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1.
Sci Rep ; 11(1): 16086, 2021 08 09.
Artículo en Inglés | MEDLINE | ID: mdl-34373542

RESUMEN

High-density lipoprotein (HDL) particles have multiple beneficial and cardioprotective roles, yet our understanding of their full structural and functional repertoire is limited due to challenges in separating HDL particles from contaminating plasma proteins and other lipid-carrying particles that overlap HDL in size and/or density. Here we describe a method for isolating HDL particles using a combination of sequential flotation density ultracentrifugation and fast protein liquid chromatography with a size exclusion column. Purity was visualized by polyacrylamide gel electrophoresis and verified by proteomics, while size and structural integrity were confirmed by transmission electron microscopy. This HDL isolation method can be used to isolate a high yield of purified HDL from a low starting plasma volume for functional analyses. This method also enables investigators to select their specific HDL fraction of interest: from the least inclusive but highest purity HDL fraction eluting in the middle of the HDL peak, to pooling all of the fractions to capture the breadth of HDL particles in the original plasma sample. We show that certain proteins such as lecithin cholesterol acyltransferase (LCAT), phospholipid transfer protein (PLTP), and clusterin (CLUS) are enriched in large HDL particles whereas proteins such as alpha-2HS-glycoprotein (A2HSG), alpha-1 antitrypsin (A1AT), and vitamin D binding protein (VDBP) are enriched or found exclusively in small HDL particles.


Asunto(s)
Lipoproteínas HDL/sangre , Lipoproteínas HDL/aislamiento & purificación , Cromatografía en Gel/métodos , Cromatografía Liquida/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Humanos , Tamaño de la Partícula , Proteínas/aislamiento & purificación , Ultracentrifugación/métodos
2.
Int J Mol Sci ; 22(6)2021 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-33799511

RESUMEN

In uremic patients, high-density lipoprotein (HDL) loses its anti-inflammatory features and can even become pro-inflammatory due to an altered protein composition. In chronic kidney disease (CKD), impaired functions of polymorphonuclear leukocytes (PMNLs) contribute to inflammation and an increased risk of cardiovascular disease. This study investigated the effect of HDL from CKD and hemodialysis (HD) patients on the CD14 expression on PMNLs. HDL was isolated using a one-step density gradient centrifugation. Isolation of PMNLs was carried out by discontinuous Ficoll-Hypaque density gradient centrifugation. CD14 surface expression was quantified by flow cytometry. The activity of the small GTPase Rac1 was determined by means of an activation pull-down assay. HDL increased the CD14 surface expression on PMNLs. This effect was more pronounced for HDL isolated from uremic patients. The acute phase protein serum amyloid A (SAA) caused higher CD14 expression, while SAA as part of an HDL particle did not. Lipid raft disruption with methyl-ß-cyclodextrin led to a reduced CD14 expression in the absence and presence of HDL. HDL from healthy subjects but not from HD patients decreased the activity of Rac1. Considering the known anti-inflammatory effects of HDL, the finding that even HDL from healthy subjects increased the CD14 expression was unexpected. The pathophysiological relevance of this result needs further investigation.


Asunto(s)
Receptores de Lipopolisacáridos/genética , Lipoproteínas HDL/farmacología , Neutrófilos/efectos de los fármacos , Insuficiencia Renal Crónica/genética , Uremia/genética , Anciano , Estudios de Casos y Controles , Femenino , Humanos , Receptores de Lipopolisacáridos/metabolismo , Lipoproteínas HDL/aislamiento & purificación , Masculino , Microdominios de Membrana/química , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/metabolismo , Persona de Mediana Edad , Neutrófilos/metabolismo , Neutrófilos/patología , Cultivo Primario de Células , Diálisis Renal , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/fisiopatología , Insuficiencia Renal Crónica/terapia , Proteína Amiloide A Sérica/genética , Proteína Amiloide A Sérica/metabolismo , Uremia/metabolismo , Uremia/fisiopatología , Uremia/terapia , beta-Ciclodextrinas/farmacología , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo
3.
Int J Mol Sci ; 21(22)2020 Nov 19.
Artículo en Inglés | MEDLINE | ID: mdl-33228032

RESUMEN

It is well known that blood lipoproteins (LPs) are multimolecular complexes of lipids and proteins that play a crucial role in lipid transport. High-density lipoproteins (HDL) are a class of blood plasma LPs that mediate reverse cholesterol transport (RCT)-cholesterol transport from the peripheral tissues to the liver. Due to this ability to promote cholesterol uptake from cell membranes, HDL possess antiatherogenic properties. This function was first observed at the end of the 1970s to the beginning of the 1980s, resulting in high interest in this class of LPs. It was shown that HDL are the prevalent class of LPs in several types of living organisms (from fishes to monkeys) with high resistance to atherosclerosis and cardiovascular disorders. Lately, understanding of the mechanisms of the antiatherogenic properties of HDL has significantly expanded. Besides the contribution to RCT, HDL have been shown to modulate inflammatory processes, blood clotting, and vasomotor responses. These particles also possess antioxidant properties and contribute to immune reactions and intercellular signaling. Herein, we review data on the structure and mechanisms of the pleiotropic biological functions of HDL from the point of view of their evolutionary role and complex dynamic nature.


