Antioxidant Capacity and NF-kB-Mediated Anti-Inflammatory Activity of Six Red Uruguayan Grape Pomaces.

Grape pomaces have a wide and diverse antioxidant phenolics composition. Six Uruguayan red grape pomaces were evaluated in their phenolics composition, antioxidant capacity, and anti-inflammatory properties. Not only radical scavenging methods as DPPH· and ABTS·+ were employed but also ORAC and FRAP analyses were applied to assess the antioxidant potency of the extracts. The antioxidant reactivity of all extracts against hydroxyl radicals was assessed with ESR. The phenol profile of the most bioactive extract was analyzed by HPLC-MS, and a set of 57 structures were determined. To investigate the potential anti-inflammatory activity of the extracts, Nuclear Factor kappa-B (NF-κB) modulation was evaluated in the human colon cancer reporter cell line (HT-29-NF-κB-hrGFP). Our results suggest that Tannat grapes pomaces have higher phenolic content and antioxidant capacity compared to Cabernet Franc. These extracts inhibited TNF-alpha mediated NF-κB activation and IL-8 production when added to reporter cells. A molecular docking study was carried out to rationalize the experimental results allowing us to propose the proactive interaction between the NF-κB, the grape extracts phenols, and their putative anti-inflammatory bioactivity. The present findings show that red grape pomace constitutes a sustainable source of phenolic compounds, which may be valuable for pharmaceutical, cosmetic, and food industry applications.

Modelling of the Citrus CCD4 Family Members: In Silico Analysis of Membrane Binding and Substrate Preference.

Coloring is one of the most important characteristics in commercial flowers and fruits, generally due to the accumulation of carotenoid pigments. Enzymes of the CCD4 family in citrus intervene in the generation of ß-citraurin, an apocarotenoid responsible for the reddish-orange color of mandarins. Citrus CCD4s enzymes could be capable of interacting with the thylakoid membrane inside chloroplasts. However, to date, this interaction has not been studied in detail. In this work, we present three new complete models of the CCD4 family members (CCD4a, CCD4b, and CCD4c), modeled with a lipid membrane. To identify the preference for substrates, typical carotenoids were inserted in the active site of the receptors and the protein-ligand interaction energy was evaluated. The results show a clear preference of CCD4s for xanthophylls over aliphatic carotenes. Our findings indicate the ability to penetrate the membrane and maintain a stable interaction through the N-terminal α-helical domain, spanning a contact surface of 2250 to 3250 Å2. The orientation and depth of penetration at the membrane surface suggest that CCD4s have the ability to extract carotenoids directly from the membrane through a tunnel consisting mainly of hydrophobic residues that extends up to the catalytic center of the enzyme.

Structural evidence of quercetin multi-target bioactivity: A reverse virtual screening strategy.

The ubiquitous flavonoid quercetin is broadly recognized for showing diverse biological and health-promoting effects, such as anti-cancer, anti-inflammatory and cytoprotective activities. The therapeutic potential of quercetin and similar compounds for preventing such diverse oxidative stress-related pathologies has been generally attributed to their direct antioxidant properties. Nevertheless, accumulated evidence indicates that quercetin is also able to interact with multiple cellular targets influencing the activity of diverse signaling pathways. Even though there are a number of well-established protein targets such as phosphatidylinositol 3 kinase and xanthine oxidase, there remains a lack of a comprehensive knowledge of the potential mechanisms of action of quercetin and its target space. In the present work we adopted a reverse screening strategy based on ligand similarity (SHAFTS) and target structure (idTarget, LIBRA) resulting in a set of predicted protein target candidates. Furthermore, using this method we corroborated a broad array of previously experimentally tested candidates among the predicted targets, supporting the suitability of this screening approach. Notably, all of the predicted target candidates belonged to two main protein families, protein kinases and poly [ADP-ribose] polymerases. They also included key proteins involved at different points within the same signaling pathways or within interconnected signaling pathways, supporting a pleiotropic, multilevel and potentially synergistic mechanism of action of quercetin. In this context we highlight the value of quercetin's broad target profile for its therapeutic potential in diseases like inflammation, neurodegeneration and cancer.

Peptidomimetic Targeting of Cavß2 Overcomes Dysregulation of the L-Type Calcium Channel Density and Recovers Cardiac Function.

BACKGROUND: L-type calcium channels (LTCCs) play important roles in regulating cardiomyocyte physiology, which is governed by appropriate LTCC trafficking to and density at the cell surface. Factors influencing the expression, half-life, subcellular trafficking, and gating of LTCCs are therefore critically involved in conditions of cardiac physiology and disease. METHODS: Yeast 2-hybrid screenings, biochemical and molecular evaluations, protein interaction assays, fluorescence microscopy, structural molecular modeling, and functional studies were used to investigate the molecular mechanisms through which the LTCC Cavß2 chaperone regulates channel density at the plasma membrane. RESULTS: On the basis of our previous results, we found a direct linear correlation between the total amount of the LTCC pore-forming Cavα1.2 and the Akt-dependent phosphorylation status of Cavß2 both in a mouse model of diabetic cardiac disease and in 6 diabetic and 7 nondiabetic cardiomyopathy patients with aortic stenosis undergoing aortic valve replacement. Mechanistically, we demonstrate that a conformational change in Cavß2 triggered by Akt phosphorylation increases LTCC density at the cardiac plasma membrane, and thus the inward calcium current, through a complex pathway involving reduction of Cavα1.2 retrograde trafficking and protein degradation through the prevention of dynamin-mediated LTCC endocytosis; promotion of Cavα1.2 anterograde trafficking by blocking Kir/Gem-dependent sequestration of Cavß2, thus facilitating the chaperoning of Cavα1.2; and promotion of Cavα1.2 transcription by the prevention of Kir/Gem-mediated shuttling of Cavß2 to the nucleus, where it limits the transcription of Cavα1.2 through recruitment of the heterochromatin protein 1γ epigenetic repressor to the Cacna1c promoter. On the basis of this mechanism, we developed a novel mimetic peptide that, through targeting of Cavß2, corrects LTCC life-cycle alterations, facilitating the proper function of cardiac cells. Delivery of mimetic peptide into a mouse model of diabetic cardiac disease associated with LTCC abnormalities restored impaired calcium balance and recovered cardiac function. CONCLUSIONS: We have uncovered novel mechanisms modulating LTCC trafficking and life cycle and provide proof of concept for the use of Cavß2 mimetic peptide as a novel therapeutic tool for the improvement of cardiac conditions correlated with alterations in LTCC levels and function.