1,721,083 research outputs found
STUDIO DELL'INTERAZIONE TRA LE PROTEINE CITOSCHELETRICHE 4.1R80 E 4.1R135 E LA PROTEINA ICLN: ASPETTI MOLECOLARI E FUNZIONALI.
Full text linkICln is a multifunctional protein that has been implicated in many cellular functions, from cell volume regulation to RNA metabolism. It interacts with many members or regulators of the cytoskeleton, like the 4.1R protein, actin, myosin light chain and the JBP1 protein. This suggests that the cytoskeleton rearrangement that follows an hypotonic challenge could be involved in the translocation of the ICln protein from the cytosol to the cell membrane and in the activation of the ICl,swell. In this work we tried to elucidate the role of ICln-4.1R interaction in the context of Regulatory Volume Decrease; we focused on the in vivo interaction between ICln and the protein 4.1R by the use of the FRET technique. We used two different 4.1R isoforms for the FRET experiments: an high molecular weight (HMW) isoform (4.1 long) and a low molecular weight (LMW) one (4.1 short, without the initial 209 aa box). The strongest FRET signal was measured between ICln and 4.1Rsh, and it was significally increased by an hypotonic shock. Moreover, when the two proteins are co-expressed and interact, it seems that the two proteins change each other localization showing a decreased nuclear and, for 4.1R, membrane localization. From a functional point of view, preliminary patch-clamp experiments suggest that the over-expression of the 4.1sh isoform in HEK cells leads to an increase of the ICl,swell (the anion current that allows RVD) activation, thus confirming a role for 4.1R in cell volume regulation
In-cell western assay: a new approach to study the hypocholesterolemic effects of food components at HepG2 cell line
No full textCardiovascular disease, such as atherosclerosis and coronary heart disease, is a leading death cause in the developed world. Significant research efforts focusing on the prevention and treatment of this disease have identified elevated plasma cholesterol as primary risk factor for cardiovascular disease. In particular, the low density lipoprotein (LDL) receptor (LDLR) is a transmembrane glycoprotein that plays a pivotal role in the binding and endocytosis of circulating LDL increasing its plasma clearance. In literature, it is widely recognized that the LDLR pathway could be implicated in the hypocholesterolemic activity of several food components, such as plant proteins (soy [1,2], lupin [3,4]) and polyphenols (resveratrol [5]) via the activation of transcription factors and the expression of lipogenic enzymes. In particular, the transcription factor Sterol Regulatory Element Binding Protein (SREBP) Pathway is directly involved in the expression regulation of some lipogenic enzymes, such as LDLR and 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase).
The aim of the present work is the development of the In-Cell Western (ICW), a novel quantitative colorimetric cell-based technique, in order to screen and to evaluate in a throughput way the effects of lupin bioactive peptides on the LDLR-SREBP2 pathway. In particular, the ICW has been optimized and validated at HepG2 cell line using lovastatin as known reference compound. The ICW optimization is a multi step process in which different parameters and conditions have been standardized at HepG2 cells. Experiments have been performed in 96-well plate format using fixed cells in order to establish the right conditions in which lovastatin is able to properly stimulate LDLR. This optimized method will be used for the studying the effects of food components. Results demonstrated that the ICW optimization and validation have provided a robust, reproducible, and easy to use assay that will permit to study the functionality of plant peptides, such as lupin bioactive peptides through the detection and quantification of LDLR protein level directly in the HepG2 cells.
References:
1) Lovati MR, Manzoni C, Gianazza E, Arnoldi A, Kurowska E, Carroll KK, and. Sirtori CR. Soy Protein Peptides Regulate Cholesterol Homeostasis in Hep G2 Cells. J Nutr. 2000;130:2543-2549.
2) Torres N, Torre-Villalvazo I, Tovar AR. Regulation of lipid metabolism by soy protein and its implication in diseases mediated by lipid disorders. J Nutr Biochem. 2006;17:365-73
3) Sirtori CR, Lovati MR, Manzoni C, Castiglioni S, Duranti M, Magni C, Morandi S, D'Agostina A, Arnoldi A. Proteins of white lupin seed, a naturally isoflavone-poor legume, reduce cholesterolemia in rats and increase LDL receptor activity in HepG2 cells. J Nutr. 2004;134:18-23.
