disrupting the pcsk9 ldlr protein protein interaction by an imidazole based minimalist peptidomimetic

We report on a tetraimidazole-based β-strand minimalist peptidomimetic as a novel inhibitor of LDLR–PCSK9 protein–protein interaction, a promising target for hypercholesterolemia

Evaluation of the multifunctional dipeptidyl-peptidase IV and angiotensin converting enzyme inhibitory properties of a casein hydrolysate using cell-free and cell-based assays

The objective of the study was the evaluation of the potential pleiotropic effect of a commercial casein hydrolysate (CH). After an analysis of the composition, the BIOPEP-UWM database suggested that these peptides contained numerous sequences with potential inhibitory activities on angiotensin converting enzyme (ACE) and dipeptidyl-peptidase IV (DPP-IV). The anti-diabetic and anti-hypertensive effects of these peptides were thus assessed using either cell-free or cell-based assays. In the cell-free system, CH displayed inhibitory properties against DPP-IV (IC50 value equal to 0.38 ± 0.01 mg/mL) and ACE (IC50 value equal to 0.39 ± 0.01 mg/mL). Further, CH reduced the DPP-IV and ACE activities expressed by human intestinal Caco-2 cells by 61.10 ± 1.70% and 76.90 ± 4.47%, respectively, versus untreated cells, after 6 h of treatment at the concentration of 5 mg/mL. This first demonstration of the multifunctional behavior of this material suggests that it may become an anti-diabetic and/or anti-hypertensive ingredient to be included in the formulation of different functional food or nutraceutics

Chemical and biological characterization of the DPP-IV inhibitory activity exerted by lupin (Lupinus angustifolius) peptides: From the bench to the bedside investigation

10 Páginas.-- 5 Figuras.-- 1 TablaDipeptidyl peptidase IV (DPP-IV) is considered a key target for the diabetes treatment, since it is involved in glucose metabolism. Although lupin protein consumption shown hypoglycemic activity, there is no evidence of its effect on DPP-IV activity. This study demonstrates that a lupin protein hydrolysate (LPH), obtained by hydrolysis with Alcalase, exerts anti-diabetic activity by modulating DPP-IV activity. In fact, LPH decreased DPP-IV activity in a cell-free and cell-based system. Contextually, Caco-2 cells were employed to identify LPH peptides that can be intestinally trans-epithelial transported. Notably, 141 different intestinally transported LPH sequences were identified using nano- and ultra-chromatography coupled to mass spectrometry. Hence, it was demonstrated that LPH modulated the glycemic response and the glucose concentration in mice, by inhibiting the DPP-IV. Finally, a beverage containing 1 g of LPH decreased DPP-IV activity and glucose levels in humans.Ministerio de Economía y Competitividad, Gobierno de España [AGL2012-40247-C02-01 and AGL2012-40247-C02-02]. Consejería de Salud [PC-0111-2016-0111], and the PAIDI Program [CTS160] from Junta de Andalucía. I.C.-C. was supported by Ministerio de Educación, Cultura y Deporte, Gobierno de España [FPU13/01210], by the Universidad de Sevilla [VIPPIT-2020-II.4], and by a postdoctoral fellowship from the Consejería de Transformación Económica, Industria, Conocimiento y Universidades, Junta de Andalucía [DOC_00587/2020]. G.S.-S. was supported by Ministerio de Educación, Cultura y Deporte, Gobierno de España [FPU16/02339], and by an Erasmus+ Mobility Programme.Peer reviewe

A Lupinus angustifolius protein hydrolysate exerts hypocholesterolemic effects in Western diet-fed ApoE-/- mice through the modulation of LDLR and PCSK9 pathways

