RESUMEN
High-density lipoproteins (HDLs) are responsible for removing cholesterol from arterial walls, through a process known as reverse cholesterol transport. The main protein in HDL, apolipoprotein A-I (ApoA-I), is essential to this process, and changes in its sequence significantly alter HDL structure and functions. ApoA-I amyloidogenic variants, associated with a particular hereditary degenerative disease, are particularly effective at facilitating cholesterol removal, thus protecting carriers from cardiovascular disease. Thus, it is conceivable that reconstituted HDL (rHDL) formulations containing ApoA-I proteins with functional/structural features similar to those of amyloidogenic variants hold potential as a promising therapeutic approach. Here we explored the effect of protein cargo and lipid composition on the function of rHDL containing one of the ApoA-I amyloidogenic variants G26R or L174S by Fourier transformed infrared spectroscopy and neutron reflectometry. Moreover, small-angle x-ray scattering uncovered the structural and functional differences between rHDL particles, which could help to comprehend higher cholesterol efflux activity and apparent lower phospholipid (PL) affinity. Our findings indicate distinct trends in lipid exchange (removal vs. deposition) capacities of various rHDL particles, with the rHDL containing the ApoA-I amyloidogenic variants showing a markedly lower ability to remove lipids from artificial membranes compared to the rHDL containing the native protein. This effect strongly depends on the level of PL unsaturation and on the particles' ultrastructure. The study highlights the importance of the protein cargo, along with lipid composition, in shaping rHDL structure, contributing to our understanding of lipid-protein interactions and their behavior.
Asunto(s)
Apolipoproteína A-I , Lipoproteínas HDL , Lipoproteínas HDL/química , Lipoproteínas HDL/metabolismo , Apolipoproteína A-I/genética , Membranas Artificiales , Colesterol/metabolismo , FosfolípidosRESUMEN
AIMS/HYPOTHESIS: Roux-en-Y gastric bypass surgery (RYGB) frequently results in remission of type 2 diabetes as well as exaggerated secretion of glucagon-like peptide-1 (GLP-1). Here, we assessed RYGB-induced transcriptomic alterations in the small intestine and investigated how they were related to the regulation of GLP-1 production and secretion in vitro and in vivo. METHODS: Human jejunal samples taken perisurgically and 1 year post RYGB (n=13) were analysed by RNA-seq. Guided by bioinformatics analysis we targeted four genes involved in cholesterol biosynthesis, which we confirmed to be expressed in human L cells, for potential involvement in GLP-1 regulation using siRNAs in GLUTag and STC-1 cells. Gene expression analyses, GLP-1 secretion measurements, intracellular calcium imaging and RNA-seq were performed in vitro. OGTTs were performed in C57BL/6j and iScd1-/- mice and immunohistochemistry and gene expression analyses were performed ex vivo. RESULTS: Gene Ontology (GO) analysis identified cholesterol biosynthesis as being most affected by RYGB. Silencing or chemical inhibition of stearoyl-CoA desaturase 1 (SCD1), a key enzyme in the synthesis of monounsaturated fatty acids, was found to reduce Gcg expression and secretion of GLP-1 by GLUTag and STC-1 cells. Scd1 knockdown also reduced intracellular Ca2+ signalling and membrane depolarisation. Furthermore, Scd1 mRNA expression was found to be regulated by NEFAs but not glucose. RNA-seq of SCD1 inhibitor-treated GLUTag cells identified altered expression of genes implicated in ATP generation and glycolysis. Finally, gene expression and immunohistochemical analysis of the jejunum of the intestine-specific Scd1 knockout mouse model, iScd1-/-, revealed a twofold higher L cell density and a twofold increase in Gcg mRNA expression. CONCLUSIONS/INTERPRETATION: RYGB caused robust alterations in the jejunal transcriptome, with genes involved in cholesterol biosynthesis being most affected. Our data highlight SCD as an RYGB-regulated L cell constituent that regulates the production and secretion of GLP-1.
