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1.
Arterioscler Thromb Vasc Biol ; 42(3): 289-304, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35045727

RESUMO

BACKGROUND: Elevated plasma Lp(a) (lipoprotein(a)) levels are associated with increased risk for atherosclerotic cardiovascular disease and aortic valve stenosis. However, the cell biology of Lp(a) biosynthesis remains poorly understood, with the locations of the noncovalent and covalent steps of Lp(a) assembly unclear and the nature of the apoB-containing particle destined for Lp(a) unknown. We, therefore, asked if apo(a) and apoB interact noncovalently within hepatocytes and if this impacts Lp(a) biosynthesis. METHODS: Using human hepatocellular carcinoma cells expressing 17K (17 kringle) apo(a), or a 17KΔLBS7,8 variant with a reduced ability to bind noncovalently to apoB, we performed coimmunoprecipitation, coimmunofluorescence, and proximity ligation assays to document intracellular apo(a):apoB interactions. We used a pulse-chase metabolic labeling approach to measure apo(a) and apoB secretion rates. RESULTS: Noncovalent complexes containing apo(a)/apoB are present in lysates from cells expressing 17K but not 17KΔLBS7,8, whereas covalent apo(a)/apoB complexes are absent from lysates. 17K and apoB colocalized intracellularly, overlapping with staining for markers of endoplasmic reticulum trans-Golgi, and early endosomes, and less so with lysosomes. The 17KΔLBS7,8 had lower colocalization with apoB. Proximity ligation assays directly documented intracellular 17K/apoB interactions, which were dramatically reduced for 17KΔLBS7,8. Treatment of cells with PCSK9 (proprotein convertase subtilisin/kexin type 9) enhanced, and lomitapide reduced, apo(a) secretion in a manner dependent on the noncovalent interaction between apo(a) and apoB. Apo(a) secretion was also reduced by siRNA-mediated knockdown of APOB. CONCLUSIONS: Our findings explain the coupling of apo(a) and Lp(a)-apoB production observed in human metabolic studies using stable isotopes as well as the ability of agents that inhibit apoB biosynthesis to lower Lp(a) levels.


Assuntos
Apolipoproteína B-100/metabolismo , Apolipoproteínas A/metabolismo , Hepatócitos/metabolismo , Lipoproteína(a)/metabolismo , Apolipoproteína B-100/química , Apolipoproteínas A/química , Apolipoproteínas A/genética , Sítios de Ligação/genética , Células Hep G2 , Humanos , Kringles/genética , Lipoproteína(a)/química , Lisina/química , Redes e Vias Metabólicas , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
2.
J Phys Chem Lett ; 12(51): 12402-12410, 2021 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-34939807

RESUMO

Apolipoprotein B-100 (apo B-100) is the protein moiety of both low- and very-low-density lipoproteins, whose role is crucial to cholesterol and triglyceride transport. Aiming at the molecular dynamics' details of apo B-100, scarcely studied, we performed elastic and quasi-elastic incoherent neutron scattering (EINS, QENS) experiments combining different instruments and time scales. Similar to classical membrane proteins, the solubilization results in remaining detergent, here Nonidet P-40 (NP40). Therefore, we propose a framework for QENS studies of protein-detergent complexes, with the introduction of a combined model, including the experimental apo B-100/NP40 ratio. Relying on the simultaneous analysis of all QENS amplitudes, this approach is sensitive enough to separate both contributions. Its application identified two points: (i) apo B-100 slow dynamics and (ii) the acceleration of NP40 dynamics in the presence of apo B-100. Direct translation of the exposed methodology now makes the investigation of more membrane proteins by neutron spectroscopy achievable.


Assuntos
Apolipoproteína B-100/química , Detergentes/química , Simulação de Dinâmica Molecular , Humanos , Nêutrons , Espalhamento a Baixo Ângulo
3.
RNA ; 27(2): 163-173, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33177188

RESUMO

Small interfering RNAs (siRNAs) can be utilized not only as functional biological research tools but also as therapeutic agents. For the clinical use of siRNA as drugs, various chemical modifications have been used to improve the activity of siRNA drugs, and further chemical modifications are expected to improve the utility of siRNA therapeutics. As the 5' nucleobase of the guide strand affects the interaction between an siRNA and AGO2 and target cleavage activity, structural optimization of this specific position may be a useful strategy for improving siRNA activity. Here, using the in silico model of the complex between human AGO2 MID domain and nucleoside monophosphates, we screened and synthesized an original adenine-derived analog, 6-(3-(2-carboxyethyl)phenyl)purine (6-mCEPh-purine), that fits better than the natural nucleotide bases into the MID domain of AGO2. Introduction of the 6-mCEPh-purine analog at the 5'-end of the siRNA guide strand significantly enhanced target knockdown activity in both cultured cell lines and in vivo animal models. Our findings can help expand strategies for rationally optimizing siRNA activity via chemical modifications of nucleotide bases.


