SCREENING AND IDENTIFICATION OF NEW FOOD DERIVED PEPTIDES WITH MULTI-TARGET CHOLESTEROL-LOWERING ACTIVITY

In recent years, there is a significant focus on the potential health benefits of food peptides. These short and medium-size peptides are produced by the hydrolysis of food proteins and may be absorbed by the body to modulate specific metabolic pathways by binding or inhibiting targeted receptors. This modulation can lead to positive effects on metabolic diseases, making it particularly noteworthy because it aligns with the trend of many individuals seeking healthier lifestyles through dietary management rather than relying on pharmaceuticals. Additionally, bioactive peptides are typically associated with very low or non-existent toxic or adverse effects. Thus, the identification and exploration of the mechanisms behind bioactive peptides present the opportunity to develop innovative functional foods and nutraceuticals. To date, eggs, meat, fish, soybean, wheat, milk, and its derivatives have been the primary sources of bioactive peptides, as they are widely available and easily accessible. Notably, recent research has also focused on alternative sources such as lupin and hempseed, which are rich in protein and have shown promising health benefits. Lupin, a legume commonly found in Mediterranean countries, has been found to contain bioactive peptides that can lower blood pressure and improve cardiovascular health. Similarly, hempseed, derived from the industrial hemp, has been shown to have anti-inflammatory and antioxidant properties, making it a potential source of bioactive peptides that could have a range of health benefits. Based on these considerations, the aim of this PhD thesis was to screen and identify new promising peptides from lupin and hempseed protein. The identified peptides were evaluated for their ability to target hypercholesterolemia, which is a medical condition characterized by high levels of cholesterol and is a major risk factor for the development of cardiovascular disease (CVD). To achieve this objective, multidisciplinary approaches were employed, involving peptidomic techniques to profile the peptide sequences, molecular modeling methods to predict potentially bioactive peptides, and biochemical and cellular tools to evaluate the bioactivity of peptides and to explore their possible mechanism of action. Briefly, in the present study, the peptides derived from lupin (Lupinus albus) and hempseed (Cannabis sativa) generally presented significant cholesterol-lowering activity. In detail, peptides derived from lupin 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 HMGCoAR. Moreover, P5 was successfully transported by differentiated human intestinal Caco-2 cells through transcytosis, and, during transport, it was partially metabolized in a breakdown fragment (LPKHSDAD, P5-met), which retained the biological activity of the parent peptide. Based on these observations, P5 analogs, including P5-Best (LYLPKHSDRD), P5-H6A (LILPKASDAD) and P5-S7A (LILPKHADAD), were computationally designed to target both PCSK9 and HMGCoAR, displaying an improved and dual hypocholesterolemic activity evaluated by biochemical assays. In addition, hydrolysates derived from hempseed were shown to have a hypocholesterolaemia effect by dropping the activity of the HMGCoAR, showing a similar cholesterol-lowering activity of statins. Since proteins are hydrolysed during digestion, the activity may be attributed to specific peptides encrypted in the protein sequences that are released by digestion and absorbed at an intestinal level. Hence, further experiments using intestinal trans-epithelial transport revealed that among the peptides contained within hempseed hydrolysates are able to pass through the mature Caco-2 cell barrier. Particularly, peptide H3 (IGFLIIWV) possess antioxidant activity in HepG2 cells by modulating the Nrf-2 and iNOS pathways, leading to the decrease of cellular H2O2-induced ROS, NO, and lipid peroxidation levels. Meanwhile, since there is a link between inflammation and oxidative stress, the evaluation of the anti-inflammatory effect of peptide H3 in HepG2 cells was carried out. As expected, peptide H3 was shown to modulate the production of pro (IFN-γ, TNF and IL-6)- and anti (IL-10)-inflammatory cytokines and NO through regulation of the NF-κB and iNOS pathways, exerting an anti-inflammatory capacity in the HepG2 cells. Furthermore, a deeper mechanistic investigation of peptide H3 was also performed in HepG2 cells. Peptide H3 can modulate the activity of the key targets involved in cholesterol metabolism, i.e., LDLR, SREBP-2, HMGCoAR, p-AMPK (Thr 172), HNF-1α, and PCSK9, and improve the capacity of HepG2 cells treated with peptide H3 to absorb extracellular LDL cholesterol. In conclusion, the investigation of bioactive peptides holds significant promise in developing novel molecular entities with hypocholesterolemic activity. This study provides valuable insights into the potential of lupin peptides (P5 and its derivatives) and hempseed peptide (H3) as peptide-based inhibitors targeting PCSK9 and HMGCoAR, making them viable candidates for hypocholesterolemic interventions. However, it is important to acknowledge that in cases where the condition's severity leads to significant pathology, food- or food-supplement-based interventions may not be effective. Additionally, it is crucial to recognize that the current evidence is primarily derived from in vitro studies, highlighting the need for further research to validate these findings in animal models and human clinical trials. Conducting rigorous clinical trials in the future will provide a more comprehensive understanding of the efficacy and safety of bioactive peptides, ultimately enhancing cholesterol management strategies and improving overall health outcomes. In summary, this study underscores the promise of investigating bioactive peptides for hypocholesterolemic interventions. Nevertheless, it is imperative to conduct additional research to validate these findings and consider the limitations of food- or food-supplement-based approaches in cases involving severe pathology. By addressing these knowledge gaps, we can advance our understanding of bioactive peptides, optimize cholesterol management strategies, and ultimately improve overall health outcomes.