Asunto(s)
Aterosclerosis/sangre , Colesterol/metabolismo , Homeostasis/fisiología , Lipoproteínas HDL/fisiología , Animales , Antiinfecciosos/sangre , Antiinfecciosos/farmacología , Antiinflamatorios/sangre , Antiinflamatorios/farmacología , Antioxidantes/metabolismo , Antioxidantes/farmacología , Aterosclerosis/genética , Aterosclerosis/fisiopatología , Transporte Biológico , Coagulación Sanguínea/efectos de los fármacos , Coagulación Sanguínea/fisiología , Colesterol/química , Humanos , Lipoproteínas HDL/química , Lipoproteínas HDL/clasificación , Lipoproteínas HDL/aislamiento & purificación , Transducción de Señal , Vasodilatadores/sangre , Vasodilatadores/farmacología , Sistema Vasomotor/efectos de los fármacos , Sistema Vasomotor/fisiología
4.
J Biol Chem ; 295(39): 13601-13616, 2020 09 25.
Artículo en Inglés | MEDLINE | ID: mdl-32737203

RESUMEN

Strong evidence suggests that dysregulated lipid metabolism involving dysfunction of the retinal pigmented epithelium (RPE) underlies the pathogenesis of age-related macular degeneration (AMD), the leading cause of irreversible blindness in the elderly. A hallmark of AMD is the overproduction of lipid- and protein-rich extracellular deposits that accumulate in the extracellular matrix (Bruch's membrane (BrM)) adjacent to the RPE. We analyzed apolipoprotein A-1 (ApoA-1)-containing lipoproteins isolated from BrM of elderly human donor eyes and found a unique proteome, distinct from high-density lipoprotein (HDL) isolated from donor plasma of the same individuals. The most striking difference is higher concentrations of ApoB and ApoE, which bind to glycosaminoglycans. We hypothesize that this interaction promotes lipoprotein deposition onto BrM glycosaminoglycans, initiating downstream effects that contribute to RPE dysfunction/death. We tested this hypothesis using two potential therapeutic strategies to alter the lipoprotein/protein profile of these extracellular deposits. First, we used short heparan sulfate oligosaccharides to remove lipoproteins already deposited in both the extracellular matrix of RPE cells and aged donor BrM tissue. Second, an ApoA-1 mimetic, 5A peptide, was demonstrated to modulate the composition and concentration of apolipoproteins secreted from primary porcine RPE cells. Significantly, in a mouse model of AMD, this 5A peptide altered the proteomic profile of circulating HDL and ameliorated some of the potentially harmful changes to the protein composition resulting from the high-fat, high-cholesterol diet in this model. Together, these results suggest that targeting HDL interactions with BrM represents a new strategy to slow AMD progression in humans.


Asunto(s)
Lipoproteínas HDL/metabolismo , Degeneración Macular/metabolismo , Animales , Apolipoproteína A-I/análisis , Apolipoproteína A-I/metabolismo , Lámina Basal de la Coroides/metabolismo , Células Cultivadas , Humanos , Lipoproteínas HDL/sangre , Lipoproteínas HDL/aislamiento & purificación , Ratones , Epitelio Pigmentado de la Retina/metabolismo , Porcinos
5.
Methods Mol Biol ; 2116: 463-483, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32221937

RESUMEN

Interest in trypanosome lytic factors (TLFs) and apolipoprotein L1, the ion channel-forming protein component of TLFs, has increased tenfold since 2010. This is due to the association of African variants of APOL1 with kidney disease such that interest has reached circles beyond parasitology. We have extensive experience purifying and working with these proteins and protein complexes. Herein we describe our detailed purification protocols to aid the new burgeoning field by providing an opportunity for consistency in reagents used across laboratories. We emphasize that it is imperative to maintain APOL1 protein intact (~42 kDa) to analyze the active ion channel-forming component/protein.