4) Bettzieche A, Brandsch C, Weisse K, Hirche F, Eder K, Stangl GI. Lupin protein influences the expression of hepatic genes involved in fatty acid synthesis and triacylglycerol hydrolysis of adult rats. Br J Nutr. 2008;99:952-62.
5) Takuya Y., Manami N., Makoto S., Jun I., Ryuichiro S. Resveratrol increases the expression and activity of the low density lipoprotein receptor in hepatocytes by the proteolytic activation of the sterol regulatory element-binding proteins. Atherosclerosis 2012 ; 220:369-38
A simple and high-throughput in-cell Western assay using HepG2 cell line for investigating the potential hypocholesterolemic effects of food components and nutraceutics
No full textSince saving time and money are critical issues while developing innovative functional foods and nutraceutics, the use of specific and high-throughput assays for the fast screening of potentially bioactive ingredients is crucial. In this context, the aim of the present investigation was the development of an in-cell Western (ICW) assay, a quantitative colorimetric cell-based technique, at the HepG2 cell line for screening and evaluating the effects of potentially bioactive compounds on the low density lipoprotein (LDL) receptor (LDLR). It is known that LDLR plays a pivotal role in the binding and endocytosis of circulating LDL, increasing its plasma clearance. The ICW was optimised and validated using monacolin K, the main hypocholesterolemic component of red yeast rice. This provided a robust and reproducible assay useful for characterising the cholesterol-lowering properties of bioactive food components. To our knowledge, this is the first application of the ICW technique in the field of functional foods and nutraceutics
Cholesterol-lowering properties of lupin peptides: molecular and functional investigations at Hepg2 cell line
No full textAlthough statins are the main drugs for the treatment of hypercholesterolemic subjects, there is still interest for nutraceutics either to enhance the potency of drugs or to avoid their undesired side effects. An important dietary component able to reduce cholesterolemia is soy protein1, whose stimulated research on other legumes, in particular on lupin. Indeed investigations in suitable animal models2;3 and in the clinics4 demonstrated that this legume is able to lower cholesterol in case of moderate hypercholesterolemia.
Having the objective of investigating the mechanism of this activity, in the present study we characterized the cholesterol lowering properties of lupin peptides, obtained after pepsin (P) and trypsin (T) hydrolysis. In particular, we performed a molecular and functional investigations of the effects on LDL receptor / SREBP2 pathway at HepG2 cell line. For the first time, we provided the experimental evidence that lupin peptides P and T are able to up-regulate the LDL receptor and SREBP2 proteins through an activation of PI3K/Akt/GSK3αβ pathways increasing the LDL-uptake at HepG2 cell line.
References
1 Sirtori, C.R.; Eberini, I.; Arnoldi, A.; Hypocholesterolaemic effects of soya proteins: results of recent studies are predictable from the Anderson meta-analysis data. Brit. J. Nutr. 2007, 97, 816-822.
2 Sirtori, C.R.; Lovati, M.R.; Manzoni, C.; et al.; Proteins of white lupin seed, a naturally isoflavone-poor legume, reduce cholesterolemia in rats and increase LDL receptor activity in HepG2 cells. J Nutr, 2004, 134, 18-23.
3 Marchesi, M.; Parolini, C.; Diani, E.; et al.; Hypolipidaemic and anti-atherosclerotic effects of lupin proteins in a rabbit model. Brit. J. Nutr., 2008, 100, 707-710.
