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1.
J Mol Biol ; 436(4): 168441, 2024 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-38199491

RESUMO

Amyloid resistance is the inability or the reduced susceptibility of an organism to develop amyloidosis. In this study we have analysed the molecular basis of the resistance to systemic AApoAII amyloidosis, which arises from the formation of amyloid fibrils from apolipoprotein A-II (ApoA-II). The disease affects humans and animals, including SAMR1C mice that express the C allele of ApoA-II protein, whereas other mouse strains are resistant to development of amyloidosis due to the expression of other ApoA-II alleles, such as ApoA-IIF. Using cryo-electron microscopy, molecular dynamics simulations and other methods, we have determined the structures of pathogenic AApoAII amyloid fibrils from SAMR1C mice and analysed the structural effects of ApoA-IIF-specific mutational changes. Our data show that these changes render ApoA-IIF incompatible with the specific fibril morphologies, with which ApoA-II protein can become pathogenic in vivo.


Assuntos
Amiloide , Amiloidose , Apolipoproteína A-II , Animais , Camundongos , Amiloide/química , Amiloide/genética , Amiloidose/genética , Amiloidose/metabolismo , Apolipoproteína A-II/química , Apolipoproteína A-II/genética , Microscopia Crioeletrônica , Alelos , Simulação de Dinâmica Molecular , Mutação , Camundongos Mutantes
2.
J Lipid Res ; 62: 100099, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34324889

RESUMO

Human high-density lipoproteins (HDLs) are a complex mixture of structurally related nanoparticles that perform distinct physiological functions. We previously showed that human HDL containing apolipoprotein A-I (APOA1) but not apolipoprotein A-II (APOA2), designated LpA-I, is composed primarily of two discretely sized populations. Here, we isolated these particles directly from human plasma by antibody affinity chromatography, separated them by high-resolution size-exclusion chromatography and performed a deep molecular characterization of each species. The large and small LpA-I populations were spherical with mean diameters of 109 Å and 91 Å, respectively. Unexpectedly, isotope dilution MS/MS with [15N]-APOA1 in concert with quantitation of particle concentration by calibrated ion mobility analysis demonstrated that the large particles contained fewer APOA1 molecules than the small particles; the stoichiometries were 3.0 and 3.7 molecules of APOA1 per particle, respectively. MS/MS experiments showed that the protein cargo of large LpA-I particles was more diverse. Human HDL and isolated particles containing both APOA1 and APOA2 exhibit a much wider range and variation of particle sizes than LpA-I, indicating that APOA2 is likely the major contributor to HDL size heterogeneity. We propose a ratchet model based on the trefoil structure of APOA1 whereby the helical cage maintaining particle structure has two "settings"-large and small-that accounts for these findings. This understanding of the determinants of HDL particle size and protein cargo distribution serves as a basis for determining the roles of HDL subpopulations in metabolism and disease states.


Assuntos
Apolipoproteína A-II/química , Apolipoproteína A-I/química , HDL-Colesterol/química , Tamanho da Partícula
3.
Cancer Lett ; 495: 112-122, 2020 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-32949679

RESUMO

We hypothesised that synthetic HDL nanoparticles carrying a gemcitabine prodrug and apolipoprotein A-II (sHDLGemA2) would target scavenger receptor-B1 (SR-B1) to preferentially and safely deliver gemcitabine into pancreatic ductal adenocarcinoma (PDAC). We designed, manufactured and characterised sHDLGemA2 nanoparticles sized ~130 nm, incorporating 20 mol% of a gemcitabine prodrug within the lipid bilayer, which strengthens on adding ApoA-II. We measured their ability to inhibit growth in cell lines and cell-derived and patient-derived murine PDAC xenografts. Fluorescent-labelled sHDLGemA2 delivered gemcitabine inside xenografts. Xenograft levels of active gemcitabine after sHDLGemA2 were similar to levels after high-dose free gemcitabine. Growth inhibition in mice receiving 4.5 mg gemcitabine/kg/d, carried in sHDLGemA2, was equivalent to inhibition after high-dose (75 mg/kg/d) free gemcitabine, and greater than inhibition after low-dose (4.5 mg/kg/d) free gemcitabine. sHDLGemA2 slowed growth in semi-resistant cells and a resistant human xenograft. sHDLGemA2 targeted xenografts more effectively than sHDLGemA1. SR-B1 was over-expressed in PDAC cells and xenografts. Targeting by ApoA-II was suppressed by anti-SR-B1. Because sHDLGemA2 provided only ~6% of the free gemcitabine dose for an equivalent response, patient side effects can be greatly reduced, and the sHDLGemA2 concept should be developed through clinical trials.


