Salivary proteomic profile of young healthy subjects.

Background: The incidence of noncommunicable diseases (NCDs) has been rapidly ramped up worldwide. Hence, there is an urgent need to non-invasively detect NCDs possibly by exploiting saliva as a 'liquid biopsy' to identify biomarkers of the health status. Since, the absence of standardized procedures of collection/analysis and the lack of normal ranges makes the use of saliva still tricky, our purpose was to outline a salivary proteomic profile which features healthy individuals. Methods: We collected saliva samples from 19 young blood donors as reference population and the proteomic profile was investigated through mass-spectrometry. Results: We identified 1,004 proteins of whose 243 proteins were shared by all subjects. By applying a data clustering approach, we found a set of six most representative proteins across all subjects including Coronin-1A, F-actin-capping protein subunit alpha, Immunoglobulin J chain, Prosaposin, 78 kDa glucose-regulated protein and Heat shock 70 kDa protein 1A and 1B. Conclusion: All of these proteins are involved in immune system activation, cellular stress responses, proliferation, and invasion thus suggesting their use as biomarkers in patients with NCDs.

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.

Lipidome Investigation of Carnosine Effect on Nude Mice Skin to Prevent UV-A Damage.

The lipid profile of skin is fundamental in the maintenance of the protective barrier against the external environment. Signaling and constitutive lipids of this large organ are involved in inflammation, metabolism, aging, and wound healing, such as phospholipids, triglycerides, FFA, and sphingomyelin. Skin exposure to ultraviolet (UV) radiation results in a photoaging process that is an accelerated form of aging. UV-A radiation deeply penetrates the dermis and promotes damage to DNA, lipids, and proteins by increasing the generation of reactive oxygen species (ROS). Carnosine, an endogenous ß-alanyl-L-histidine dipeptide, demonstrated antioxidant properties that prevent photoaging and modification of skin protein profiling, making carnosine a compelling ingredient to consider for use in dermatology. The aim of this research was to investigate the modification of skin lipidome after UV-A treatment in presence or not of topic administration of carnosine. Quantitative analyses based on high-resolution mass spectrometry of nude mice skin-extracted lipids resulted in several modifications of barrier composition after UV-A radiation, with or without carnosine treatment. In total, 328 out of 683 molecules showed significant alteration-262 after UV-A radiation and 126 after UV-A and carnosine treatment versus controls. Importantly, the increased oxidized TGs after UV-A radiation, responsible of dermis photoaging, were completely reverted by carnosine application to prevent the UV-A damage. Network analyses also showed that the production of ROS and the calcium and TNF signaling were modulated by UV-A and carnosine. In conclusion, lipidome analyses attested the carnosine activity to prevent the UV-A damage, reducing the lipid oxidation, the inflammation, and the dysregulation of lipid skin barrier.

Antioxidant Effect Assessment and Trans Epithelial Analysis of New Hempseed Protein Hydrolysates.

Hempseed (Cannabis sativa) is one of the most promising sources of plant proteins. It contains approximately 24% (w/w) protein, and edestin accounts for approximately 60-80% (w/w) of its total proteins. In a framework of research aimed at fostering the proteins recovered from the press cake by-products generated after the extraction of hempseed oil, two hempseed protein hydrolysates (HH1 and HH2) were produced at an industrial level using a mixture of different enzymes from Aspergillus niger, Aspergillus oryzae, and Bacillus licheniformis for different times (5 h and 18 h). Using a combination of different direct antioxidant tests (DPPH, TEAC, FRAP, and ORAC assays, respectively), it has been demonstrated that HHs exert potent, direct antioxidant activity. A crucial feature of bioactive peptides is their intestinal bioavailability; for this reason, in order to solve this peculiar issue, the ability of HH peptides to be transported by differentiated human intestinal Caco-2 cells has been evaluated. Notably, by using mass spectrometry analysis (HPLC Chip ESI-MS/MS), the stable peptides transported by intestinal cells have been identified, and dedicated experiments confirmed that the trans-epithelial transported HH peptide mixtures retain their antioxidant activity, suggesting that these hempseed hydrolysates may be considered sustainable antioxidant ingredients to be exploited for further application, i.e., nutraceutical and/or food industries.

The Effect of Carnosine on UVA-Induced Changes in Intracellular Signaling of Human Skin Fibroblast Spheroids.