Asunto(s)
Apolipoproteína L1/aislamiento & purificación , Lipoproteínas HDL/aislamiento & purificación , Tripanosomiasis Africana/sangre , Apolipoproteína L1/sangre , Apolipoproteína L1/química , Apolipoproteína L1/metabolismo , Humanos , Enfermedades Renales/sangre , Enfermedades Renales/inmunología , Lipoproteínas HDL/química , Lipoproteínas HDL/metabolismo , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Trypanosoma/inmunología , Tripanosomiasis Africana/complicaciones , Tripanosomiasis Africana/inmunología , Tripanosomiasis Africana/parasitología
6.
Biomed Chromatogr ; 34(1): e4693, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31465544

RESUMEN

High-density lipoproteins (HDLs) have anti-inflammatory and antioxidant properties and are potentially cardio-protective. Defective HDL function is caused by alterations in both the proteome and lipidome of HDL particles. As potential biomarkers, the development of analytical methods is necessary for the enrichment of HDLs. Therefore, a method for selective enrichment of HDLs using immobilized metal ion affinity chromatography (IMAC) and metal oxide affinity chromatography (MOAC) is presented. SPE-based isolation of HDLs from whole serum is adopted as an alternative to traditional ultracentrifugation methods followed by SDS-PAGE. The enrichment mechanism relies on isoelectric points of lipoproteins and metal oxide. Negatively charged lipoprotein particles interact with positively charged metal oxides and IMAC affinity, which acts as a cation. Identified proteins from HDL through MALDI-MS analysis are apo AI, AII, AIV, CI, CIII, E, J, M, H, serum amyloid A and other nonapoproteins that are part of HDL particles and perform cellular functions. This serum-based proteomics approach gives insight into the functional role of HDL. HDL-associated phospholipids have also been analyzed by LDI-MS. Results suggest that the adopted analytical strategy is a feasible idea to extract lipoproteins from serum. A comparative study of healthy and diseased samples using this approach will provide valuable information in future.


Asunto(s)
Cromatografía de Afinidad/métodos , Lipoproteínas HDL/sangre , Fosfolípidos/sangre , Proteoma/análisis , Humanos , Lipoproteínas HDL/aislamiento & purificación , Fosfolípidos/aislamiento & purificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
7.
Nat Commun ; 10(1): 5813, 2019 12 20.
Artículo en Inglés | MEDLINE | ID: mdl-31862950

RESUMEN

Gut microbiota has been implicated in major diseases affecting the human population and has also been linked to triglycerides and high-density lipoprotein levels in the circulation. Recent development in metabolomics allows classifying the lipoprotein particles into more details. Here, we examine the impact of gut microbiota on circulating metabolites measured by Nuclear Magnetic Resonance technology in 2309 individuals from the Rotterdam Study and the LifeLines-DEEP cohort. We assess the relationship between gut microbiota and metabolites by linear regression analysis while adjusting for age, sex, body-mass index, technical covariates, medication use, and multiple testing. We report an association of 32 microbial families and genera with very-low-density and high-density subfractions, serum lipid measures, glycolysis-related metabolites, ketone bodies, amino acids, and acute-phase reaction markers. These observations provide insights into the role of microbiota in host metabolism and support the potential of gut microbiota as a target for therapeutic and preventive interventions.


Asunto(s)
Microbioma Gastrointestinal/fisiología , Interacciones Microbiota-Huesped/fisiología , Metaboloma/fisiología , Proteínas de Fase Aguda/aislamiento & purificación , Proteínas de Fase Aguda/metabolismo , Adulto , Aminoácidos/sangre , Aminoácidos/aislamiento & purificación , Aminoácidos/metabolismo , Bacterias/genética , Bacterias/aislamiento & purificación , Estudios de Cohortes , ADN Bacteriano/aislamiento & purificación , Heces/microbiología , Femenino , Glucólisis/fisiología , Humanos , Lipoproteínas HDL/sangre , Lipoproteínas HDL/aislamiento & purificación , Lipoproteínas HDL/metabolismo , Masculino , Metabolómica/métodos , Persona de Mediana Edad , Países Bajos , Espectroscopía de Protones por Resonancia Magnética , ARN Ribosómico 16S/genética , Análisis de Regresión , Triglicéridos/sangre , Triglicéridos/aislamiento & purificación , Triglicéridos/metabolismo
8.
Expert Rev Proteomics ; 16(9): 749-760, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31382838

RESUMEN

Introduction: High-density lipoprotein (HDL) particles are heterogeneous and their proteome is complex and distinct from HDL cholesterol. However, it is largely unknown whether HDL proteins are associated with cardiovascular protection. Areas covered: HDL isolation techniques and proteomic analyses are reviewed. A list of HDL proteins reported in 37 different studies was compiled and the effects of different isolation techniques on proteins attributed to HDL are discussed. Mass spectrometric techniques used for HDL analysis and the need for precise and robust methods for quantification of HDL proteins are discussed. Expert opinion: Proteins associated with HDL have the potential to be used as biomarkers and/or help to understand HDL functionality. To achieve this, large cohorts must be studied using precise quantification methods. Key factors in HDL proteome quantification are the isolation methodology and the mass spectrometry technique employed. Isolation methodology affects what proteins are identified in HDL and the specificity of association with HDL particles needs to be addressed. Shotgun proteomics yields imprecise quantification, but the majority of HDL studies relied on this approach. Few recent studies used targeted tandem mass spectrometry to quantify HDL proteins, and it is imperative that future studies focus on the application of these precise techniques.