Asunto(s)
Diabetes Mellitus Tipo 2 , Derivación Gástrica , Humanos , Animales , Ratones , Péptido 1 Similar al Glucagón/metabolismo , Derivación Gástrica/métodos , Células L , Diabetes Mellitus Tipo 2/metabolismo , ARN , Ratones Endogámicos C57BL , Análisis de Secuencia de ARN , Colesterol , ARN Mensajero , Glucemia/metabolismoRESUMEN
There is a close relationship between the SARS-CoV-2 virus and lipoproteins, in particular high-density lipoprotein (HDL). The severity of the coronavirus disease 2019 (COVID-19) is inversely correlated with HDL plasma levels. It is known that the SARS-CoV-2 spike (S) protein binds the HDL particle, probably depleting it of lipids and altering HDL function. Based on neutron reflectometry (NR) and the ability of HDL to efflux cholesterol from macrophages, we confirm these observations and further identify the preference of the S protein for specific lipids and the consequent effects on HDL function on lipid exchange ability. Moreover, the effect of the S protein on HDL function differs depending on the individuals lipid serum profile. Contrasting trends were observed for individuals presenting low triglycerides/high cholesterol serum levels (LTHC) compared to high triglycerides/high cholesterol (HTHC) or low triglycerides/low cholesterol serum levels (LTLC). Collectively, these results suggest that the S protein interacts with the HDL particle and, depending on the lipid profile of the infected individual, it impairs its function during COVID-19 infection, causing an imbalance in lipid metabolism.
Asunto(s)
COVID-19 , Lipoproteínas HDL , Humanos , Glicoproteína de la Espiga del Coronavirus , SARS-CoV-2/metabolismo , Colesterol , TriglicéridosRESUMEN
Cyanogenic glucosides are important defense molecules in plants with useful biological activities in animals. Their last biosynthetic step consists of a glycosylation reaction that confers stability and increases structural diversity and is catalyzed by the UDP-dependent glycosyltransferases (UGTs) of glycosyltransferase family 1. These versatile enzymes have large and varied substrate scopes, and the structure-function relationships controlling scope and specificity remain poorly understood. Here, we report substrate-bound crystal structures and rational engineering of substrate and stereo-specificities of UGT85B1 from Sorghum bicolor involved in biosynthesis of the cyanogenic glucoside dhurrin. Substrate specificity was shifted from the natural substrate (S)-p-hydroxymandelonitrile to (S)-mandelonitrile by combining a mutation to abolish hydrogen bonding to the p-hydroxyl group with a mutation to provide steric hindrance at the p-hydroxyl group binding site (V132A/Q225W). Further, stereo-specificity was shifted from (S) to (R) by substituting four rationally chosen residues within 6 Å of the nitrile group (M312T/A313T/H408F/G409A). These activities were compared to two other UGTs involved in the biosynthesis of aromatic cyanogenic glucosides in Prunus dulcis (almond) and Eucalyptus cladocalyx. Together, these studies enabled us to pinpoint factors that drive substrate and stereo-specificities in the cyanogenic glucoside biosynthetic UGTs. The structure-guided engineering of the functional properties of UGT85B1 enhances our understanding of the evolution of UGTs involved in the biosynthesis of cyanogenic glucosides and will enable future engineering efforts towards new biotechnological applications.
Asunto(s)
Aminoácidos , Nitrilos , Animales , Glucósidos/metabolismo , Glicósidos , Glicosiltransferasas , Nitrilos/metabolismo , Uridina DifosfatoRESUMEN
Amyloidoses are characterized by the accumulation and aggregation of misfolded proteins into fibrils in different organs, leading to cell death and consequent organ dysfunction. The specific substitution of Leu 75 for Pro in Apolipoprotein A-I protein sequence (ApoA-I; L75P-ApoA-I) results in late onset amyloidosis, where deposition of extracellular protein aggregates damages the normal functions of the liver. In this work, we describe that the autophagic process is inhibited in the presence of the L75P-ApoA-I amyloidogenic variant in stably transfected human hepatocyte carcinoma cells. The L75P-ApoA-I amyloidogenic variant alters the redox status of the cells, resulting into excessive mitochondrial stress and consequent cell death. Moreover, L75P-ApoA-I induces an impairment of the autophagic flux. Pharmacological induction of autophagy or transfection-enforced overexpression of the pro-autophagic transcription factor EB (TFEB) restores proficient proteostasis and reduces oxidative stress in these experimental settings, suggesting that pharmacological stimulation of autophagy could be a promising target to alleviate ApoA-I amyloidosis.