Dermis fibroblasts are very sensitive to penetrating UVA radiation and induce photo-damage. To protect skin cells against this environmental damage, there is an urgent need for effective compounds, specifically targeting UVA-induced mitochondrial injury. This study aimed to analyze the effect of carnosine on the proteome of UVA-irradiated human skin fibroblast, cultured in a three-dimensional (3D) biological system recapitulating dermal compartment as a test system to investigate the altered cellular pathways after 48 h and 7 days of culture with or without carnosine treatment. The obtained results indicate that UVA dysregulates Oxidative Phosphorylation, the Fibrosis Signaling Pathway, Glycolysis I and Nrf2-mediated Oxidative Stress Response. Carnosine exercises provide a protective function against the harmful effects of UVA radiation by activating the Nrf2 pathway with the upregulations of some ROS-detoxifying enzymes such as the glutathione S-transferase (GST) protein family. Additionally, carnosine regulates the activation of the Epithelial Adherens Junction and Wound Healing Signaling Pathway by mediating the activation of structural proteins such as vinculin and zyxin as well as fibronectin 1 and collagen type XVIII alpha 1 chain against UVA-induced changes.

Quality Assessment of the Protein Ingredients Recovered by Ultrasound-Assisted Extraction from the Press Cakes of Coconut and Almond Beverage Preparation.

The manufacture of vegetal beverages has the drawback of producing large amounts of press cakes that are generally used as feed components. This work had the objective of valorizing the press cakes deriving from almond and coconut drinks production by using ultrasound-assisted extraction (UAE) to obtain protein ingredients for human use. Starting from coconut and almond press cakes, whose initial protein contents were 19.7% and 18.6%, respectively, the UAE treatment allowed liquid fractions to be obtained that were then freeze-dried: the extraction yields were 24.4 g dry extract/100 g press cake in case of coconut and 49.3 g dry extract/100 g press cake in case of almond. The protein contents of these dried materials were 30.10% and 22.88%, respectively. The quality of the extracted protein ingredients was assessed in term of phytic acid content, protein profile, techno-functional features, and antioxidant properties. The sonication had also a favorable effect on digestibility.

Preparation, Characterization and In Vitro Stability of a Novel ACE-Inhibitory Peptide from Soybean Protein.

A soy protein isolate was hydrolyzed with Alcalase®, Flavourzyme® and their combination, and the resulting hydrolysates (A, F and A + F) were ultrafiltered and analyzed through SDS-PAGE. Fractions with MW < 1 kDa were investigated for their ACE-inhibitory activity, and the most active one (A < 1 kDa) was purified by semi-preparative RP-HPLC, affording three further subfractions. NMR analysis and Edman degradation of the most active subfraction (A1) enabled the identification of four putative sequences (ALKPDNR, VVPD, NDRP and NDTP), which were prepared by solid-phase synthesis. The comparison of their ACE-inhibitory activities suggested that the novel peptide NDRP might be the main agent responsible for A1 fraction ACE inhibition (ACE inhibition = 87.75 ± 0.61%; IC50 = 148.28 ± 9.83 µg mL−1). NDRP acts as a non-competitive inhibitor and is stable towards gastrointestinal simulated digestion. The Multiple Reaction Monitoring (MRM) analysis confirmed the presence of NDRP in A < 1 kDa.

Protein Profiling of a Cellular Model of NAFLD by Advanced Bioanalytical Approaches.

Advanced quantitative bioanalytical approaches in combination with network analyses allow us to answer complex biological questions, such as the description of changes in protein profiles under disease conditions or upon treatment with drugs. In the present work, three quantitative proteomic approaches-either based on labelling or not-in combination with network analyses were applied to a new in vitro cellular model of nonalcoholic fatty liver disease (NAFLD) for the first time. This disease is characterized by the accumulation of lipids, inflammation, fibrosis, and insulin resistance. Hepatic G2 cells were used as model, and NAFLD was induced by a complex of oleic acid and bovine albumin. The development of the disease was verified by lipid vesicle staining and by the increase in the expression of perilipin-2-a protein constitutively present in the vesicles during NAFLD. The nLC-MS/MS analyses of peptide samples obtained from three different proteomic approaches resulted in accurate and reproducible quantitative data of protein fold-change expressed in NAFLD versus control cells. The differentially regulated proteins were used to evaluate the involved and statistically enriched pathways. Network analyses highlighted several functional and disease modules affected by NAFLD, such as inflammation, oxidative stress defense, cell proliferation, and ferroptosis. Each quantitative approach allowed the identification of similar modulated pathways. The combination of the three approaches improved the power of statistical network analyses by increasing the number of involved proteins and their fold-change. In conclusion, the application of advanced bioanalytical approaches in combination with pathway analyses allows the in-depth and accurate description of the protein profile of an in vitro cellular model of NAFLD by using high-resolution quantitative mass spectrometry data. This model could be extremely useful in the discovery of new drugs to modulate the equilibrium NAFLD health state.