Asunto(s)
HDL-Colesterol/aislamiento & purificación , Lipoproteínas HDL/aislamiento & purificación , Proteoma/genética , Proteómica/métodos , Biomarcadores/metabolismo , HDL-Colesterol/genética , Humanos , Lipoproteínas HDL/genética , Espectrometría de Masas , Proteoma/aislamiento & purificación
9.
Anal Bioanal Chem ; 411(3): 777-786, 2019 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-30470915

RESUMEN

High- and low-density lipoproteins (HDL and LDL) are attractive targets for biomarker discovery. However, ultracentrifugation (UC), the current methodology of choice for isolating HDL and LDL, is tedious, requires large sample volume, results in sample loss, and does not readily provide information on particle size. In this work, human plasma HDL and LDL are separated and collected using semi-preparative asymmetrical flow field-flow fractionation (SP-AF4) and UC. The SP-AF4 and UC separation conditions, sample throughput, and liquid chromatography/mass spectrometry (LC/MS) lipidomic results are compared. Over 600 µg of total proteins is recovered in a single SP-AF4 run, and Western blot results confirm apoA1 pure and apoB100 pure fractions, consistent with HDL and LDL, respectively. The SP-AF4 separation requires ~ 60 min per sample, thus providing a marked improvement over UC which can span hours to days. Lipidome analysis of SP-AF4-prepared HDL and LDL fractions is compared to UC-prepared HDL and LDL samples. Over 270 lipids in positive MS mode and over 140 lipids in negative MS mode are identified by both sample preparation techniques with over 98% overlap between the lipidome. Additionally, lipoprotein size distributions are determined using analytical scale AF4 coupled with multiangle light scattering (MALS) and dynamic light scattering (DLS) detectors. These developments position SP-AF4 as a sample preparation method of choice for lipoprotein biomarker characterization and identification. Graphical abstract ᅟ.


Asunto(s)
Fraccionamiento de Campo-Flujo/métodos , Lipoproteínas HDL/sangre , Lipoproteínas LDL/sangre , Western Blotting , Cromatografía Liquida/métodos , Dispersión Dinámica de Luz/métodos , Humanos , Lipoproteínas HDL/aislamiento & purificación , Lipoproteínas LDL/aislamiento & purificación , Tamaño de la Partícula , Manejo de Especímenes , Espectrometría de Masas en Tándem/métodos , Ultracentrifugación
10.
Biosci Rep ; 38(5)2018 10 31.
Artículo en Inglés | MEDLINE | ID: mdl-30279204

RESUMEN

Dihydro-sphingosine 1-phosphate (DH-S1P) is an analog of sphingosine 1-phosphate (S1P), which is a potent lysophospholipid mediator. DH-S1P has been proposed to exert physiological properties similar to S1P. Although S1P is known to be carried on HDL via apolipoprotein M (apoM), the association between DH-S1P and HDL/apoM has not been fully elucidated. Therefore, in the present study, we aimed to elucidate this association and to compare it with that of S1P and HDL/apoM. First, we investigated the distributions of S1P and DH-S1P among lipoproteins and lipoprotein-depleted fractions in human serum and plasma samples and observed that both S1P and DH-S1P were detected on HDL; furthermore, elevated amounts of DH-S1P in serum samples were distributed to the lipoprotein-depleted fraction to a greater degree than to the HDL fraction. Concordantly, a preference for HDL over albumin was only observed for S1P, and not for DH-S1P, when the molecules were secreted from platelets. Regarding the association with HDL, although both S1P and DH-S1P prefer to bind to HDL, HDL preferentially accepts S1P over DH-S1P. For the association with apoM, S1P was not detected on HDL obtained from apoM knockout mice, while DH-S1P was detected. Moreover, apoM retarded the degradation of S1P, but not of DH-S1P. These results suggest that S1P binds to HDL via apoM, while DH-S1P binds to HDL in a non-specific manner. Thus, DH-S1P is not a mere analog of S1P and might possess unique clinical significance.


Asunto(s)
Apolipoproteínas M/sangre , Lipoproteínas HDL/sangre , Lisofosfolípidos/sangre , Esfingosina/análogos & derivados , Animales , Apolipoproteínas M/aislamiento & purificación , Plaquetas/citología , Plaquetas/metabolismo , Proteínas Portadoras , Células Cultivadas , Eritrocitos/citología , Eritrocitos/metabolismo , Células Hep G2 , Humanos , Cinética , Lipoproteínas HDL/clasificación , Lipoproteínas HDL/aislamiento & purificación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Unión Proteica , Albúmina Sérica/metabolismo , Esfingosina/sangre , Ultracentrifugación
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