Asunto(s)
Amiloidosis , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas , Amiloidosis/genética , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Autofagia/genética , Humanos , Agregado de ProteínasRESUMEN
Apolipoprotein A-I (ApoA-I) amyloidosis is a rare protein misfolding disease where fibrils of the N-terminal domain of the protein accumulate in several organs, leading to their failure. Although ApoA-I amyloidosis is systemic, the different amyloidogenic variants show a preferential tissue accumulation that appears to correlate with the location of the mutation in the protein sequence and with the local extracellular microenvironment. However, the factors leading to cell/tissues damage, as well as the mechanisms behind the observed organ specificity are mostly unknown. Therefore, we investigated the impact of ApoA-I variants on cell physiology and the mechanisms driving the observed tissue specificity. We focused on four ApoA-I amyloidogenic variants and analyzed their cytotoxicity as well as their ability to alter redox homeostasis in cell lines from different tissues (liver, kidney, heart, skin). Moreover, variant-specific interactions with extracellular matrix (ECM) components were measured by synchrotron radiation circular dichroism and enzyme-linked immunosorbent assay. Data indicated that ApoA-I variants exerted a cytotoxic effect in a time and cell-type-specific manner that seems to be due to protein accumulation in lysosomes. Interestingly, the ApoA-I variants exhibited specific preferential binding to the ECM components, reflecting their tissue accumulation pattern in vivo. While the binding did not to appear to affect protein conformations in solution, extended incubation of the amyloidogenic variants in the presence of different ECM components resulted in different aggregation propensity and aggregation patterns.
Asunto(s)
Amiloidosis , Apolipoproteína A-I , Humanos , Apolipoproteína A-I/metabolismo , Especificidad de Órganos/genética , Amiloidosis/metabolismo , Mutación , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Amiloide/metabolismoRESUMEN
Apolipoprotein E (ApoE), an important mediator of lipid transportation in plasma and the nervous system, plays a large role in diseases such as atherosclerosis and Alzheimer's. The major allele variants ApoE3 and ApoE4 differ only by one amino acid. However, this difference has major consequences for the physiological behaviour of each variant. In this paper, we follow (i) the initial interaction of lipid-free ApoE variants with model membranes as a function of lipid saturation, (ii) the formation of reconstituted High-Density Lipoprotein-like particles (rHDL) and their structural characterisation, and (iii) the rHDL ability to exchange lipids with model membranes made of saturated lipids in the presence and absence of cholesterol [1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) with and without 20 mol% cholesterol]. Our neutron reflection results demonstrate that the protein variants interact differently with the model membranes, adopting different protein conformations. Moreover, the ApoE3 structure at the model membrane is sensitive to the level of lipid unsaturation. Small-angle neutron scattering shows that the ApoE containing lipid particles form elliptical disc-like structures, similar in shape but larger than nascent or discoidal HDL based on Apolipoprotein A1 (ApoA1). Neutron reflection shows that ApoE-rHDL do not remove cholesterol but rather exchange saturated lipids, as occurs in the brain. In contrast, ApoA1-containing particles remove and exchange lipids to a greater extent as occurs elsewhere in the body.