Assuntos
Adenina/farmacologia , Proteínas Argonautas/genética , Interferência de RNA/efeitos dos fármacos , RNA de Cadeia Dupla/genética , RNA Interferente Pequeno/agonistas , Complexo de Inativação Induzido por RNA/agonistas , Adenina/análogos & derivados , Adenina/síntese química , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Animais , Apolipoproteína B-100/antagonistas & inibidores , Apolipoproteína B-100/sangue , Apolipoproteína B-100/química , Apolipoproteína B-100/genética , Proteínas Argonautas/metabolismo , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Colesterol/sangue , Células HeLa , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Masculino , Metilação , Camundongos , Camundongos Knockout , Modelos Moleculares , Ligação Proteica , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Complexo de Inativação Induzido por RNA/genética , Complexo de Inativação Induzido por RNA/metabolismo , Uridina Monofosfato/química , Uridina Monofosfato/metabolismo
4.
Artigo em Inglês | MEDLINE | ID: mdl-32919080

RESUMO

We have reported that acrolein-conjugated low-density lipoprotein (Acro-LDL) uptake by scavenger receptor class A type 1 (SR-A1) can mediate macrophage foam cell formation. The purpose of this study was to determine which amino acid residues of apoB protein in LDL are conjugated with acrolein. Acro-apoB was prepared by incubation of LDL with acrolein (10 to 60 µM) at 37 °C for 7 days. Identification of acrolein-conjugated amino acid residues in apoB was performed using LC-MS/MS. The levels of acrolein-conjugated amino acid residues of apoB as well as crosslinking apoB increased in proportion to acrolein concentration. The level of LDL uptake by macrophages was parallel with the acrolein-conjugated monomer apoB. Acrolein-conjugated amino acid residues in apoB were C212, K327, K742, K949, K1087, H1923, K2634, K3237 and K3846. The NH2-teriminal four amino acid residues (C212, K327, K742 and K949) were located at the scavenger receptor SR-A1 recognition site, suggesting that these four acrolein-conjugated amino acids are involved in the rapid uptake of Acro-LDL by macrophages. It is proposed that the rapid uptake of LDL by macrophages is dependent on acrolein conjugation of four amino acids residues at the scavenger receptor recognition site of apoB in LDL.


Assuntos
Acroleína/química , Apolipoproteína B-100/química , LDL-Colesterol/química , Fatores de Processamento de Serina-Arginina/química , Sequência de Aminoácidos , Apolipoproteína B-100/genética , Apolipoproteína B-100/metabolismo , Sítios de Ligação , LDL-Colesterol/metabolismo , Expressão Gênica , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Células THP-1
5.
J Lipid Res ; 61(3): 455-463, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31888979

RESUMO

Elevated levels of triglyceride-rich lipoproteins (TRLs), both fasting and postprandial, are associated with increased risk for atherosclerosis. However, guidelines for treatment are defined solely by fasting lipid levels, even though postprandial lipids may be more informative. In the postprandial state, circulating lipids consist of dietary fat transported from the intestine in chylomicrons (CMs; containing ApoB48) and fat transported from the liver in VLDL (containing ApoB100). Research into the roles of endogenous versus dietary fat has been hindered because of the difficulty in separating these particles by ultracentrifugation. CM fractions have considerable contamination from VLDL (purity, 10%). To separate CMs from VLDL, we produced polyclonal antibodies against ApoB100 and generated immunoaffinity columns. TRLs isolated by ultracentrifugation of plasma were applied to these columns, and highly purified CMs were collected (purity, 90-94%). Overall eight healthy unmedicated adult volunteers (BMI, 27.2 ± 1.4 kg/m2; fasting triacylglycerol, 102.6 ± 19.5 mg/dl) participated in a feeding study, which contained an oral stable-isotope tracer (1-13C acetate). We then used this technique on plasma samples freshly collected during an 8 h human feeding study from a subset of four subjects. We analyzed fractionated lipoproteins by Western blot, isolated and derivatized triacylglycerols, and calculated fractional de novo lipogenesis. The results demonstrated effective separation of postprandial lipoproteins and substantially improved purity compared with ultracentrifugation protocols, using the immunoaffinity method. This method can be used to better delineate the role of dietary sugar and fat on postprandial lipids in cardiovascular risk and explore the potential role of CM remnants in atherosclerosis.


