ABSTRACT
Alzheimer's disease (AD) is the most common neurodegenerative disease. The morbidity of Alzheimer's disease is currently on the rise worldwide, but no effective treatment is available. Cornus officinalis is an herb and edible plant used in traditional Chinese medicine, whose extract has neuroprotective properties. In this investigation, we endeavored to refine a systems pharmacology strategy combining bioinformatics analysis, drug prediction, network pharmacology, and molecular docking to screen tetrahydroalstonine (THA) from Cornus officinalis as a therapeutic component for AD. Subsequent in vitro experiments were validated using MTT assay, Annexin V-PI flow cytometry, Western blotting, and immunofluorescence analysis. In Palmitate acid-induced SK-N-MC cells, THA restored the impaired PI3K/AKT signaling pathway, regulated insulin resistance, and attenuated BACE1 and GSK3ß activity. In addition, THA significantly reduced cell apoptosis rate, down-regulated relative levels of p-JNK/JNK, Bax/Bcl-2, cytochrome C, active caspase-3 and caspase-3, and attenuated Palmitate acid-induced Aß1-42 and Tau generation. THA may regulate the phenotype of AD and reduce cell apoptosis by modulating the PI3K/AKT signaling pathway. This systematic analysis provides new ramifications concerning the therapeutic utility of tetrahydroalstonine for AD.
Subject(s)
Alzheimer Disease , Neurodegenerative Diseases , Humans , Alzheimer Disease/drug therapy , Alzheimer Disease/genetics , Proto-Oncogene Proteins c-akt/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Palmitic Acid/toxicity , Amyloid Precursor Protein Secretases/metabolism , Caspase 3/metabolism , Amyloid beta-Peptides/metabolism , Molecular Docking Simulation , Aspartic Acid Endopeptidases/metabolism , Aspartic Acid Endopeptidases/pharmacology , Aspartic Acid Endopeptidases/therapeutic use , Signal Transduction , Palmitates/pharmacologyABSTRACT
This study aimed to demonstrate the potential benefits of donepezil (DPZ) and vitamin D (Vit D) in combination to counteract the neurodegenerative disorders induced by CuSO4 intake in experimental rats. Neurodegeneration (Alzheimer-like) was induced in twenty-four male Wistar albino rats by CuSO4 supplement to drinking water (10 mg/L) for 14 weeks. AD rats were divided into four groups: untreated AD group (Cu-AD) and three treated AD groups; orally treated for 4 weeks with either DPZ (10 mg/kg/day), Vit D (500 IU/kg/day), or DPZ + Vit D starting from the 10th week of CuSO4 intake. Another six rats were used as normal control (NC) group. The hippocampal tissue content of ß-amyloid precursor protein cleaving enzyme 1 (BACE1), phosphorylated Tau (p-tau), clusterin (CLU), tumor necrosis factor-α (TNF-α), caspase-9 (CAS-9), Bax, and Bcl-2 and the cortical content of acetylcholine (Ach), acetylcholinesterase (AChE), total antioxidant capacity (TAC), and malondialdehyde (MDA) were measured. Cognitive function tests (Y-maze) and histopathology studies (hematoxylin and eosin and Congo red stains) and immunohistochemistry for neurofilament. Vit D supplementation alleviated CuSO4-induced memory deficits including significant reduction hippocampal BACE1, p-tau, CLU, CAS-9, Bax, and TNF-α and cortical AChE and MDA. Vit D remarkably increased cortical Ach, TAC, and hippocampal Bcl-2. It also improved neurobehavioral and histological abnormalities. The effects attained by Vit D treatment were better than those attained by DPZ. Furthermore, Vit D boosted the therapeutic potential of DPZ in almost all AD associated behavioral and pathological changes. Vit D is suggested as a potential therapy to retard neurodegeneration.