RESUMEN
Metabolic control is mediated by the dynamic assemblies and function of multiple redox enzymes. A key element in these assemblies, the P450 oxidoreductase (POR), donates electrons and selectively activates numerous (>50 in humans and >300 in plants) cytochromes P450 (CYPs) controlling metabolism of drugs, steroids and xenobiotics in humans and natural product biosynthesis in plants. The mechanisms underlying POR-mediated CYP metabolism remain poorly understood and to date no ligand binding has been described to regulate the specificity of POR. Here, using a combination of computational modeling and functional assays, we identify ligands that dock on POR and bias its specificity towards CYP redox partners, across mammal and plant kingdom. Single molecule FRET studies reveal ligand binding to alter POR conformational sampling, which results in biased activation of metabolic cascades in whole cell assays. We propose the model of biased metabolism, a mechanism akin to biased signaling of GPCRs, where ligand binding on POR stabilizes different conformational states that are linked to distinct metabolic outcomes. Biased metabolism may allow designing pathway-specific therapeutics or personalized food suppressing undesired, disease-related, metabolic pathways.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Ligandos , Redes y Vías Metabólicas , Aromatasa/metabolismo , Línea Celular , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Pruebas de Enzimas , Transferencia Resonante de Energía de Fluorescencia , Humanos , Liposomas/metabolismo , Simulación del Acoplamiento Molecular , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Imagen Individual de Molécula , Esteroide 17-alfa-Hidroxilasa/metabolismo , Esteroide 21-Hidroxilasa/metabolismo , Especificidad por SustratoRESUMEN
Apolipoprotein A-I (ApoA-I) of high density lipoproteins (HDLs) is essential for the transportation of cholesterol between peripheral tissues and the liver. However, specific mutations in ApoA-I of HDLs are responsible for a late-onset systemic amyloidosis, the pathological accumulation of protein fibrils in tissues and organs. Carriers of these mutations do not exhibit increased cardiovascular disease risk despite displaying reduced levels of ApoA-I/HDL cholesterol. To explain this paradox, we show that the HDL particle profiles of patients carrying either L75P or L174S ApoA-I amyloidogenic variants show a higher relative abundance of the 8.4-nm versus 9.6-nm particles and that serum from patients, as well as reconstituted 8.4- and 9.6-nm HDL particles (rHDL), possess increased capacity to catalyze cholesterol efflux from macrophages. Synchrotron radiation circular dichroism and hydrogen-deuterium exchange revealed that the variants in 8.4-nm rHDL have altered secondary structure composition and display a more flexible binding to lipids than their native counterpart. The reduced HDL cholesterol levels of patients carrying ApoA-I amyloidogenic variants are thus balanced by higher proportion of small, dense HDL particles, and better cholesterol efflux due to altered, region-specific protein structure dynamics.
Asunto(s)
Apolipoproteína A-IRESUMEN
The increase of plasma levels of high-density lipoproteins and Apolipoprotein A-I (ApoA-I), its main protein component, has been shown to have a positive action on glucose disposal in type 2 diabetic patients. The current study investigates the unexplored function of ApoA-I to prime beta cells for improved insulin secretion. INS-1E rat clonal beta cells as well as isolated murine islets were used to study the effect of ApoA-I on responsiveness of the beta cells to high glucose challenge. Confocal and transmission electron microscopy were used to dissect ApoA-I mechanisms of action. Chemical endocytosis blockers were used to understand the role of ApoA-I internalization in mediating its positive effect. Pre-incubation of beta cells and isolated murine islets with ApoA-I augmented glucose stimulated insulin secretion. This effect appeared to be due to an increased reservoir of insulin granules at the cell membrane, as confirmed by confocal and transmission electron microscopy. Moreover, ApoA-I induced pancreatic and duodenal homeobox 1 (PDX1) shuttling from the cytoplasm to the nucleus, with the subsequent increase in the proinsulin processing enzyme protein convertase 1 (PC1/3). Finally, the blockade of ApoA-I endocytosis in beta cells resulted in a loss of ApoA-I positive action on insulin secretion. The proposed mechanisms of the phenomenon here described include ApoA-I internalization into beta cells, PDX1 nuclear translocation, and increased levels of proinsulin processing enzymes. Altogether, these events lead to an increased number of insulin granules.