Assuntos
Apolipoproteína B-100/química , Quilomícrons/isolamento & purificação , Lipoproteínas/isolamento & purificação , Triglicerídeos/isolamento & purificação , Cromatografia de Afinidade , Quilomícrons/química , Feminino , Voluntários Saudáveis , Humanos , Imunoprecipitação , Lipoproteínas/química , Masculino , Período Pós-Prandial , Triglicerídeos/química
6.
Sci Rep ; 9(1): 17391, 2019 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-31757993

RESUMO

Cardiovascular diseases form the most common cause of death worldwide, with atherosclerosis as main etiology. Atherosclerosis is marked by cholesterol rich lipoprotein deposition in the artery wall, evoking a pathogenic immune response. Characteristic for the disease is the pathogenic accumulation of macrophages in the atherosclerotic lesion, which become foam cells after ingestion of large quantities of lipoproteins. We hypothesized that, by inducing a CD8 T cell response towards lipoprotein derived apolipoprotein-B100 (ApoB100), lesional macrophages, that are likely to cross-present lipoprotein constituents, can specifically be eliminated. Based on in silico models for protein processing and MHC-I binding, 6 putative CD8 T cell epitopes derived from ApoB100 were synthesized. HLA-A2 binding was confirmed for all peptides by T2 cell binding assays and recall responses after vaccination with the peptides proved that 5 of 6 peptides could induce CD8 T cell responses. Induction of ApoB100 specific CD8 T cells did not impact plaque size and cellular composition in HLA-A2 and human ApoB100 transgenic LDLr-/- mice. No recall response could be detected in cultures of cells isolated from the aortic arch, which were observed in cell cultures of splenocytes and mesenteric lymph nodes, suggesting that the atherosclerotic environment impairs CD8 T cell activation.


Assuntos
Apolipoproteína B-100/imunologia , Aterosclerose/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígeno HLA-A2/imunologia , Animais , Anticorpos Monoclonais Humanizados/metabolismo , Apolipoproteína B-100/química , Aterosclerose/genética , Aterosclerose/patologia , Células Cultivadas , Modelos Animais de Doenças , Epitopos de Linfócito T/imunologia , Antígeno HLA-A2/genética , Antígeno HLA-A2/metabolismo , Humanos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Peptídeos/imunologia , Receptores de LDL/genética , Receptores de LDL/metabolismo
7.
Int J Nanomedicine ; 14: 7431-7446, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31686815

RESUMO

BACKGROUND: Low density lipoprotein (LDL) has been regarded as a promising antitumor drug vehicle. However some problems, such as rare source, difficulty of large-scale production, and potential safety concerns, hinder its clinical application. PURPOSE: The objective of this study is to develop a biomimetic LDL nanocarrier by replacing the native apolipoprotein B-100 (apoB-100) with an artificial amphipathic peptide and demonstrate its antitumor efficacy. METHODS: The amphipathic hybrid peptide (termed as FPL) consisting of a lipid binding motif of apoB-100 (LBMapoB)-polyethylene glycol (PEG)-folic acid (FA) was synthesized and characterized by 1H NMR and circular dichroism. FPL decorated lipoprotein-mimic nanoparticles (termed as FPLM NPs) were prepared by a modified solvent emulsification method. Paclitaxel (PTX) was incorporated into NPs and its content was quantified by HPLC analysis. The morphology of NPs was observed by transmission electron microscopy (TEM), and the particle size and zeta potential of NPs were determined by dynamic light scattering (DLS). The colloidal stability of FPLM NPs was evaluated in PBS containing bovine serum albumin (BSA). In vitro release of PTX loaded FPLM NPs was evaluated using the dialysis method. Cellular uptake and cytotoxity assayswere evaluated on human cervical cancer cells (HeLa) and lung cancer cells (A549). Tumor inhibition in vivo was investigated in M109 tumor-bearing mice via tail vein injection of Taxol formulation and PTX loaded NPs. RESULTS: The composition of FPLM NPs, including cholesteryl oleate, glyceryl trioleate, cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and FPL peptides, was optimized to be 5:1:1:3:10 (w/w). FPLM NPs had a spherical shape with a mean diameter of 83 nm and a negative charge (-12 mV). FPLM NPs with optimum formulation had good colloidal stability in BSA solution.The release of PTX from FPLM NPs was slow and sustained. The uptake of FPLM NPs was higher in folate receptor (FR) overexpressing tumor cells (HeLa cells) than in FR deficient tumor cells (A549 cells). The intracellular distribution indicated that FPLM NPs had the lysosome escape capacity. The internalization mechanism of FPLM NPs was involved with clathrin- and caveolae-mediated endocytosis and FR played a positive role in the internalization of FPLM NPs. The CCK-8 assay demonstrated that FPLM NPs exhibited notably better anti-tumor effect than Taxol formulation in vitro. Moreover, PTX loaded FPLM NPs produced very marked anti-tumor efficiency in M109 tumor-bearing mice in vivo. CONCLUSION: FPLM NPs is a promising nanocarrier which can improve the therapeutic effect and reduce the side effects of antitumor drugs.