Subject(s)
Alzheimer Disease , Brain Injuries , Cognitive Dysfunction , Rats , Male , Animals , Donepezil/adverse effects , Copper , Copper Sulfate/adverse effects , Copper Sulfate/metabolism , Alzheimer Disease/chemically induced , Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , Amyloid Precursor Protein Secretases/metabolism , Amyloid Precursor Protein Secretases/pharmacology , Amyloid Precursor Protein Secretases/therapeutic use , Vitamin D/pharmacology , Vitamin D/therapeutic use , Acetylcholinesterase/metabolism , Sulfates/metabolism , Sulfates/pharmacology , Sulfates/therapeutic use , bcl-2-Associated X Protein/metabolism , Tumor Necrosis Factor-alpha/metabolism , Rats, Wistar , Aspartic Acid Endopeptidases/metabolism , Aspartic Acid Endopeptidases/pharmacology , Aspartic Acid Endopeptidases/therapeutic use , Brain Injuries/metabolism , Antioxidants/pharmacology , Antioxidants/therapeutic use , Antioxidants/metabolism , Vitamins/pharmacology , Brain , Cognitive Dysfunction/chemically inducedABSTRACT
In this article, we report the cloning of an aspartic protease (AP) from flowers of Arctium minus (Hill) Bernh. (Asteraceae) along with the use of depigmented aqueous flower extracts, as a source of APs, for the hydrolysis of whey proteins. The isolated cDNA encoded a protein product with 509 amino acids called arctiumisin, with the characteristic primary structure organization of typical plant APs. Bovine whey protein hydrolysates, obtained employing the enzyme extracts of A. minus flowers, displayed inhibitory angiotensin-converting enzyme (ACE) and antioxidant activities. Hydrolysates after 3 and 5 h of reaction (degree of hydrolysis 2.4 and 5.6, respectively) and the associated peptide fraction with molecular weight below 3 kDa were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, matrix-assisted laser desorption ionization/time of flight mass spectrometry, and reverse phase-high-performance liquid chromatography. The results obtained in this study demonstrate the viability of using proteases from A. minus to increase the antioxidant and inhibitory ACE capacity of whey proteins.
Subject(s)
Arctium/chemistry , Aspartic Acid Endopeptidases/chemistry , Aspartic Acid Endopeptidases/genetics , Aspartic Acid Endopeptidases/pharmacology , Aspartic Acid Proteases/chemistry , Hydrolysis/drug effects , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Proteins/pharmacology , Whey Proteins/chemistry , Angiotensin-Converting Enzyme Inhibitors/chemistry , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Animals , Antioxidants/chemistry , Antioxidants/pharmacology , Cattle , Chromatography, High Pressure Liquid/methods , DNA, Complementary , Flowers/chemistry , Mass Spectrometry/methods , Peptide Fragments/genetics , Plant Extracts/chemistryABSTRACT
Artichoke (Cynara scolymus L.) flower extract was assayed with the aim of replacing animal rennet in the manufacture of Gouda-type cheeses from bovine milk. Floral extract coagulated milk within a suitable time for use on an industrial scale, while the yield of cheese obtained was equal to that achieved with bovine abomasum. Five proteolytic fractions with milk-clotting activity were isolated in a two-step purification protocol, three belonging to the cardosin group. Cheeses made with C. scolymus proteases must be brined for a longer period (40 h) to prevent overproteolysis and avoid the development of a background flavor. The type of coagulant (bovine or vegetable) had no significant effect on the cheeses' chemical parameters analyzed throughout ripening, and no significant organoleptic differences were detected between those manufactured with C. scolymus or animal rennet. The results indicate that C. scolymus flower extract is suitable for replacing animal rennet in the production of Gouda-type cheeses.
