3 research outputs found
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 IC<sub>50</sub> 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<sup>α</sup>‑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<sup>α</sup>-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<sup>α</sup>-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<sup>α</sup>-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