Assuntos
Materiais Biomiméticos/química , Sistemas de Liberação de Medicamentos , Lipídeos/química , Lipoproteínas LDL/química , Nanopartículas/química , Neoplasias/tratamento farmacológico , Paclitaxel/uso terapêutico , Peptídeos/química , Células A549 , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Apolipoproteína B-100/química , Coloides/química , Liberação Controlada de Fármacos , Endocitose , Ácido Fólico/química , Células HeLa , Humanos , Camundongos Endogâmicos BALB C , Nanopartículas/ultraestrutura , Paclitaxel/farmacologia , Tamanho da Partícula , Polietilenoglicóis/química , Eletricidade Estática
9.
Sci Rep ; 9(1): 6728, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31040323

RESUMO

Cationic Host Defense Peptides (HDPs) are endowed with a broad variety of activities, including direct antimicrobial properties and modulatory roles in the innate immune response. Even if it has been widely demonstrated that bacterial membrane represents the main target of peptide antimicrobial activity, the molecular mechanisms underlying membrane perturbation by HDPs have not been fully clarified yet. Recently, two cryptic HDPs have been identified in human apolipoprotein B and found to be endowed with a broad-spectrum antimicrobial activity, and with anti-biofilm, wound healing and immunomodulatory properties. Moreover, ApoB derived HDPs are able to synergistically act in combination with conventional antibiotics, while being not toxic for eukaryotic cells. Here, by using a multidisciplinary approach, including time killing curves, Zeta potential measurements, membrane permeabilization assays, electron microscopy analyses, and isothermal titration calorimetry studies, the antimicrobial effects of ApoB cryptides have been analysed on bacterial strains either susceptible or resistant to peptide toxicity. Intriguingly, it emerged that even if electrostatic interactions between negatively charged bacterial membranes and positively charged HDPs play a key role in mediating peptide toxicity, they are strongly influenced by the composition of negatively charged bacterial surfaces and by defined extracellular microenvironments.


Assuntos
Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Apolipoproteína B-100/química , Anti-Infecciosos/química , Peptídeos Catiônicos Antimicrobianos/química , Calorimetria , Permeabilidade da Membrana Celular/efeitos dos fármacos , Humanos , Lipopolissacarídeos/química , Lipopolissacarídeos/farmacologia , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Transmissão , Pseudomonas aeruginosa/efeitos dos fármacos
10.
Med Hypotheses ; 126: 20-22, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31010493

RESUMO

Carbamylation (or carbamoylation) is a non-enzymatic post-translational modification process of lysine residues and protein N-termini, which occurs throughout the lifespan of both various plasma proteins and low-density lipoprotein (LDL) particles. Carbamylation results from the binding of isocyanates spontaneously derived from high levels of blood urea, environmental pollutants, nutritional sources and leads to the formation of potentially atherogenic carbamylated-LDL (c-LDL) particles. The carbamylation of LDL apolipoproteins is associated unfavorable downstream effects. Ornithine is a non-proteinogenic amino acid, which plays a central role at the urea cycle function. The primary use of ornithine in supplements is to support athletic performance, liver function and wound recovery. Ornithine is structurally highly similar to lysine, and is only one carbon atom shorter in its side-chain. Therefore, we hypothesize that supplemented ornithine could compete with ε-amino groups of lysine residues found in apolipoproteins of native LDL particles in their binding to isocyanates and decrease c-LDL formation. This issue still remains unresolved in current literature and needs to be elucidated in experimental studies.