Assuntos
Apolipoproteína A-II/administração & dosagem , Carcinoma Ductal Pancreático/tratamento farmacológico , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/tratamento farmacológico , Pró-Fármacos/administração & dosagem , Receptores Depuradores Classe B/metabolismo , Animais , Apolipoproteína A-II/química , Apolipoproteína A-II/farmacologia , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Desoxicitidina/administração & dosagem , Desoxicitidina/química , Desoxicitidina/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Humanos , Lipoproteínas HDL/química , Masculino , Camundongos , Nanopartículas , Tamanho da Partícula , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Gencitabina
4.
Clin Biochem ; 50(13-14): 777-783, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28366823

RESUMO

BACKGROUND: Apolipoprotein E (apoE) is closely involved in the pathogenesis of apoE-related diseases, such as Alzheimer's disease and cardiovascular disease. The redox modulation of cysteine-thiols in a protein is involved in various pathophysiological regulations; however, that of apoE has not been studied in detail. Herein, we devised an analytical method to determine the redox status of serum apoE and assessed its relation to serum cholesterol levels and apoE phenotype. METHODS: The present method was based on a band shift assay, using a photocleavable maleimide-conjugated polyethylene glycol. RESULTS: The basic characteristics of the present method were found to be satisfactory to determine the redox status of serum apoE quantitatively. Serum apoE was separated into its reduced-form (r-), non-reduced-form (nr-), apoE-AII complex, and homodimer using this method. R-apoE could be detected as a 40-kDa band, whereas nr-apoE remained as monomeric apoE. R-apoE displayed a preference for VLDL; however, the levels showed the correlation with HDL-cholesterol levels (p<0.005). Redox status of serum apoE was significantly different among apoE phenotypes. The quantitative ratios of nr-apoE to total apoE in serum from subjects with apoE4/E3 were higher than in serum from subjects with apoE3/E3 (p<0.0001) and apoE3/E2 (p<0.001). CONCLUSION: The redox status of serum apoE might be related to the synthesis of HDL. The information concerning the redox status of serum apoE provided by the present method may be a potent indicator to evaluate various apoE-related diseases.


Assuntos
Apolipoproteínas E/sangue , HDL-Colesterol/sangue , Apolipoproteína A-II/sangue , Apolipoproteína A-II/química , Apolipoproteína A-II/isolamento & purificação , Apolipoproteína E2/sangue , Apolipoproteína E2/química , Apolipoproteína E2/isolamento & purificação , Apolipoproteína E3/sangue , Apolipoproteína E3/química , Apolipoproteína E3/isolamento & purificação , Apolipoproteína E4/sangue , Apolipoproteína E4/química , Apolipoproteína E4/isolamento & purificação , Apolipoproteínas E/química , Apolipoproteínas E/isolamento & purificação , HDL-Colesterol/química , Cisteína/química , Diamida/química , Dimerização , Ditiotreitol/química , Ensaio de Desvio de Mobilidade Eletroforética , Células HEK293 , Humanos , Indicadores e Reagentes/química , Peso Molecular , Oxirredução , Processos Fotoquímicos , Polietilenoglicóis/química , Solubilidade , Reagentes de Sulfidrila/química , Raios Ultravioleta
5.
J Colloid Interface Sci ; 488: 1-9, 2017 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-27816633

RESUMO

HYPOTHESIS: Biodistribution is a key issue when it comes to medical applications of nanomaterials. Hollow mesoporous silica nanoparticles (HMSNs) loaded with fluorine compounds can be applied as positive magnetic resonance imaging (MRI) contrast agents (CAs). These CAs exhibit an unusual biodistribution which is influenced by the cargo and which could be linked to their serum protein adsorption behaviour. EXPERIMENTS: HMSNs were post-synthetically loaded with perfluoro-15-crown-5-ether (PFCE). The 19F signal was quantified with MRI in a murine model. Furthermore protein adsorption tests were performed in full serum. FINDINGS: Quantitative analysis of the 19F-signal revealed that the particles were exclusively accumulating in the liver 24h post-injection, and no accumulation in other reticuloendothelial system (RES) organs like spleen or lung was observed. The protein corona around non-loaded and loaded particles was therefore analysed, and more proteins adsorbed on PFCE-loaded particles as compared to the bare particles, and importantly, the amount of apolipoproteins A-1 and A-2, was clearly elevated for the PFCE-loaded particles. The results underline that the type of cargo may have major influences on the biodistribution of mesoporous silica drug vectors.