Investigation of the intestinal trans-epithelial transport and antioxidant activity of two hempseed peptides WVSPLAGRT (H2) and IGFLIIWV (H3).

A preceding paper has shown that a hempseed peptic hydrolysate displays a cholesterol-lowering activity with a statin-like mechanism of action in HepG2 cells and a potential hypoglycemic activity by the inhibition of dipeptidyl peptidase-IV in Caco-2 cells. In the framework of a research aimed at fostering the multifunctional behavior of hempseed peptides, we present here the identification and evaluation of some antioxidant peptides from the same hydrolysate. After evaluation of its diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, a trans-epithelial transport experiment was performed using differentiated Caco-2 cells that permitted the identification of five transported peptides that were synthesized and evaluated by measuring the oxygen radical absorbance capacity (ORAC), the ferric reducing antioxidant power (FRAP), and the 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic) acid (ABTS), and diphenyl-2-picrylhydrazyl radical DPPH assays. The most active peptides, i.e. WVSPLAGRT (H2) and IGFLIIWV (H3), were then tested in cell assays. Both peptides were able to reduce the H2O2-induced reactive oxygen species (ROS), lipid peroxidation, and nitric oxide (NO) production levels in HepG2 cells, via the modulation of Nrf-2 and iNOS pathways, respectively.

Oxidative Stress Modulation by Carnosine in Scaffold Free Human Dermis Spheroids Model: A Proteomic Study.

Carnosine is an endogenous ß-alanyl-L-histidine dipeptide endowed with antioxidant and carbonyl scavenger properties, which is able to significantly prevent the visible signs of aging and photoaging. To investigate the mechanism of action of carnosine on human skin proteome, a 3D scaffold-free spheroid model of primary dermal fibroblasts from a 50-year-old donor was adopted in combination with quantitative proteomics for the first time. The label free proteomics approach based on high-resolution mass spectrometry, integrated with network analyses, provided a highly sensitive and selective method to describe the human dermis spheroid model during long-term culture and upon carnosine treatment. Overall, 2171 quantified proteins allowed the in-depth characterization of the 3D dermis phenotype during growth and differentiation, at 14 versus 7 days of culture. A total of 485 proteins were differentially regulated by carnosine at 7 days, an intermediate time of culture. Of the several modulated pathways, most are involved in mitochondrial functionality, such as oxidative phosphorylation, TCA cycle, extracellular matrix reorganization and apoptosis. In long-term culture, functional modules related to oxidative stress were upregulated, inducing the aging process of dermis spheroids, while carnosine treatment prevented this by the downregulation of the same functional modules. The application of quantitative proteomics, coupled to advanced and relevant in vitro scaffold free spheroids, represents a new concrete application for personalized therapies and a novel care approach.

Composition of the Protein Ingredients from Insoluble Oat Byproducts Treated with Food-Grade Enzymes, Such as Amylase, Cellulose/Xylanase, and Protease.

The manufacture of plant-based drinks has the drawback of a huge production of underexploited press cakes. In particular, the oat press cake is mainly used in feed formulation, whereas added-value applications in human nutrition are scarce. Considering that enzymatic treatments may be useful to improve the nutritional quality of these insoluble byproducts, this study aimed to evaluate whether the treatment with some food-grade enzymes, such as amylase, cellulase/xylanase, protease, and their combination, may be useful to achieve this goal. Proteomic and peptidomic studies showed that the enzymatic treatments improved the protein extraction yields and induced a release of low molecular weight (LMW) peptides that were demonstrated to provide a useful antioxidant activity. In the treated oat press cake proteins, the concentration of the bound phenolic compounds was decreased, with the exception of caffeic acid, which was increased, and avenanthramides, which remained unchanged. Finally, the enzymatic treatment decreased the concentration of phytic acid. All these results indicate that the enzymatic treatments may be useful to ameliorate the nutritional profile of these protein ingredients, before their inclusion in different food products.

Integratomics of Human Dermal Fibroblasts Treated with Low Molecular Weight Hyaluronic Acid.

