Multifunctional Peptides from Spanish Dry-Cured Pork Ham: Endothelial Responses and Molecular Modeling Studies

Food peptides contain a very wide range of diversified structures, which explains their diverse range of functional activities. Proatherogenic endothelium is related to vasoconstriction, inflammation, and oxidative stress. In this line, four synthetic bioactive peptides from dry-cured pork ham, previously identified according to their Angiotensin I Converting Enzyme (ACE) inhibitory capacity and high bioavailability, were tested. Among them, KPVAAP displayed an estimated IC50 of 59.22 µM for human ACE inhibition, and docking simulations demonstrated the consistency of the noncompetitive binding with the protein. The addition of synthetic peptides to human endothelial cells significantly prevents the expression of genes related to endothelial dysfunction and inflammation (eNOS, ICAM-1, VCAM-1, IL-6) and lowers NF-κB activation (all p < 0.05). In silico dockings showed that the four bioactive peptides interact with the regulatory subunit NEMO of the NF-κB transcription factor at the same site as other characterized inhibitors (CC2-LZ region). This is the first study linking experimental and computational approaches that shows NF-κB to be the target of biopeptides of food origin. These multifunctional peptides from dry-cured pork ham make them good candidates for further research into their therapeutic or preventive use to attenuate the inflammatory atherosclerotic process.


Cell Viability
The tetrazolium assay (MTT) was used to estimate the cell viability in the presence of synthetic peptides (up to 600 µM) and 300 µM H2O2. Exponentially growing cells were seeded into a 96-well plate at a density of 1 × 10 4 cells/well. After 48 h of treatment, cells were incubated with MTT solution at a final concentration of 1 mg/mL for 4 h at 37 °C. The formazan crystals formed in the intact cells were dissolved in 200 µL dimethyl sulfoxide (DMSO) for 30 min and the absorbance was measured in a microplate reader at 570 nm and 620 nm of reference. Results were calculated as cell viability (%) = average optic density (OD) of wells/average OD of control wells.

Western Blot Analysis
Protease inhibition cocktail (P8340 Sigma-Aldrich Chemical Co., Saint Louis, MO, USA), and phosphatase inhibitors (NaF and Na3VO4) were added to the cell protein extracts. The homogenates were centrifuged (13,000× g, 20 min, 4 °C) and the protein concentrations in the supernatants were determined by the BioRad Protein Assay (BioRad, Hercules, CA, USA) using BSA as standard (Sigma-Aldrich Chemical Co., Saint Louis, USA). Protein extracts (10 µg) were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene difluoride (PVDF) membranes using the Trans-Blot ® Turbo™ Transfer System (Bio-Rad, Hercules, CA, USA), and then identified by immunoblot analysis. Rabbit anti-ICAM-1 and VCAM-1 at a dilution of 1:1000 (Santa Cruz Biotechnology, Texas, TX, USA) were used. Mouse antiactin (Sigma-Aldrich Chemical Co., Saint Louis, MO, USA) at a dilution of 1:5000 was used for reference.

Protein Carbonylation with Oxyblot
Protein carbonyl content is the most general and well-used biomarker of severe oxidative protein damage [1]. Cells were treated with synthetic peptides for 16 h before adding 300 µM H2O2 and leaving for additional 30 min. Protein carbonyl groups levels were determined by immunoblotting using the Oxyblot Protein Oxidation Detection Kit (Millipore Merck, Temecula, CA, USA) according to the manufacturer's protocol. PVDF membranes were blocked with 1% BSA in PBS-0.05% Tween 20 (v/v) (PBS-T) for 1 h at room temperature. The blots were incubated overnight at 20 °C with the polyclonal antibody. Subsequently, blots were washed (3-5 times) with PBS-T and incubated with goat anti-rabbit IgG/HRP conjugate (1∶300 dilution in PBS/BSA) (Sigma-Aldrich Chemical Co., Saint Louis, MO, USA) for 1 h at room temperature. An enhanced chemiluminescence kit (Pierce ECL detection kit, ThermoFisher Scientific Inc., Rockford, IL, USA) was used for detection. To exclude nonspecific reactions, the 2,4-dinitrophenylhydrazine (DNPH) incubation step was omitted in a control experiment. In this condition, no signal was detected in the PVDF membranes (data not shown). The emitted chemiluminescent signals were detected using a digital imaging system (ImageQuant LAS500, Thermo FisherScientific Inc., Rockford, IL, USA) after automatic exposition time. Quantitation of the densitometry plots for chemiluminescence detection was carried out using ImageJ software for the analysis of 1D gels. Peaks were drawn from the distinct western-blot bands, the area were calculated and normalized to control conditions.

Supplementary Figure Legends
Table S1. Apoptotic and necrosis rate measured by FACS in oxidative conditions. AnV: Annexin V; PI: Propidium Iodide; BP: Bioactive peptide; NS: Nonsignificant. Data from three independent experiments are expressed as mean ± SD. H2O2 stimulated cells are compared to control conditions, and preincubations with 300 µM synthetic bioactive peptides are compared to 300 µM H2O2 alone.

Apoptosis (AnV+/PI-)
Necrosis (     The results showed that the MFI in TNF-α-treated cells for ICAM-1 (FL2) was significantly higher than in control conditions. VCAM-1 protein expression (FL·3) was undetected even in the presence of 100 ng/mL TNF-α. D) Western blot analysis. Band intensity was used to measure differences in protein expression of ICAM-1 in the cell culture after the 100 ng/mL TNF-α treatment. TNF-α induced the expression of 82-85 kDa protein, corresponding to ICAM-1. Actin was used as a loading control. The asterisk * indicates a statistically significant difference compared to stimulated cells (p < 0.05).  Supplementary Figure S6. Oxidized protein levels after treatment with 300 µM synthetic peptides and then 300 µM H2O2. A) Western blot. Band intensity was used to measure differences in protein carbonylation levels in the cell culture after H2O2 treatment. B) Representative blots. Data represent averaged values of triplicate measurements. # indicates a statistically significant difference from unstimulated cells (p < 0.05). The asterisks (* or **) indicate a statistically significant difference compared to stimulated cells (p < 0.05 or 0.01, respectively).