Assuntos
Apolipoproteína A-II/sangue , Apolipoproteína A-I/sangue , Meios de Contraste/química , Éteres de Coroa/química , Fígado/diagnóstico por imagem , Nanopartículas/química , Dióxido de Silício/química , Adsorção , Animais , Apolipoproteína A-I/química , Apolipoproteína A-II/química , Meios de Contraste/farmacocinética , Éteres de Coroa/farmacocinética , Composição de Medicamentos , Flúor/química , Flúor/farmacocinética , Imagem por Ressonância Magnética de Flúor-19 , Fígado/metabolismo , Camundongos , Nanopartículas/ultraestrutura , Porosidade , Dióxido de Silício/farmacocinética , Distribuição Tecidual
6.
J Proteomics ; 146: 184-94, 2016 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-27385375

RESUMO

UNLABELLED: Given their association with cardiovascular disease protection, there has been intense interest in understanding the biology of high density lipoproteins (HDL). HDL is actually a family of diverse particle types, each made up of discrete - but as yet undetermined - combinations of proteins drawn from up to 95 lipophilic plasma proteins. The abundant apolipoproteins (apo) of the A class (apoA-I, apoA-II and apoA-IV) have been proposed to act as organizing platforms for auxiliary proteins, but this concept has not been systematically evaluated. We assessed the impact of genetic knock down of each platform protein on the particle size distribution of auxiliary HDL proteins. Loss of apoA-I or apoA-II massively reduced HDL lipids and changed the plasma size pattern and/or abundance of several plasma proteins. Surprisingly though, many HDL proteins were not affected, suggesting they assemble on lipid particles in the absence of apoA-I or apoA-II. In contrast, apoA-IV ablation had minor effects on plasma lipids and proteins, suggesting that it forms particles that largely exclude other apolipoproteins. Overall, the data indicate that distinct HDL subpopulations exist that do not contain, nor depend on, apoA-I, apoA-II or apoA-IV and these contribute substantially to the proteomic diversity of HDL. BIOLOGICAL SIGNIFICANCE: Plasma levels of high density lipoproteins (HDL) are inversely correlated with cardiovascular disease. These particles are becoming known as highly heterogeneous entities that have diverse compositions and functions that may impact disease. Unfortunately, we know little about the forces that maintain the composition of each particle in plasma. It has been suggested that certain 'scaffold' proteins, such as apolipoprotein (apo) A-I, apoA-II and apoA-IV, may act as organizing centers for the docking of myriad accessory proteins. To test this hypothesis, we took advantage of the genetic tractability of the mouse model and ablated these three proteins individually. We then tracked the abundance and size profile of the remaining HDL proteins by gel filtration chromatography combined with mass spectrometry. The results clearly show that certain cohorts of proteins depend on each scaffold molecule to assemble normal sized HDL particles under wild-type conditions. This work forms the basis for more detailed studies that will define the specific compositions of HDL subspecies with the possibility of connecting them to specific functions or roles in disease.


Assuntos
Apolipoproteínas/química , Deleção de Genes , Lipoproteínas HDL/química , Animais , Apolipoproteína A-I/química , Apolipoproteína A-I/genética , Apolipoproteína A-II/química , Apolipoproteína A-II/genética , Apolipoproteínas/análise , Apolipoproteínas/genética , Apolipoproteínas A/química , Apolipoproteínas A/genética , Cromatografia em Gel , Espectrometria de Massas , Camundongos , Tamanho da Partícula , Proteômica/métodos
7.
J Lipid Res ; 57(6): 969-79, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27018443