Hyaluronic acid (HA) is a glycosaminoglycan very common in commercial products from pharmaceuticals to cosmetics due to its widespread distribution in humans and its diversified physico-chemical proprieties. Despite its extended use and preliminary evidence showing even also opposite activities to the native form, the precise cellular effects of HA at low-molecular-weight (LWM-HA) are currently unclear. The 'omics sciences currently in development offer a new and combined perspective on the cellular and organismal environment. This work aims to integrate lipidomics analyses to our previous quantitative proteomics one for a multi-omics vision of intra- and extra-cellular impact of different concentrations (0.125, 0.25, and 0.50%) of LMW-HA (20-50 kDa) on normal human dermal fibroblasts by LC-high resolution mass spectrometry (LC-HRMS). Untargeted lipidomics allowed us to identify 903 unique lipids mostly represented by triacylglycerols, ceramides, and phosphatidylcholines. According to proteomics analyses, LMW-HA 0.50% was the most effective concentration also in the lipidome rearrangement especially stimulating the synthesis of ceramides involved in skin hydration and reparation, cell signaling, and energy balance. Finally, integrative analyses showed 25 nodes covering several intra- and extra-cellular functions. The more complete comprehension of intra- and extra-cellular effects of LMW-HA here pointed out will be useful to further exploit its features and improve current formulations even though further studies on lipids biosynthesis and degradation are necessary.

Investigation of Chlorella pyrenoidosa Protein as a Source of Novel Angiotensin I-Converting Enzyme (ACE) and Dipeptidyl Peptidase-IV (DPP-IV) Inhibitory Peptides.

Chlorella pyrenoidosa (C. pyrenoidosa) is a microalgae species with a remarkably high protein content that may potentially become a source of hypotensive and hypoglycemic peptides. In this study, C. pyrenoidosa proteins were extracted and hydrolyzed overnight with pepsin and trypsin with final degrees of hydrolysis of 18.7% and 35.5%, respectively. By LC-MS/MS, 47 valid peptides were identified in the peptic hydrolysate (CP) and 66 in the tryptic one (CT). At the concentration of 1.0 mg/mL, CP and CT hydrolysates inhibit in vitro the angiotensin-converting enzyme (ACE) activity by 84.2 ± 0.37% and 78.6 ± 1.7%, respectively, whereas, tested at cellular level at the concentration of 5.0 mg/mL, they reduce the ACE activity by 61.5 ± 7.7% and 69.9 ± 0.8%, respectively. At the concentration of 5.0 mg/mL, they decrease in vitro the DPP-IV activity by 63.7% and 69.6% and in Caco-2 cells by 38.4% and 42.5%, respectively. Short peptides (≤10 amino acids) were selected for investigating the potential interaction with ACE and DPP-IV by using molecular modeling approaches and four peptides were predicted to block both enzymes. Finally, the stability of these peptides was investigated against gastrointestinal digestion.

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.

Assessment of the Physicochemical and Conformational Changes of Ultrasound-Driven Proteins Extracted from Soybean Okara Byproduct.

This study was aimed at the valorization of the okara byproduct deriving form soy food manufacturing, by using ultrasound at different temperatures for extracting the residual proteins. The physicochemical and conformational changes of the extracted proteins were investigated in order to optimize the procedure. Increasing the temperature from 20 up to 80 °C greatly enhanced the yields and the protein solubility without affecting the viscosity. The protein secondary and tertiary structures were also gradually modified in a significant way. After the ultrasonication at the highest temperature, a significant morphological transition from well-defined single round structures to highly aggregated ones was observed, which was confirmed by measuring the water contact angles and wettability. After the ultrasound process, the improvement of peptides generation and the different amino acid exposition within the protein led to an increase of the antioxidant properties. The integrated strategy applied in this study allows to foster the okara protein obtained after ultrasound extraction as valuable materials for new applications.

Analysis of Narrow-Leaf Lupin Proteins in Lupin-Enriched Pasta by Untargeted and Targeted Mass Spectrometry.

The supplementation of different food items with grain legumes and, in particular, with lupin has been demonstrated to provide useful health benefits, especially in the area of cardiovascular disease prevention. In this work, label free quantitative untargeted and targeted approaches based on liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) for investigating the protein profile of three pasta samples containing different percentages of narrow-leaf lupin flour were carried out. The untargeted method permitted the identification of the main acidic globulins (α-conglutin, ß-conglutin, and δ-conglutin) and the comparison of their profile with raw lupin flour. The targeted method, based on High-performance liquid chromatography electrospray ionization tandem mass spectrometry HPLC-Chip-Multiple Reaction Monitoring (MRM) mode, allowed the quantification of γ-conglutin, the main hypoglycemic component of lupin protein: its concentration was around 2.25 mg/g in sample A, 2.16 mg/g in sample D, and 0.57 mg/g in sample F.

