, whereas pinocembrin and naringenin performed inhibition activity at 76.8 and 72.6%, respectively. According to the molecular docking analysis, pinocembrin, naringenin, and eriodictyol showed -CDOCKER_energy values of 15.22, 16.95, and 21.49, respectively, reflecting that eriodictyol could bind with the binding site better than the other two flavonoids. Interestingly, eriodictyol had a remarkably different pose to bind with the kinase as a result of the two hydroxyl groups on its B-ring, which consequently contributed to greater antioxidant activity over pinocembrin and naringenin

Improved N^α‑Acetylated Peptide Enrichment Following Dimethyl Labeling and SCX

Protein N-terminal acetylation is one of the most common modifications occurring co- and post-translationally on either eukaryote or prokaryote proteins. However, compared to other protein modifications, the physiological role of protein N-terminal acetylation is relatively unclear. To explore the biological functions of protein N-terminal acetylation, a robust and large-scale method for qualitative and quantitative analysis of this modification is required. Enrichment of N^α-acetylated peptides or depletion of the free N-terminal and internal tryptic peptides prior to analysis by mass spectrometry are necessary based on current technologies. This study demonstrated a simple strong cation exchange (SCX) fractionation method to selectively enrich N^α-acetylated tryptic peptides via dimethyl labeling without the need for tedious protective labeling and depleting procedures. This method was introduced for the comprehensive analysis of N-terminal acetylated proteins from HepG2 cells. Several hundred N-terminal acetylation sites were readily identified in a single SCX flow-through fraction. Moreover, the N^α-acetylated peptides of some protein isoforms were simultaneously observed in the SCX flow-through fraction, which indicated that this approach can be utilized to discriminate protein isoforms with very similar full sequences but different N-terminal sequences, such as β-actin/γ-actin, ERK1/ERK2, α-centractin/β-centractin, and ADP/ATP translocase 2 and 3. Compared to other methods, this method is relatively simple and can be directly implemented in a two-dimensional separation (SCX-RP)-mass spectrometry scheme for quantitative N-terminal proteomics using stable-isotope dimethyl labeling

Proteomic Study Reveals a Co-occurrence of Gallic Acid-Induced Apoptosis and Glycolysis in B16F10 Melanoma Cells

Gallic acid (GA) has long been associated with a wide range of biological activities. In this study, its antitumor effect against B16F10 melanoma cells was demonstrated by MTT assay, cell migration assay, wound-healing assay, and flow cytometric analysis. GA with a concentration >200 μM shows apoptotic activity toward B16F10 cells. According to Western blotting data, overexpressions of cleaved forms of caspase-9, caspase-3, and PARP-1 and pro-apoptotic Bax and Bad, accompanied by underexpressed anti-apoptotic Bcl-2 and Bcl-xL indicate that GA induces B16F10 cell apoptosis via mitochondrial pathway. The 2-DE based comparative proteomics was further employed in B16F10 cells with and without GA treatment for a large-scale protein expression profiling. A total of 41 differential protein spots were quantified, and their identities were characterized using LC-MS/MS analysis and database matching. In addition to some regulated proteins that were associated with apoptosis, interestingly, some identified proteins involved in glycolysis such as glucokinase, α-enolase, aldolase, pyruvate kinase, and GAPDH were simultaneously up-regulated, which reveals that the GA-induced cellular apoptosis in B16 melanoma cells is associated with metabolic glycolysis