Asunto(s)
Apolipoproteína A-I/metabolismo , Glucosa/metabolismo , Secreción de Insulina/fisiología , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Animales , Línea Celular Tumoral , Diabetes Mellitus Tipo 2/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Lipoproteínas HDL/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , RatasRESUMEN
The Wall Gecko shows heterogeneous colour pattern, which may vary among individuals, depending on the time of day and on the habitat segregation. Nocturnal pale geckos live exclusively on walls. Diurnal dark geckos preferentially live on olive tree trunks, demonstrating an ability to change skin colour that is superior to that of the pale gecko and allows diurnal geckos becoming camouflaged on the diverse substrates occupied during the day. In our study, the nocturnal/pale/wall and diurnal/dark/trunk geckos could be considered the extremes of an ecological cline of morphological variation on which divergent selection may be acting. Combining the effect of balancing selection on nocturnal geckos and disruptive selection between two sympatric populations could lead to speciation. All geckos analysed here belong to the same species, as confirmed by genetic characterization, however diurnal and nocturnal gecko populations seem to be in an early stage of incipient speciation. These two different morphs still combine genes, as revealed by neutral genetic markers, yet they show complete separation according to the analyses of mtDNA coding genes. Experimental results show that diurnal and nocturnal geckos do not swap their niches, likely because the predation pressure causes severe selection for background matching. Genomic analysis of complete mtDNA suggests that nocturnal geckos seem to be under balancing selection perhaps due to the narrow niche in which they live, whereas the daytime population has more opportunity in fitting into the multiple available niches, and they experience positive selection. Here we hypothesize that the ecological segregation that we are witnessing between the nocturnal and diurnal geckos, can lead to a ecological speciation.
Asunto(s)
Adaptación Fisiológica , ADN Mitocondrial/genética , Especiación Genética , Lagartos/genética , AnimalesRESUMEN
Apolipoprotein A-I (ApoA-I)-related amyloidosis is a rare disease caused by missense mutations in the APOA1 gene. These mutations lead to protein aggregation and abnormal accumulation of ApoA-I amyloid fibrils in heart, liver, kidneys, skin, nerves, ovaries, or testes. Consequently, the carriers are at risk of single- or multi-organ failure and of need of organ transplantation. Understanding the basic molecular structure and function of ApoA-I amyloidogenic variants, as well as their biological effects, is, therefore, of great interest. However, the intrinsic low stability of this type of proteins makes their overexpression and purification difficult. To overcome this barrier, we here describe an optimized production and purification procedure for human ApoA-I amyloidogenic proteins that efficiently provides between 46 mg and 91 mg (depending on the protein variant) of pure protein per liter of Escherichia coli culture. Structural integrity of the amyloidogenic and native ApoA-I proteins were verified by circular dichroism spectroscopy and intrinsic fluorescence analysis, and preserved functionality was demonstrated by use of a lipid clearance assay as well as by reconstitution of high-density lipoprotein (HDL) particles. In conclusion, the use of the described high-yield protein production system to obtain amyloidogenic ApoA-I proteins, and their native counterpart, will enable molecular and cellular experimental studies aimed to explain the molecular basis for this rare disease.
Asunto(s)
Apolipoproteína A-I/biosíntesis , Escherichia coli/metabolismo , Variación Genética , Proteínas Recombinantes/biosíntesis , Apolipoproteína A-I/genética , Apolipoproteína A-I/aislamiento & purificación , Escherichia coli/genética , Variación Genética/genética , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Exposure to UV light triggers the rapid generation and accumulation of reactive oxygen species (ROS) in skin cells, with consequent increase in oxidative stress and thus in photoaging. Exogenous supplementation with dietary antioxidants and/or skin pretreatment with antioxidant-based lotions before sun exposure might be a winning strategy against age-related skin pathologies. In this context, plants produce many secondary metabolites to protect themselves from UV radiations and these compounds can also protect the skin from photoaging. Phenolic compounds, ascorbic acid and carotenoids, derived from different plant species, are able to protect the skin by preventing UV penetration, reducing inflammation and oxidative stress, and influencing several survival signalling pathways. In this review, we focus our attention on the double role of oxidants in cell metabolism and on environmental and xenobiotic agents involved in skin photoaging. Moreover, we discuss the protective role of dietary antioxidants from fruits and vegetables and report their antiaging properties related to the reduction of oxidative stress pathways.