Assuntos
Aminoácidos/metabolismo , Aterosclerose/metabolismo , Aterosclerose/terapia , Lipoproteínas LDL/metabolismo , Ornitina/uso terapêutico , Carbamilação de Proteínas , Apolipoproteína B-100/química , Apolipoproteínas/química , Aterosclerose/fisiopatologia , Humanos , Lisina/química , Modelos Biológicos , Ornitina/química
11.
Nanomedicine (Lond) ; 13(20): 2657-2668, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30334470

RESUMO

AIM: We analyzed the protein corona of thermoresponsive, poly(N-isopropylacrylamide)- or poly(N-isopropylmethacrylamide)-based nanogels. MATERIALS & METHODS: Traces of protein corona detected after incubation in human serum were characterized by proteomics and dynamic light scattering in undiluted serum. RESULTS: Apolipoprotein B-100 and albumin were the main components of the protein coronae. For dendritic polyglycerol-poly(N-isopropylacrylamide) nanogels at 37°C, an increase in adsorbed immunoglobulin light chains was detected, followed by partially reversible nanogel aggregation. All nanogels in their hydrophilic state are colloidally stable in serum and bear a dysopsonin-rich protein corona. CONCLUSION: We observed strong changes in NG stability upon slight alterations in the composition of the protein coronae according to nanogel solvation state. Nanogels in their hydrophilic state possess safe protein coronae.


Assuntos
Apolipoproteína B-100/química , Nanopartículas/química , Coroa de Proteína/química , Proteômica , Acrilamidas/química , Apolipoproteína B-100/genética , Difusão Dinâmica da Luz , Humanos , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Nanogéis , Nanopartículas/administração & dosagem , Polietilenoglicóis/química , Polietilenoimina/química , Pele/efeitos dos fármacos
12.
Protein J ; 37(6): 548-571, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30259240

RESUMO

LDL, VLDL and other members of the low-density lipoparticles (LLPs) enter cells through a large family of receptors. The actual receptor ligand(s) in apolipoprotein B100, one of the main proteins of LLP, remain(s) unknown. The objective of this study was to identify true receptor ligand(s) in apo B100, a molecule of 4563 residues. Apo B100 contains 33 analogues of Cardin-Weintraub arginine/lysine-based receptor ligand motifs and shares key lysine motifs and sequence similarity with the LDL receptor-associated protein, MESD, and heat shock proteins. Eleven FITC-labeled synthetic peptides of 21-42 residues, with at least one ligand, were tested for binding and internalization using HeLa cells. All peptides bind but display different binding capacities and patterns. Peptides B0013, B0582, B2366, and B2932 mediate endocytosis and appear in distinct sites in the cytoplasm. B0708 and B3181 bind and remain on the cell surface as aggregates/clusters. Peptides B3119 (Site A) and B3347 (Site B), the putative ligands, showed low binding and no cell entry capacity. Apo B100 regions in this study share similarities with related proteins of known function including chaperone proteins and Apo BEC stimulating protein, and not directly related proteins, e.g., the DNA-binding domain of interferon regulatory factors, MSX2-interacting protein, and snake venom Zinc metalloproteinase-disintegrin-like proteins.


Assuntos
Apolipoproteína B-100 , Endocitose/efeitos dos fármacos , Peptídeos , Receptores de LDL , Motivos de Aminoácidos , Apolipoproteína B-100/química , Apolipoproteína B-100/farmacologia , Células HeLa , Humanos , Peptídeos/química , Peptídeos/farmacologia , Domínios Proteicos , Receptores de LDL/agonistas , Receptores de LDL/metabolismo
13.
J Inorg Biochem ; 188: 29-37, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30119015