Assessment of the Multifunctional Behavior of Lupin Peptide P7 and Its Metabolite Using an Integrated Strategy.

LTFPGSAED (P7) is a multifunctional hypocholesterolemic and hypoglycemic lupin peptide. While assessing its angiotensin-converting enzyme (ACE) inhibitory activity, it was more effective in intestinal Caco-2 cells (IC50 of 13.7 µM) than in renal HK-2 cells (IC50 of 79.6 µM). This discrepancy was explained by the metabolic transformation mediated by intestinal peptidases, which produced two main detected peptides, TFPGSAED and LTFPG. Indeed LTFPG, dynamically generated by intestinal dipeptidyl peptidase IV as well as its parent peptide P7 were linearly absorbed by mature Caco-2 cells. An in silico study demonstrated that the metabolite was a better ligand of the ACE enzyme than P7. These results are in agreement with an in vivo study, previously performed by Aluko et al., which has shown that LTFPG is an effective hypotensive peptide. Our work highlights the dynamic nature of bioactive food peptides that may be modulated by the metabolic activity of intestinal cells.

Phycobiliproteins from Arthrospira Platensis (Spirulina): A New Source of Peptides with Dipeptidyl Peptidase-IV Inhibitory Activity.

Arthrospira platensis (spirulina) is a cyanobacterium, which contains mainly two phycobiliproteins (PBP), i.e., C-phycocyanin (C-PC) and allophycocyanin (APC). In this study, PBP were hydrolyzed using trypsin, and the composition of the hydrolysate was characterized by HPLC-ESI-MS/MS. Furthermore, the potential anti-diabetic activity was assessed by using either biochemical or cellular techniques. Findings suggest that PBP peptides inhibit DPP-IV activity in vitro with a dose-response trend and an IC50 value falling in the range between 0.5 and 1.0 mg/mL. A lower inhibition of the DPP-IV activity expressed by Caco-2 cells was observed, which was explained by a secondary metabolic degradation exerted by the same cells.

Recent Advances in Microalgae Peptides: Cardiovascular Health Benefits and Analysis.

There is now great interest in food protein hydrolysates and food-derived peptides, because they may provide numerous health benefits. Among other foodstuffs, microalgae appear to be sustainable sources of proteins and bioactive peptides that can be exploited in foods and functional formulations. This review considers protein hydrolysates and individual peptides that may be relevant in cardiovascular disease prevention because they mimic the functions of mediators involved in pathologic processes that represent relevant risk factors for cardiovascular disease development, such as hypercholesterolemia, hypertension, diabetes, inflammation, and oxidative status. Some of these peptides are also multifunctional (i.e., they offer more than one benefit). Moreover, the most efficient techniques for protein extraction and hydrolyzation are commented on, as well as the best methodologies for high-throughput detection and quantification. Finally, current challenges and critical issues are discussed.

Soybean Peptides Exert Multifunctional Bioactivity Modulating 3-Hydroxy-3-Methylglutaryl-CoA Reductase and Dipeptidyl Peptidase-IV Targets in Vitro.

This study was aimed at evaluating the cellular mechanism through which peptic (P) and tryptic (T) soybean hydrolysates modulate the targets involved in hypocholesterolemic pathways in HepG2 and antidiabetic pathways in Caco-2 cells. Both hydrolysates (tested in the concentration range of 0.5-2.5 mg/mL) inhibited the 3-hydroxy-3-methylglutaryl-CoA reductase activity in HepG2 cells. In addition, Soybean P increased LDLR protein levels on HepG2 membranes by 51.5 ± 11.6% and 63.0 ± 6.9% (0.5-1.0 mg/mL) whereas Soybean T increased them by 55.2 ± 9.7% and 85.8 ± 21.5% (0.5-1.0 mg/mL) vs the control, with a final improved HepG2 capacity in the uptake of extracellular LDL. Soybean P reduced in vitro the dipeptidyl peptidase-IV activity by 16.3 ± 3.0% and 31.4 ± 0.12% (1.0 and 2.5 mg/mL), whereas Soybean T reduced it by 15.3 ± 11.0% and 11.0 ± 0.30% (1.0 and 2.5 mg/mL) vs the control. Finally, both hydrolysates inhibited dipeptidyl peptidase-IV activity in situ in human intestinal Caco-2 cells. This investigation may help to explain the activities observed in experimental and clinical studies.