Asunto(s)
Antioxidantes/uso terapéutico , Plantas/química , Envejecimiento de la Piel/efectos de los fármacos , Enfermedades de la Piel/tratamiento farmacológico , Antioxidantes/farmacología , Humanos , Especies Reactivas de OxígenoRESUMEN
Prolonged hyperglycemia in poorly controlled diabetes leads to an increase in reactive glucose metabolites that covalently modify proteins by non-enzymatic glycation reactions. Apolipoprotein A-I (apoA-I) of high-density lipoprotein (HDL) is one of the proteins that becomes glycated in hyperglycemia. The impact of glycation on apoA-I protein structure and function in lipid and glucose metabolism were investigated. ApoA-I was chemically glycated by two different glucose metabolites (methylglyoxal and glycolaldehyde). Synchrotron radiation and conventional circular dichroism spectroscopy were used to study apoA-I structure and stability. The ability to bind lipids was measured by lipid-clearance assay and native gel analysis, and cholesterol efflux was measured by using lipid-laden J774 macrophages. Diet induced obese mice with established insulin resistance, L6 rat and C2C12 mouse myocytes, as well as INS-1E rat insulinoma cells, were used to determine in vivo and in vitro glucose uptake and insulin secretion. Site-specific, covalent modifications of apoA-I (lysines or arginines) led to altered protein structure, reduced lipid binding capability and a reduced ability to catalyze cholesterol efflux from macrophages, partly in a modification-specific manner. The stimulatory effects of apoA-I on the in vivo glucose clearance were negatively affected when apoA-I was modified with methylglyoxal, but not with glycolaldehyde. The in vitro data showed that both glucose uptake in muscle cells and insulin secretion from beta cells were affected. Taken together, glycation modifications impair the apoA-I protein functionality in lipid and glucose metabolism, which is expected to have implications for diabetes patients with poorly controlled blood glucose.
Asunto(s)
Apolipoproteína A-I/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glucosa/metabolismo , Metabolismo de los Lípidos , Acetaldehído/análogos & derivados , Acetaldehído/farmacología , Animales , Apolipoproteína A-I/química , Glucemia/efectos de los fármacos , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/prevención & control , Línea Celular , Colesterol/metabolismo , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/etiología , Modelos Animales de Enfermedad , Glicosilación/efectos de los fármacos , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Macrófagos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Células Musculares/efectos de los fármacos , Células Musculares/metabolismo , Unión Proteica/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Piruvaldehído/farmacología , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Specific mutations in APOA1 gene lead to systemic, hereditary amyloidoses. In ApoA-I related amyloidosis involving the heart, amyloid deposits are mainly constituted by the 93-residue N-terminal region of the protein, here indicated as [1-93]ApoA-I. Oxidative stress is known to be an enhancing factor for protein aggregation. In healthy conditions, humans are able to counteract the formation and the effects of oxidative molecules. However, aging and atmospheric pollution increase the concentration of oxidative agents, such as metal ions. As the main effect of iron deregulation is proposed to be an increase in oxidative stress, we analysed the effects of iron on [1-93]ApoA-I aggregation. By using different biochemical approaches, we demonstrated that Fe(II) is able to reduce the formation of [1-93]ApoA-I fibrillar species, probably by stabilizing its monomeric form, whereas Fe(III) shows a positive effect on polypeptide fibrillogenesis. We hypothesize that, in healthy conditions, Fe(III) is reduced by the organism to Fe(II), thus inhibiting amyloid formation, whereas during ageing such protective mechanisms decline, thus exposing the organism to higher oxidative stress levels, which are also related to an increase in Fe(III). This alteration could contribute to the pathogenesis of amyloidosis.