RESUMO

[Fe(NO)2] - modified nanoparticles of low-density protein (DNICLDL) can serve as conveyors of iron in the form of stable complexes with ApoB100 protein. As reported recently, in human hepatoma cells DNICLDL significantly increased the total iron content, while showing low toxicity. In the present work, we focused on the effects of internalization of DNIC-modified lipoproteins in macrophages, with special regards to cytotoxicity. DNICLDL was administered to a model macrophage cell line, RAW 264.7. Administration of DNICLDL considerably increased total iron content. High increase of iron was accompanied by moderate toxicity. As shown by in vitro plasmid nicking assay, chelation of iron in the form of DNIC strongly reduced the iron-related reactive oxygen species (ROS) -induced DNA damage. In addition, DNICLDL, plausibly due to its NO-donating activity, did not induce inducible nitric oxide synthase (iNOS) expression, as opposed to other forms of low-density protein (LDL).


Assuntos
Ferro , Lipoproteínas LDL , Macrófagos/metabolismo , Óxidos de Nitrogênio , Animais , Apolipoproteína B-100/química , Apolipoproteína B-100/farmacologia , Ferro/química , Ferro/farmacologia , Lipoproteínas LDL/química , Lipoproteínas LDL/farmacologia , Macrófagos/citologia , Camundongos , Óxidos de Nitrogênio/química , Óxidos de Nitrogênio/farmacologia , Células RAW 264.7
14.
Biosci Rep ; 38(2)2018 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-29545317

RESUMO

Estimation of the function as well as the amount of high-density lipoprotein (HDL) is required to predict the risk of cardiovascular disease development. Cholesterol efflux capacity (CEC) is the key metric for determining the antiatherosclerotic function of HDL. However, the assay methods currently used to calculate CEC are not ideal for clinical use as they require the culture of cells. In the present study, we developed a novel CEC assay using immobilized liposome-bound gel beads (ILGs), containing fluorescently labeled cholesterol, as a substitute for cultured cells. When apolipoprotein B-100 depleted serum, obtained by polyethylene glycol precipitation, was used as the cholesterol acceptors, the basic properties of this method, such as the available range of HDL-cholesterol, efflux temperature and time, and normalization parameters, indicate that this method is sufficient to estimate CEC. Furthermore, the CEC values obtained with this ILG method were also correlated with those obtained with a conventional method using THP-1 macrophages derived foam cells and 3H-cholesterol as a tracer (r = 0.932). Overall, this novel cholesterol efflux assay method is a realistic and effective alternative to current methods in the field while also being easier to use in clinical laboratories as neither cell culture, radioisotope nor ultracentrifugation is required.


Assuntos
Apolipoproteína B-100/química , Colesterol/análise , Lipossomos/química , Polietilenoglicóis/química , Apolipoproteína B-100/metabolismo , Colesterol/metabolismo , Células Espumosas/metabolismo , Células Espumosas/patologia , Humanos , Polietilenoglicóis/metabolismo , Células THP-1
15.
Biochemistry ; 56(31): 4084-4094, 2017 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-28702990

RESUMO

Our previous studies demonstrated that the first 1000 amino acid residues (the ßα1 domain) of human apolipoprotein (apo) B-100, termed apoB:1000, are required for the initiation of lipoprotein assembly and the formation of a monodisperse stable phospholipid (PL)-rich particle. The objectives of this study were (a) to assess the effects on the properties of apoB truncates undergoing sequential inclusion of the amphipathic ß strands in the 700 N-terminal residues of the ß1 domain of apoB-100 and (b) to identify the subdomain in the ß1 domain that is required for the formation of a microsomal triglyceride transfer protein (MTP)-dependent triacylglycerol (TAG)-rich apoB-containing particle. Characterization of particles secreted by stable transformants of McA-RH7777 cells demonstrated the following. (1) The presence of amphipathic ß strands in the 200 N-terminal residues of the ß1 domain resulted in the secretion of apoB truncates (apoB:1050 to apoB:1200) as both lipidated and lipid-poor particles. (2) Inclusion of residues 300-700 of the ß1 domain led to the secretion of apoB:1300, apoB:1400, apoB:1500, and apoB:1700 predominantly as lipidated particles. (3) Particles containing residues 1050-1500 were all rich in PL. (4) There was a marked increase in the lipid loading capacity and TAG content of apoB:1700-containing particles. (5) Only the level of secretion of apoB:1700 was markedly diminished by MTP inhibitor BMS-197636. These results suggest that apoB:1700 marks the threshold for the formation of a TAG-rich particle and support the concept that MTP participates in apoB assembly and secretion at the stage where particles undergo a transition from PL-rich to TAG-rich.