4 Sirtori, C.R.; Triolo, M.; Bosisio, R.; et al.; Hypocholesterolaemic effects of lupin protein and pea protein/fibre combinations in moderately hypercholesterolaemic individuals. Brit. J. Nutr., 2012, 107, 1176-1183
In-Cell Western Assay: a tool to study the hypocholesterolemic effects of potentially bioactive food components at HepG2 cell line
No full textSince reducing time and saving money are very important issues while developing innovative functional foods and nutraceutics, it is crucial to use specific and highly throughput assays for the fast screening of potentially bioactive ingredients. The In-Cell Western (ICW) assay, a quantitative colorimetric cell-based technique, has all these characteristics. Having the final goal of sorting out novel hypocholesterolemic food components, the aim of the present investigation was the development of the In-Cell Western (ICW) at the HepG2 cell line for screening and evaluating the effects of potentially bioactive compounds at the low density lipoprotein (LDL) receptor (LDLR). This receptor plays a pivotal role in the binding and endocytosis of circulating LDL, increasing its plasma clearance. The ICW was optimized and validated by using monacolin K, the main hypocholesterolemic component of red yeast rice. Experiments were performed using fixed cells in order to establish the best conditions for a proper stimulation of the LDLR protein level. Subsequently, the ICW was used to investigate the activity of peptide mixtures obtained by pepsin or trypsin hydrolysis of lupin protein. The ICW optimization and validation provided a robust and reproducible assay useful for characterizing the cholesterol-lowering properties of bioactive food components. It is important to underline that this is the first application of the ICW technique in the field of functional foods and nutraceutics
IAVPGEVA, IAVPTGVA, and LPYP, three peptides from soy glycinin, modulate cholesterol metabolism in HepG2 cells through the activation of the LDLR-SREBP2 pathway
No full textAlthough the hypocholesterolemic activity of soy foods is well known from decades, its molecular mechanism is still poorly understood. The aim of this study was a detailed molecular characterization of the activity of IAVPGEVA, IAVPTGVA, and LPYP, three peptides from soy glycinin hydrolysis, which in the past had been shown to be competitive inhibitors of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCoAR). HepG2 cells were treated with these peptides and the samples were analyzed with a combination of molecular techniques. The experiments demonstrated that they are able to interfere with the catalytic activity of HMGCoAR and to modulate the cholesterol metabolism, through the activation of the LDLR-SREBP2 pathway, increasing the ability of HepG2 cells to uptake the LDL. This involves also the activation of AMPK and ERK 1/2. For the first time, this study provides a characterization of the molecular mechanism
Functional foods for human health benefits: focus on lupin
No full textIn the last decades, numerous studies have reported the beneficial effects of soy proteins and in 1999, the America Food and Drug Administration has approved the health claim that soy protein is useful to reduce the risk of cardiovascular disease, since it is able to control the serum cholesterol level. Subsequently, nutritionists have now a main interest in soy and other legume proteins not only for their nutritional role, but also for the positive effects that they may exert on the human body, including hypotensive, hypoglycemic, hypolipidemic, anti-carcinogenic and anti-obesity activities. Numerous teams around the world have started to investigate the health benefits deriving from the consumption of different legumes, such as lupin, chickpea, common bean, faba bean, pea, lentils, etc.
In this scenario, lupin is a very special case, since its seed contain up to 35-40% protein as in the case of soybean, but are completely devoid of isoflavones1. Nowadays, four Mediterranean species of lupin are cultivated, i.e. Lupinus albus, L. angustifolius, L. luteus and L. mutabilis.
Numerous teams have studied the biological activities of lupin proteins and investigated the possible health benefits derived from their consumption. In particular, studies have evaluated either in animal model (rats) or in clinical study the hypocholesterolemic, hypoglycemic and anti-hypertensive effect of lupin. Another study has shown, in a rabbit model, a protective activity of lupin protein against atherosclerosis progression. In parallel, the mechanism of action at molecular level is under investigation
Investigation of the hypocholesterolemic mechanism of action of soy and lupin peptides
No full textThe investigations on the mechanism of action of bioactive components of functional foods and nutraceutics are not very frequent and in general not very detailed. We have recently established a cell culture lab aimed to fill this gap, starting our work from grain legumes. In fact, the seeds of grain legumes, besides being major foodstuffs in most countries, provide also some health benefits, in particular in the area of hypercholesterolemia and hypertension prevention. The main objective of the present work was to provide evidences that peptides generated by the digestion of soy and lupin proteins may be responsible of the hypocholesterolemic activity observed in vivo and to investigate the mechanism of action. In particular, a deepened study was carried out in order to characterize the hypocholesterolemic effects of pure peptides from soy proteins as well as peptide mixtures obtained by pepsin and trypsin hydrolysis of lupin proteins. The hepatic cell line HepG2 was treated with these peptides and molecular and functional investigations were performed on the LDL receptor / SREBP2 pathway. For the first time, this report provides the experimental evidence that, interfering with the HMGCoAR activity, legume peptides are able to up-regulate the LDL receptor and SREBP2 proteins via the activation of specific phosphorylation pathways and increasing the LDL-uptake at HepG2 cell line
- …