Asunto(s)
Amiloidosis Familiar/metabolismo , Apolipoproteína A-I/genética , Hierro/metabolismo , Miocardio/metabolismo , Envejecimiento/genética , Envejecimiento/metabolismo , Envejecimiento/patología , Amiloidosis Familiar/genética , Amiloidosis Familiar/patología , Apolipoproteína A-I/química , Humanos , Hierro/química , Mutación , Miocardio/patología , Estrés Oxidativo/genética , Péptidos/química , Péptidos/metabolismo , Placa Amiloide/genética , Placa Amiloide/metabolismo , Placa Amiloide/fisiopatología , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/fisiopatologíaRESUMEN
Twenty Apolipoprotein A-I (ApoA-I) variants are responsible for a systemic hereditary amyloidosis in which protein fibrils can accumulate in different organs, leading to their failure. Several ApoA-I amyloidogenic mutations are also associated with hypoalphalipoproteinemia, low ApoA-I and high-density lipoprotein (HDL)-cholesterol plasma levels; however, subjects affected by ApoA-I-related amyloidosis do not show a higher risk of cardiovascular diseases (CVD). The structural features, the lipid binding properties and the functionality of four ApoA-I amyloidogenic variants were therefore inspected in order to clarify the paradox observed in the clinical phenotype of the affected subjects. Our results show that ApoA-I amyloidogenic variants are characterized by a different oligomerization pattern and that the position of the mutation in the ApoA-I sequence affects the molecular structure of the formed HDL particles. Although lipidation increases ApoA-I proteins stability, all the amyloidogenic variants analyzed show a lower affinity for lipids, both in vitro and in ex vivo mouse serum. Interestingly, the lower efficiency at forming HDL particles is compensated by a higher efficiency at catalysing cholesterol efflux from macrophages. The decreased affinity of ApoA-I amyloidogenic variants for lipids, together with the increased efficiency in the cholesterol efflux process, could explain why, despite the unfavourable lipid profile, patients affected by ApoA-I related amyloidosis do not show a higher CVD risk.
Asunto(s)
Apolipoproteína A-I/química , Apolipoproteína A-I/metabolismo , HDL-Colesterol/metabolismo , Colesterol/metabolismo , Dislipidemias/metabolismo , Amiloidosis/metabolismo , Animales , Apolipoproteína A-I/genética , Sitios de Unión , Colesterol/sangre , Humanos , Hipoalfalipoproteinemias/metabolismo , Metabolismo de los Lípidos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de Proteína , Relación Estructura-ActividadRESUMEN
The human cathelicidin peptide LL-37 has antimicrobial and anti-biofilm functions, but LL-37 may also damage the host by triggering inflammation and exerting a cytotoxic effect, thereby reducing host cell viability. Human plasma mitigates LL-37-induced host cell cytotoxicity but the underlying mechanisms are not completely understood. Apolipoprotein A-I (ApoA-I) is a plasma protein endowed with atheroprotective effects. Here, we investigate the interaction between ApoA-I and LL-37 by biochemical techniques, and furthermore assess if ApoA-I protects against LL-37-evoked cytotoxicity in human umbilical vein endothelial cells (HUVEC). Our results demonstrated that ApoA-I effectively binds LL-37. The binding of ApoA-I to LL-37 resulted in a structural rearrangement of the protein, but this interaction did not cause lower ApoA-I stability. Recombinant ApoA-I protected against LL-37-induced cytotoxicity in HUVEC and endogenous ApoA-I knockdown in HepG2 cells made the cells more sensitive to LL-37-evoked cytotoxicity. We conclude that ApoA-I physically interacts with LL-37 and antagonizes LL-37-induced down-regulation of endothelial cell viability suggesting that this mechanism counteracts endothelial cell dysfunction.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/administración & dosificación , Péptidos Catiónicos Antimicrobianos/química , Apolipoproteína A-I/química , Apolipoproteína A-I/metabolismo , Apoptosis/fisiología , Células Endoteliales/metabolismo , Apoptosis/efectos de los fármacos , Sitios de Unión , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Humanos , Unión Proteica , CatelicidinasRESUMEN
UV-A radiations are known to induce cellular oxidative stress, leading to premature skin aging. Consumption of açai fruit (Euterpe oleracea Martius) is known to have many health benefits due to its high level of antioxidants. Herein, we analyzed the ability of phenolic compounds extracted from this fruit to attenuate UV-A-induced oxidative stress in immortalized fibroblast. A methanol/water açai extract was fractionated by HPLC and each fraction tested for anti-oxidant stress activity. Immortalized fibroblasts were pre-incubated with açai fractions and then exposed to UV-A radiations. Açai extract was found to be able to strongly protect cells from oxidative stress. In particular, reactive oxygen species (ROS) production, GSH depletion, lipid peroxidation and no increase in the phosphorylation levels of proteins involved in the oxidative stress pathway was observed in cells pre-incubated with the extract and then irradiated by UV-A. Mass spectrometry analyses of HPLC fractionated extract led us to the identification of malvidin and cyanidin derivatives as the most active molecules able to counteract the negative effects induced by UV-A irradiation. Our results indicate, for the first time, that açai fruit is a valuable natural source for malvidin and cyanidin to be used as anti-stress molecules and represent good candidates for dietary intervention in the prevention of age related skin damage.