6 Figuras.-- 2 TablasLupin protein hydrolysates (LPHs) are gaining attention in the food and nutraceutical industries due to their several beneficial health effects. Recently, we have shown that LPH treatment reduces liver cholesterol and triglyceride levels in hypercholesterolemic mice. The aim of this study was to elucidate the effects of LPH treatment on the molecular mechanism underlying liver cholesterol metabolism in ApoE-/- mice fed the Western diet. After identifying the composition of the peptide within the LPH mixture and determining its ability to reduce HMGCoAR activity in vitro, its effect on the LDLR and PCSK9 pathways was measured in liver tissue from the same mice. Thus, the LPH reduced the protein levels of HMGCoAR and increased the phosphorylated inactive form of HMGCoAR and the pHMGCoAR/HMGCoAR ratio, which led to the deactivation of de novo cholesterol synthesis. Furthermore, the LPH decreased the protein levels of SREBP2, a key upstream transcription factor involved in the expression of HMGCoAR and LDLR. Consequently, LDLR protein levels decreased in the liver of LPH-treated animals. Interestingly, the LPH also increased the protein levels of pAMPK responsible for HMGCoAR phosphorylation. Furthermore, the LPH controlled the PSCK9 signal pathway by decreasing its transcription factor, the HNF1-α protein. Consequently, lower PSCK9 protein levels were found in the liver of LPH-treated mice. This is the first study elucidating the molecular mechanism at the basis of the hypocholesterolemic effects exerted by the LPH in an in vivo model. All these findings point out LPHs as a future lipid-lowering ingredient to develop new functional foods.This research was funded by the Andalusian Government Ministry of Health (PC-0111-2016-0111), and the PAIDI Program from the Andalusian Government (CTS160). I. C.-C. was supported by the VI Program of Inner Initiative for Research and Transfer of University of Seville (VIPPIT-2020-II.4) and a postdoctoral fellowship from the Andalusian Government Ministry of Economy, Knowledge, Business, and University (DOC_00587/2020). G. S.-S. was supported by a FPU grant from the Spanish Ministerio de Educación, Cultura y Deporte (FPU16/02339), and by an Erasmus+ Mobility Programme. The authors gratefully acknowledge the Carlo Sirtori Foundation (Milan, Italy) for having provided part of the equipment used in this experimentation. We thank all the staff from the Instituto de Biomedicina de Sevilla (IBiS) Animal Facility for their valuable assistance.Peer reviewe

Virgin Olive Oil Extracts Reduce Oxidative Stress and Modulate Cholesterol Metabolism: Comparison between Oils Obtained with Traditional and Innovative Processes

This study was aimed at demonstrating the substantial equivalence of two extra virgin olive oil samples extracted from the same batch of Coratina olives with (OMU) or without (OMN) using ultrasound technology, by performing chemical, biochemical, and cellular investigations. The volatile organic compounds compositions and phenolic profiles were very similar, showing that, while increasing the extraction yields, the innovative process does not change these features. The antioxidant and hypocholesterolemic activities of the extra virgin olive oil (EVOO) phenol extracts were also preserved, since OMU and OMN had equivalent abilities to scavenge the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radicals in vitro and to protect HepG2 cells from oxidative stress induced by H2O2, reducing intracellular reactive oxygen species (ROS) and lipid peroxidation levels. In addition, by inhibiting 3-hydroxy-3-methylglutarylcoenzyme a reductase, both samples modulated the low-density lipoprotein receptor (LDLR) pathway leading to increased LDLR protein levels and activity

Innovative Functional Methods Useful for New Cellular Target Identification and for Receptor Ligand Characterization