Subject(s)
Aspartic Acid Endopeptidases/pharmacology , Cheese , Chymosin/pharmacology , Cynara scolymus/chemistry , Plant Extracts/pharmacology , Amino Acid Sequence , Animals , Caseins/metabolism , Cattle , Cheese/analysis , Flowers , Molecular Sequence DataABSTRACT
Specific roles of glycosylation appear to be protein-dependent. Plant aspartic proteases (APs) contain two or more consensus N-glycosylation sites; however, the importance of them is not well understood. StAPs (Solanum tuberosum aspartic proteases) are bifunctional proteins with both proteolytic and antimicrobial activities. These proteins are accumulated into the intercellular washing fluid of potato tubers and leaves after wounding or infection. In this paper we investigated the importance of glycosylation on the StAPs apoplast accumulation, biochemical parameters, and fungicidal activity. Assays to evaluate the importance of StAPs glycosylation groups by using glycosylation inhibitors demonstrate that carbohydrate portions are essential to StAPs accumulation into the apoplast of tubers and leaves after wounding or detachment, respectively. Bifunctional activity of StAPs is differentially affected by this post-translational modification. Results obtained show that not significant changes were produced in the physicochemical properties after StAPs deglycosylation (pH and thermal-optimum activity and index of protein surface hydrophobicity). Otherwise, StAPs antifungal activity is affected by deglycosylation. Deglycosylated StAPs (dgStAPs) fungicidal activity is lower than native StAPs at all concentrations and times assayed. In summary, glycosylation has not a significant role on the StAPs conformational structure. However, it is involved in the StAPs subcellular accumulation and antifungal activity suggesting that it could be necessary for StAPs membrane and/or protein interactions and subsequently its biological function(s).
Subject(s)
Antifungal Agents/pharmacology , Aspartic Acid Endopeptidases/metabolism , Aspartic Acid Endopeptidases/pharmacology , Plant Roots/enzymology , Solanum tuberosum/enzymology , Glycosylation , Kinetics , Plant Leaves/enzymology , Surface Properties , Wound HealingABSTRACT
Solanum tuberosum aspartic proteases (StAPs) with antimicrobial activity are induced after abiotic and biotic stress. In this study the ability of StAPs to produce a direct antimicrobial effect was investigated. Viability assays demonstrated that StAPs are able to kill spores of Fusarium solani and Phytophthora infestans in a dose-dependent manner. Localization experiments with FITC-labelled StAPs proved that the proteins interact directly with the surface of spores and hyphae of F. solani and P. infestans. Moreover, incubation of spores and hyphae with StAPs resulted in membrane permeabilization, as shown by the uptake of the fluorescent dye SYTOX Green. It is concluded that the antimicrobial effect of StAPs against F. solani and P. infestans is caused by a direct interaction with the microbial surfaces followed by membrane permeabilization.
Subject(s)
Antifungal Agents/pharmacology , Aspartic Acid Endopeptidases/pharmacology , Plant Proteins/pharmacology , Solanum tuberosum/enzymology , Cell Membrane Permeability , Dose-Response Relationship, Drug , Fusarium/drug effects , Osmolar Concentration , Phytophthora/drug effects , Reactive Oxygen SpeciesABSTRACT
The emergence and worldwide spreading of Plasmodium falciparum strains that shown to be resistant to traditional drugs is considered a very serious health problem, given the high mortality and morbidity rate of Malaria. In the search for new drugs against this parasite, Hb hydrolyzing enzymes, such as Plasmepsin II (Plm II), have been classified as very promising targets for therapeutic attacks. In this work, it is developed a cheap and high-throughput heterogeneous enzymatic assay for measuring Plasmepsin II activity in order to use it as a tool in the discovery of new inhibitors of this enzyme. In this assay, Plasmepsin II acts upon a solid-phase bound synthetic peptide (DU2) whose sequence comprises the cleavage site F(33)-L(34) present in Hb alpha-chain. The peptide surface density is quantified by means of a classical ELISA-based procedure. In order to estimate the kinetic constants of the system and to quantify both, enzymatic and inhibitory activity, it was used a model for the kinetics of enzyme quasi-saturable systems previously developed by our group, that fitted very well to the experimental data. It was used Pepstatin as a model inhibitor of Plasmepsin II and the resulting dose-response relation agreed with the expected behavior for the Pepstatin-Plasmepsin II pair under the employed experimental conditions.