Assuntos
Apolipoproteína B-100/química , Proteínas de Transporte/metabolismo , Hepatócitos/metabolismo , Lipoproteínas VLDL/metabolismo , Animais , Apolipoproteína B-100/genética , Apolipoproteína B-100/metabolismo , Transporte Biológico/efeitos dos fármacos , Proteínas de Transporte/antagonistas & inibidores , Linhagem Celular Tumoral , Fluorenos/farmacologia , Hepatócitos/efeitos dos fármacos , Humanos , Isoindóis/farmacologia , Lipoproteínas VLDL/antagonistas & inibidores , Lipoproteínas VLDL/química , Peso Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosfolipídeos/análise , Fosfolipídeos/metabolismo , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteólise/efeitos dos fármacos , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Triglicerídeos/análise , Triglicerídeos/metabolismo
16.
J Biol Phys ; 43(3): 381-395, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28647778

RESUMO

The secondary structure of apolipoprotein B-100 is studied within the bulk phase and at the air/water interface. In these "in viro" experiments, infrared reflection absorption spectroscopy (IRRAS) study was performed at the air/water interface while circular dichroism (CD) was conducted in the bulk phase. In the bulk phase, the conformational structure containing a significant amount of ß-structure, whereas varying amount of α-helix, unordered structures, and ß-sheet were observed at the air/water interface depending on the low-density lipoprotein (LDL) film interfacial pressure. The present IRRAS results demonstrate the importance of interfacial pressure-induced structural conformations on the apoB-100. A correlation between the secondary structure of the apoB-100 protein and the monomolecular film elasticity at the air/water interface was also established. The orientation of apoB-100 with respect to the LDL film-normal was found to depend on the interfacial pressure exhibited by the monomolecular film. These results may shed light on LDL's pivotal role in the progression of atherosclerotic coronary artery disease as demonstrated previously by clinical trials.


Assuntos
Ar , Lipoproteínas LDL/química , Reologia , Água/química , Apolipoproteína B-100/química , Elasticidade , Pressão , Estrutura Secundária de Proteína , Propriedades de Superfície
17.
Proteomics Clin Appl ; 11(7-8)2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28296203

RESUMO

PURPOSE: Apolipoprotein A-I (ApoA-I) and apolipoprotein B-100 (ApoB-100) are amphipathic proteins that are strong predictors of cardiovascular disease risk. The traceable calibration of apolipoprotein assays is a persistent challenge, especially for ApoB-100, which cannot be solubilized in purified form. EXPERIMENTAL DESIGN: A simultaneous quantitation method for ApoA-I and ApoB-100 was developed using tryptic digestion without predigestion reduction and alkylation, followed by LC separation coupled with isotope dilution MS analysis. The accuracy of the method was assured by selecting structurally exposed signature peptides, optimal choice of detergent, protein:enzyme ratio, and incubation time. Peptide calibrators were value assigned by isobaric tagging isotope dilution MS amino acid analysis. RESULTS: The method reproducibility was validated in technical repeats of three serum samples, giving 2-3% intraday CVs (N = 5) and <7% interday CVs (N = 21). The repeated analysis of interlaboratory harmonization standards showed -1% difference for ApoA-I and -12% for ApoB-100 relative to the assigned value. The applicability of the method was demonstrated by repeated analysis of 24 patient samples with a wide range of total cholesterol and triglyceride levels. CONCLUSIONS AND CLINICAL RELEVANCE: The method is applicable for simultaneous analysis of ApoA-I and ApoB-100 in patient samples, and for characterization of serum pool calibrators for other analytical platforms.


Assuntos
Apolipoproteína A-I/química , Apolipoproteína B-100/química , Análise Química do Sangue/métodos , Espectrometria de Massas , Fragmentos de Peptídeos/sangue , Proteólise , Tripsina/metabolismo , Sequência de Aminoácidos , Apolipoproteína A-I/metabolismo , Apolipoproteína B-100/metabolismo , Calibragem , Cromatografia Líquida , Humanos , Isótopos/química , Modelos Lineares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Reprodutibilidade dos Testes
18.
Anal Biochem ; 518: 25-34, 2017 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-27984014