G-protein coupled receptors (GPCRs) are a large family of receptor proteins that play important roles in many physiological and pathological conditions. In drug discovery, they are one of the most important drug targets. In particular, adenosine receptors, classified as A1, A2A, A2B and A3 (A1AR, A2AAR, A2BAR, and A3AR) subtypes, have been actively studied as potential therapeutic targets in several disorders such as cardiovascular and CNS pathologies.\ud The aim of the present work is the development of innovative and non-radioactive screening systems in order to study a large number of adenosine P1 receptor ligands. On the other hand, since new assays could be useful to contribute to receptor deorphanization, the task work has been focused to characterize the putative guanosine receptor and the novel dual uracil nucleotide/cysteinyl-leukotriene receptor, GPR17. In particular, functional cAMP and DELFIA GTP-Eu binding assays were used.\ud The cAMP immuno-competitive assay, performed in homogeneous time resolved fluorescence (HTRF), was optimized and validated using as reference agonists NECA at A2AAR and A2BAR, and Cl-IBMECA at A3AR. Hence, after this assay validation, the biological profile of a series of 8-substituted 9-ethyladenines was assessed at A2AAR. Results showed that all compounds behave as A2AAR antagonists.\ud On the other hand, an innovative luminescence cAMP assay, that uses a genetically modified form of firefly luciferase containing a cAMP-binding protein moiety, was developed for ARs. Two new improved plasmids containing the gene for the modified form of firefly luciferase were optimized and validated at ARs in order to verify their efficacy at Gαs and Gαi systems. In particular, the experiments were carried out by transiently expressing these new vectors at CHO cell line stably transfected with A2A and A3 ARs. Results showed that both plasmids can be used at ARs and since this assay demonstrated to be robust and reproducible, it was used as new tool to investigate the novel “dual” GPR17. Furthermore, the better evaluation of the “dual” nature of the human GPR17 could be carried out in 1321N1 cells since they do not constitutively express nucleotide and cysteinyl-leukotriene receptors. In particular, different transient transfection methods such as calcium phosphate, lipofectamine, fugene, arrest-InTM and microporation, have been investigated. Experiments were performed using also in parallel GFP as reporter gene and results analyzed by RT-PCR suggested that microporator as well as Arrest-In are both the best method to promote an enriched hgpr17 delivery into 1321N1 cell line.\ud Moreover, DELFIA GTP-Eu binding assay was carried out using CHO-A3 membranes. The standardization of DELFIA GTP-Eu assay is a long process, in which the determination of the specific receptor buffer stimulation was the first crucial step investigated. The assay validation was carried out using know ligands and subsequently, a new series of 2-alkynyl-N6-methyl-5’-N-methylcarboxamidoAdo derivatives were screened and results suggested that they are full agonists at A3AR. Subsequently, the assay potentialities were extended at native tissue. In particular, rat brain membranes were prepared from Male Wistar rat and used to investigate putative guanosine receptor by testing known and novel ligands. Results obtained suggested the presence of a G-protein coupled receptor able to be activated by guanosine and some guanosine derivatives.\ud The AGC is a family of kinase that is composed of 60 different enzymes including PKC isoforms, PKB/AKT, S6K and their upstream kinase, the phosphoinositide-dependent protein kinase 1 (PDK1). In particular, located in the small loop of the kinase domain is present a pocket called PIF-binding pocket that is involved in the regulation of the intrinsic activity of these kinases. The molecular mechanism of allosteric activation of PDK1 by small molecular weight prodrugs and their effects in C2C12 cells were evaluated. In particular experiments were carried out incubating C2C12 with PS423 and PS0403, small chemical entities synthesized by the PhosphoSites research group, and after cell insulin stimulation the PDK1 pathway was investigated. Results obtained suggested that these compounds were able to bind the PIF-binding pocket inhibiting the S6K phosphorylation without affecting the PKB activity. On the other hand, the in cell western was optimized at C2C12, as new assay to screen and to evaluate in a throughput way the effects of several PS-compounds on the S6K phosphorylation by PDK1. Results demonstrated that the in cell western is a robust, reproducible and easy to use assay to screen directly in the cell several compounds in the same experiments

Three Peptides from Soy Glycinin Modulate Glucose Metabolism in Human Hepatic HepG2 Cells

Ile-Ala-Val-Pro-Gly-Glu-Val-Ala (IAVPGEVA), Ile-Ala-Val-Pro-Thr-Gly-Val-Ala (IAVPTGVA) and Leu-Pro-Tyr-Pro (LPYP), three peptides deriving from soy glycinin hydrolysis, are known to regulate cholesterol metabolism in human hepatic HepG2 cells. We have recently demonstrated that the mechanism of action involves the activation of adenosine monophosphate-activated protein kinase (AMPK). This fact suggested a potential activity of the same peptides on glucose metabolism that prompted us to also investigate this aspect in the same cells. After treatment with IAVPGEVA, IAVPTGVA and LPYP, HepG2 cells were analyzed using a combination of molecular techniques, including western blot analysis, glucose uptake experiments and fluorescence microscopy evaluation. The results showed that these peptides are indeed able to enhance the capacity of HepG2 cells to uptake glucose, via glucose transporter 1 GLUT1 and glucose transporter 4 GLUT4 activation, through the stimulation of protein kinase B Akt and adenosine monophosphate-activated protein kinase AMPK pathways, both involved in glucose metabolism

Computational Design and Biological Evaluation of Analogs of Lupin Peptide P5 Endowed with Dual PCSK9/HMG-CoAR Inhibiting Activity