Asunto(s)
Antocianinas/farmacología , Euterpe/química , Estrés Oxidativo/efectos de los fármacos , Rayos Ultravioleta , Células 3T3 , Animales , Antocianinas/química , Antocianinas/aislamiento & purificación , Antioxidantes/química , Antioxidantes/aislamiento & purificación , Antioxidantes/farmacología , Supervivencia Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Cromatografía de Fase Inversa , Euterpe/metabolismo , Frutas/química , Frutas/metabolismo , Espectrometría de Masas , Ratones , Estrés Oxidativo/efectos de la radiación , Fosforilación/efectos de los fármacos , Fosforilación/efectos de la radiación , Extractos Vegetales/química , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: Lipophilic antioxidants in tomato (Solanum lycopersicum) fruits exert important functions in reducing the risk of human diseases. Here the effect of thermal processing on the antioxidant activity of lipophilic extracts from the commercial tomato hybrid 'Zebrino' was analysed. Carotenoid content and lipophilic antioxidant activity were determined and the ability of tomato extracts in rescuing cells from oxidative stress was assessed. RESULTS: Lipophilic antioxidant activity was completely retained after heat treatment and extracts were able to mitigate the detrimental effect induced by oxidative stress on different cell lines. Lycopene alone was able to rescue cells from oxidative stress, even if to a lower extent compared with tomato extracts. These results were probably due to the synergistic effect of tomato compounds in protecting cells from oxidative stress injury. CONCLUSION: The current study provides valuable insights into the health effect of the dietary carotenoids present in fresh and processed tomato fruits. © 2016 Society of Chemical Industry.
Asunto(s)
Carotenoides/farmacología , Manipulación de Alimentos/métodos , Estrés Oxidativo/efectos de los fármacos , Solanum lycopersicum/química , Animales , Antioxidantes/farmacología , Línea Celular Tumoral , Frutas/química , Humanos , Licopeno , Extractos Vegetales/farmacología , RatasRESUMEN
UVA radiations contribute up to 95% of the total UV exposure and are known to induce cell damage, leading to apoptosis. Since the benefic effects of ascorbic acid on human health are well known, a new tomato genotype (named DHO4), highly rich in ascorbic acid, has been recently obtained. Here, we compared the effects of ascorbic acid and hydrophilic DHO4 extracts in protecting human keratinocytes exposed to UVA stress. Keratinocytes were pre-incubated with ascorbic acid or with extracts from the ascorbic acid enriched tomato genotype and irradiated with UVA light. Then, ROS production, intracellular GSH and lipid peroxidation levels were quantified. Western blots were carried out to evaluate mitogen-activated protein kinases cascade, activation of caspase-3 and inflammation levels. We demonstrated that ROS, GSH and lipid peroxidation levels were not altered in cell exposed to UVA stress when cells were pre-treated with ascorbic acid or with tomato extracts. In addition, no evidence of apoptosis and inflammation were observed in irradiated pre-treated cells. Altogether, we demonstrated the ability of an ascorbic acid enriched tomato genotype to counteract UVA-oxidative stress on human keratinocytes. This protective effect is due to the high concentration of vitamin C that acts as free radical scavenger. This novel tomato genotype may be used as genetic material in breeding schemes to produce improved varieties with higher antioxidant levels.