Lupin Peptide T9 (GQEQSHQDEGVIVR) Modulates the Mutant PCSK9D374Y Pathway: in vitro Characterization of its Dual Hypocholesterolemic Behavior

GQEQSHQDEGVIVR (T9) is a peptide originated by the tryptic digestion of lupin β-conglutin that is absorbed in human intestinal Caco-2 cells. A previous study has shown that T9 impairs the protein–protein interaction between mutant D374Y Proprotein Convertase Subtilisin/Kexin 9 (PCSK9D374Y) and the low-density lipoprotein receptor (LDLR), thus exerting a hypocholesterolemic effect. Moreover, a bioinformatic study predicting that T9 may potentially act as an inhibitor of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCoAR), has suggested a complementary cholesterol-lowering activity. The present study demonstrates that T9 inhibits in vitro the HMGCoAR functionality with an IC50 value of 99.5 ± 0.56 µM. Through the inhibition of either HMGCoAR or PCSK9D374Y activities, T9 enhances the LDLR protein levels leading to an improved ability of HepG2 cells transfected with the mutant PCSK9D374Y-FLAG plasmid to uptake extracellular LDL with a final cholesterol-lowering effect. In addition, T9 modulates the PCSK9D374Y signaling pathway in transfected HepG2 cells leading to a decrease of PCSK9D374Y and HNF-1α protein levels. All these results indicate that the hypocholesterolemic effects of T9 are due to a dual mechanism of action involving either the modulation of the PCSK9D374Y or LDLR pathways. This may represent an added value from a therapeutic point of view.


HMGCoAR Activity Assay
The assay buffer, NADPH, substrate solution, and HMGCoAR were provided in the HMGCoAR Assay Kit (Sigma). The experiments were carried out following the manufacturer's instructions and conditions, which were reported in the Supplementary materials. Briefly, peptide T9 was tested in a concentration range from 10 −5 to 10 −3 M or vehicle (C), at 37 °C. The absorbance at time 0 and 10 min were recorded. The HMGCoAR-dependent oxidation of NADPH and the inhibition properties of lupin peptides were measured by the absorbance reduction at 340 nm read by a microplate reader Synergy H1 from Biotek.

Cell Culture Conditions and Transfection
HepG2 cell line was bought from ATCC (HB-8065, ATCC from LGC Standards, Milan, Italy) and was cultured following the conditions previously described [4]. Detailed information is reported in the Supplementary materials. A total of 3 × 10 4 HepG2 cells/well were seeded in 96-well plates, respectively. The following day, cells at 70-90% of confluence were transfected with the mixture containing 1.0 µ g pcDNA3+PCSK9 D374Y -FLAG plasmid and 2.0 μL TurboFect Transfection Reagent in 100 µ L of serum-free DMEM for 48 h. After 24 h, transfected HepG2 cells were treated with peptide T9 (100 µ M) and incubated for 24 h at 37 °C under 5% CO2 atmosphere.

Western Blot Analysis
Transfected HepG2 cells (1.5 × 10 5 cells/well) were treated with 100 μM T9 for 24 h. After each treatment, cells were collected and lysated in 30.0 µ L ice-cold lysis buffer containing: RIPA buffer + inhibitor cocktail + 1:100 PMSF + 1:100 Na-orthovanadate. After centrifugation at 16,060 g for 15 min at 4 °C, the supernatant was recovered and total proteins were quantified by the Bradford method. A total of 50.0 μg of total proteins were loaded on a pre-cast 7.5% Sodium Dodecyl Sulphate-Polyacrylamide (SDS-PAGE) gel at 130 V for 45 min. Subsequently, the proteins were transferred to a nitrocellulose membrane (Mini nitrocellulose Transfer Packs,) using a Trans-Blot Turbo at 1.3 A, 25 V for 7 min. Target proteins, on milk blocked membrane, were detected by primary antibodies as follows: rabbit anti-SREBP-2, rabbit anti-LDLR, anti-HMGCoAR, anti-HNF1-α, anti-FLAG (for detecting PCSK9 D374Y ), and anti-β-actin. Secondary antibodies conjugated with HRP and a chemiluminescent reagent were used to visualize target proteins and their signal was quantified using the Image Lab Software (Biorad). The internal control β-actin was used to normalize loading variations.

In Cell-Western
Transfected HepG2 cells were treated with or w/o 100 µ M T9 and vehicle (H2O) for 2 h at 37 °C under 5% CO2 atmosphere. Treated HepG2 cells were fixed in 4% paraformaldehyde for 20 min at room temperature (RT). Cells were washed 5 times with 100 µ L of PBS/well (each wash was for 5 min at RT) and the endogenous peroxides activity was quenched adding 3% H2O2 in PBS for 20 min at RT. Non-specific sites were blocked with 100 µ L/well of 5% BSA in PBS for 1.5 h at RT. LDLR primary antibody solution (1:3000 in 5% BSA in PBS, 25 µ L/well) was incubated O/N at 4 °C. Subsequently, the primary antibody solution was discarded and each sample was washed 5 times with 100 µ L/well of PBS (each wash was for 5 min at RT). Goat anti-rabbit Ig-HRP secondary antibody solution (1:6000 in 5% BSA in PBS, 50 µ L/well) was added and incubated 1 h at RT. The secondary antibody solution was washed 5 times with 100 µ L/well of PBS (each wash for 5 min at RT). Freshly prepared TMB substrate (100 µ L/well), was added and the plate was incubated at RT until the color was developed. The reaction was then stopped with 2 M H2SO4 and the absorbance at 450 nm was measured using the Synergy H1 fluorescent plate reader from Biotek. Cells were stained by adding 1 × Janus green stain, incubating for 5 min at RT. The dye was removed and the sample washed 5 times with water. Afterward 0.1 mL 0.5 M HCl per well were added and incubated for 10 min. After 10 s shaking, the OD at 595 nm was measured using the Synergy H1 fluorescent plate reader from Biotek.

Assay for Evaluation of Fluorescent LDL Uptake by HepG2 Cells
After transfection, a total of 3 × 10 4 HepG2 cells/well were seeded in 96-well and treated with 100 μM T9 or vehicle (H2O) for 24 h. At the end of the treatment period, the culture medium was replaced with 75 μL/well LDL-DyLight™ 550 working solution. The cells were additionally incubated for 2 h at 37 °C and then the culture medium was aspirated and replaced with PBS (100 μL/well). The degree of LDL uptake was measured using the Synergy H1 fluorescent plate reader from Biotek (excitation and emission wavelengths 540 and 570 nm, respectively).

Statistically analysis
Statistical analyses were carried out by One-way ANOVA (Graphpad Prism 6) followed by Dunnett's test. Values were expressed as means ± SD; P-values < 0.05 were considered to be significant. Figure S1. Effect of T9 on the PCSK9-WT and LDLR protein level variations. T9 peptide induces increases of LDLR protein level in untrasfected cells without affecting the PCSK9 intracellular pathway. (***) p < 0.0001