(1) Background: Proprotein convertase subtilisin/kexin 9 (PCSK9) is responsible for the degradation of the hepatic low-density lipoprotein receptor (LDLR), which regulates the circulating cholesterol level. In this field, we discovered natural peptides derived from lupin that showed PCSK9 inhibitory activity. Among these, the most active peptide, known as P5 (LILPHKSDAD), reduced the protein-protein interaction between PCSK9 and LDLR with an IC50 equals to 1.6 µM and showed a dual hypocholesterolemic activity, since it shows complementary inhibition of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoAR). (2) Methods: In this study, by a computational approach, the P5 primary structure was optimized to obtain new analogs with improved affinity to PCSK9. Then, biological assays were carried out for fully characterizing the dual cholesterol-lowering activity of the P5 analogs by using both biochemical and cellular techniques. (3) Results: A new peptide, P5-Best (LYLPKHSDRD) displayed improved PCSK9 (IC50 0.7 µM) and HMG-CoAR (IC50 88.9 µM) inhibitory activities. Moreover, in vitro biological assays on cells demonstrated that, not only P5-Best, but all tested peptides maintained the dual PCSK9/HMG-CoAR inhibitory activity and remarkably P5-Best exerted the strongest hypocholesterolemic effect. In fact, in the presence of this peptide, the ability of HepG2 cells to absorb extracellular LDL was improved by up to 254%. (4) Conclusions: the atomistic details of the P5-Best/PCSK9 and P5-Best/HMG-CoAR interactions represent a reliable starting point for the design of new promising molecular entities endowed with hypocholesterolemic activity

Nanostructure, Self-Assembly, Mechanical Properties, and Antioxidant Activity of a Lupin-Derived Peptide Hydrogel

Naturally occurring food peptides are frequently used in the life sciences due to their beneficial effects through their impact on specific biochemical pathways. Furthermore, they are often leveraged for applications in areas as diverse as bioengineering, medicine, agriculture, and even fashion. However, progress toward understanding their self-assembling properties as functional materials are often hindered by their long aromatic and charged residue-enriched sequences encrypted in the parent protein sequence. In this study, we elucidate the nanostructure and the hierarchical self-assembly propensity of a lupin-derived peptide which belongs to the α-conglutin (11S globulin, legumin-like protein), with a straightforward N-terminal biotinylated oligoglycine tag-based methodology for controlling the nanostructures, biomechanics, and biological features. Extensive characterization was performed via Circular Dichroism (CD) spectroscopy, Fourier Transform Infrared spectroscopy (FT-IR), rheological measurements, and Atomic Force Microscopy (AFM) analyses. By using the biotin tag, we obtained a thixotropic lupin-derived peptide hydrogel (named BT13) with tunable mechanical properties (from 2 to 11 kPa), without impairing its spontaneous formation of β-sheet secondary structures. Lastly, we demonstrated that this hydrogel has antioxidant activity. Altogether, our findings address multiple challenges associated with the development of naturally occurring food peptide-based hydrogels, offering a new tool to both fine tune the mechanical properties and tailor the antioxidant activities, providing new research directions across food chemistry, biochemistry, and bioengineering

Assessment of the Cholesterol-Lowering Effect of MOMAST^®: Biochemical and Cellular Studies

MOMAST® is a patented phenolic complex derived from the olive oil vegetation water, a by-product of the olive oil supply chain, in which hydroxytyrosol (OH-Tyr) and tyrosol (Tyr) and verbascoside are the main compounds. This study was aimed at investigating its hypocholesterolemic effect by assessing the ability to modulate the low-density lipoprotein (LDL) receptor (LDLR)/sterol regulatory element-binding protein 2 (SREBP-2), and proprotein convertase subtilisin/kexin type 9 (PCSK9) pathways. MOMAST® inhibits the in vitro activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCOAR) with a dose-response trend. After the treatment of HepG2 cells, MOMAST® increases the SREBP-2, LDLR, and HMGCoAR protein levels leading, from a functional point of view to an improved ability of hepatic cells to up-take LDL from the extracellular environment with a final cholesterol-lowering effect. Furthermore, MOMAST® decreased the PCSK9 protein levels and its secretion in the extracellular environment, presumably via the reduction of the hepatic nuclear factor 1-α (HNF1-α). The experiments were performed in parallel, using pravastatin as a reference compound. Results demonstrated that MOMAST® may be exploited as a new ingredient for the development of functional foods and/or nutraceuticals for cardiovascular disease prevention