Trans-Epithelial Transport, Metabolism, and Biological Activity Assessment of the Multi-Target Lupin Peptide LILPKHSDAD (P5) and Its Metabolite LPKHSDAD (P5-Met).

P5 (LILPKHSDAD) is a hypocholesterolemic peptide from lupin protein with a multi-target activity, since it inhibits both 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoAR) and proprotein convertase subtilisin/kexin type-9 (PCSK9). This work shows that, during epithelial transport experiments, the metabolic transformation mediated by intestinal peptidases produces two main detected peptides, ILPKHSDAD (P5-frag) and LPKHSDAD (P5-met), and that both P5 and P5-met are linearly absorbed by differentiated human intestinal Caco-2 cells. Extensive comparative structural, biochemical, and cellular characterizations of P5-met and the parent peptide P5 demonstrate that both peptides have unique characteristics and share the same mechanisms of action. In fact, they exert an intrinsically multi-target behavior being able to regulate cholesterol metabolism by modulating different pathways. The results of this study also highlight the dynamic nature of bioactive peptides that may be modulated by the biological systems they get in contact with.

Aromatic interactions in the binding of ligands to HMGCoA reductase.

3-Hydroxy-3-methyglutaryl-coenzyme A (HMGCoA) reductase is the enzyme that catalyzes the rate-determining step in cholesterol synthesis; it is also the target for statin drugs, which are competitive inhibitors of the enzyme. We examine potentially important enzyme-ligand interactions currently not incorporated into statin drug design: weak, induction/dispersion interactions between ligands and residue tyrosine 479 in the HMGCoA reductase active site. HMGCoA is a large molecule with a long coenzyme A "tail", and in order to study the interactions of interest, it was necessary to find the smallest possible portion of the HMGCoA molecule that would serve as a reasonable model for the entire molecule. Using this minimal model, we calculated BSSE-corrected electronic interaction energies between the residue and the ligand molecule using several DFT methods (local, hybrid, and gradient-corrected DFT methods) as well as MP2. We also performed several in silico mutations of the tyrosine 479 residue to determine the potential effects of these changes on protein-ligand interaction energies. Our work shows that this previously unexploited protein-ligand interaction between tyrosine residue 479 and HMGCoA can be important in the design of future statin drugs. Per our previous work, our results show that local DFT methods more closely match MP2 energy values for aromatic binding than do hybrid or gradient-corrected DFT methods.

Five hourly measurements of serum cholesterol levels: a new methodology to assess and evaluate stress, good health and disease.

Research has developed a concrete link between psychological/emotional stress and life-threatening diseases such as heart disease and cancer. Here we present a technique to assess the magnitude of stress from cholesterol variation number (CVN). This number is the difference between the highest and the lowest cholesterol concentrations that relates to the five hourly cholesterol measurements performed over a five hour span. Since cholesterol in serum arises from the liver, the CVN is equated with the fluctuations in hepatic biosynthesis. This relationship is explained on the basis of the rhythmic hormonal secretions associated with cholesterol biosynthesis. Whenever stress-induced aberrations in timing of hormonal secretions occur, CVN changes. Individuals with lower CVN would have overall better health than persons with higher CVN. Thus by utilizing CVN, physicians may be able to differentiate cardiovascular health of individuals with the same or very similar serum cholesterol concentrations.