RESUMO

Two complementary instrumental techniques were used, and the data generated was processed with advanced numerical tools to investigate the interactions between anti-human apoB-100 monoclonal antibody (anti-apoB-100 Mab) and apoB-100 containing lipoproteins. Partial Filling Affinity Capillary Electrophoresis (PF-ACE) combined with Adsorption Energy Distribution (AED) calculations provided information on the heterogeneity of the interactions without any a priori model assumptions. The AED calculations evidenced a homogenous binding site distribution for the interactions. Quartz Crystal Microbalance (QCM) studies were used to evaluate thermodynamics and kinetics of the Low-Density Lipoprotein (LDL) and anti-apoB-100 Mab interactions. High affinity and selectivity were observed, and the emerging data sets were analysed with so called Interaction Maps. In thermodynamic studies, the interaction between LDL and anti-apoB-100 Mab was found to be predominantly enthalpy driven. Both techniques were also used to study antibody interactions with Intermediate-Density (IDL) and Very Low-Density (VLDL) Lipoproteins. By screening affinity constants for IDL-VLDL sample in a single injection we were able to distinguish affinity constants for both subpopulations using the numerical Interaction Map tool.


Assuntos
Anticorpos Monoclonais Murinos/química , Apolipoproteína B-100/química , Modelos Químicos , Termodinâmica , Animais , Humanos , Cinética , Camundongos
19.
Sci Rep ; 6: 36324, 2016 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-27824107

RESUMO

Acidification in the endosome causes lipoprotein release by promoting a conformational change in the LDLR allowing its recycling and degradation of LDL. Notwithstanding conformational changes occurring in the LDLR have expanded considerably, structural changes occurring in LDL particles have not been fully explored yet. The objectives of the present work were to study structural changes occurring in apoB100 by infrared spectroscopy (IR) and also LDL size and morphology by dynamic light scattering (DLS) and electron microscopy (EM) at both pH 7.4 and 5.0. We determined by IR that pH acidification from 7.4 to 5.0, resembling that occurring within endosomal environment, induces a huge reversible structural rearrangement of apoB100 that is characterized by a reduction of beta-sheet content in favor of alpha-helix structures. Data obtained from DLS and EM showed no appreciable differences in size and morphology of LDL. These structural changes observed in apoB100, which are likely implied in particle release from lipoprotein receptor, also compromise the apoprotein stability what would facilitate LDL degradation. In conclusion, the obtained results reveal a more dynamic picture of the LDL/LDLR dissociation process than previously perceived and provide new structural insights into LDL/LDLR interactions than can occur at endosomal low-pH milieu.


Assuntos
Apolipoproteína B-100/química , Apolipoproteína B-100/metabolismo , Lipoproteínas LDL/metabolismo , Difusão Dinâmica da Luz , Endossomos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Lipoproteínas LDL/química , Microscopia Eletrônica , Modelos Moleculares , Ligação Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína
20.
J Mass Spectrom ; 50(12): 1386-92, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26634972

RESUMO

Phospholipids are major components of cell membranes and lipoprotein complexes. They are prone to oxidation by endogenous and exogenous reactive oxygen species yielding a large variety of modified lipids including small aliphatic and phospholipid bound aldehydes and ketones. These carbonyls are strong electrophiles that can modify proteins and, thereby, alter their structures and functions triggering various pathophysiological conditions. The analysis of lipid-protein adducts by liquid chromatography-MS is challenged by their mixed chemical nature (polar peptide and hydrophobic lipid), low abundance in biological samples, and formation of multiple isomers. Thus, we investigated traveling wave ion mobility mass spectrometry (TWIMS) to analyze lipid-peptide adducts generated by incubating model peptides corresponding to the amphipathic ß1 sheet sequence of apolipoprotein B-100 with 1-palmitoyl-2-(oxo-nonanoyl)-sn-glycerophosphatidylcholine (PONPC). The complex mixture of peptides, lipids, and peptide-lipid adducts was separated by TWIMS, which was especially important for the identification of two mono-PONPC-peptide isomers containing Schiff bases at different lysine residues. Moreover, TWIMS separated structural conformers of one peptide-lipid adduct possessing most likely different orientations of the hydrophobic sn-1 fatty acyl residue and head group of PONPC, relative to the peptide backbone.


Assuntos
Lipídeos/química , Espectrometria de Massas/métodos , Modelos Químicos , Peptídeos/química , Apolipoproteína B-100/química , Isomerismo , Fosfatidilcolinas/química
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