RESUMO

The acute phase (AP) reactant serum amyloid A (SAA), an HDL apolipoprotein, exhibits pro-inflammatory activities, but its physiological function(s) are poorly understood. Functional differences between SAA1.1 and SAA2.1, the two major SAA isoforms, are unclear. Mice deficient in either isoform were used to investigate plasma isoform effects on HDL structure, composition, and apolipoprotein catabolism. Lack of either isoform did not affect the size of HDL, normally enlarged in the AP, and did not significantly change HDL composition. Plasma clearance rates of HDL apolipoproteins were determined using native HDL particles. The fractional clearance rates (FCRs) of apoA-I, apoA-II, and SAA were distinct, indicating that HDL is not cleared as intact particles. The FCRs of SAA1.1 and SAA2.1 in AP mice were similar, suggesting that the selective deposition of SAA1.1 in amyloid plaques is not associated with a difference in the rates of plasma clearance of the isoforms. Although the clearance rate of SAA was reduced in the absence of the HDL receptor, scavenger receptor class B type I (SR-BI), it remained significantly faster compared with that of apoA-I and apoA-II, indicating a relatively minor role of SR-BI in SAA's rapid clearance. These studies enhance our understanding of SAA metabolism and SAA's effects on AP-HDL composition and catabolism.


Assuntos
HDL-Colesterol/metabolismo , Lipoproteínas HDL/sangue , Isoformas de Proteínas/genética , Proteína Amiloide A Sérica/genética , Reação de Fase Aguda/metabolismo , Animais , Apolipoproteína A-I/sangue , Apolipoproteína A-I/química , Apolipoproteína A-II/sangue , Apolipoproteína A-II/química , Apolipoproteína A-II/metabolismo , Humanos , Lipoproteínas HDL/química , Camundongos , Isoformas de Proteínas/química , Receptores Depuradores Classe B/sangue , Receptores Depuradores Classe B/química , Proteína Amiloide A Sérica/química , Proteína Amiloide A Sérica/metabolismo
8.
Proc Natl Acad Sci U S A ; 112(8): E836-45, 2015 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-25675489

RESUMO

In murine senile amyloidosis, misfolded serum apolipoprotein (apo) A-II deposits as amyloid fibrils (AApoAII) in a process associated with aging. Mouse strains carrying type C apoA-II (APOA2C) protein exhibit a high incidence of severe systemic amyloidosis. Previously, we showed that N- and C-terminal sequences of apoA-II protein are critical for polymerization into amyloid fibrils in vitro. Here, we demonstrate that congenic mouse strains carrying type F apoA-II (APOA2F) protein, which contains four amino acid substitutions in the amyloidogenic regions of APOA2C, were absolutely resistant to amyloidosis, even after induction of amyloidosis by injection of AApoAII. In vitro fibril formation tests showed that N- and C-terminal APOA2F peptides did not polymerize into amyloid fibrils. Moreover, a C-terminal APOA2F peptide was a strong inhibitor of nucleation and extension of amyloid fibrils during polymerization. Importantly, after the induction of amyloidosis, we succeeded in suppressing amyloid deposition in senile amyloidosis-susceptible mice by treatment with the C-terminal APOA2F peptide. We suggest that the C-terminal APOA2F peptide might inhibit further extension of amyloid fibrils by blocking the active ends of nuclei (seeds). We present a previously unidentified model system for investigating inhibitory mechanisms against amyloidosis in vivo and in vitro and believe that this system will be useful for the development of novel therapies.


Assuntos
Amiloide/metabolismo , Apolipoproteína A-II/química , Apolipoproteína A-II/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Amiloide/ultraestrutura , Amiloidose/sangue , Amiloidose/patologia , Animais , Colesterol/sangue , Lipoproteínas HDL/sangue , Camundongos Endogâmicos C57BL , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Polimerização , Relação Estrutura-Atividade
9.
FEBS Lett ; 588(6): 845-50, 2014 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-24561203

RESUMO

ApoA-II is the second-major protein of high-density lipoproteins. C-terminal extension in human apoA-II or point substitutions in murine apoA-II cause amyloidosis. The molecular mechanism of apolipoprotein misfolding, from the native predominantly α-helical conformation to cross-ß-sheet in amyloid, is unknown. We used 12 sequence-based prediction algorithms to identify two ten-residue segments in apoA-II that probably initiate ß-aggregation. Previous studies of apoA-II fragments experimentally verify this prediction. Together, experimental and bioinformatics studies explain why the C-terminal extension in human apoA-II causes amyloidosis and why, unlike murine apoA-II, human apoA-II normally does not cause amyloidosis despite its unusually high sequence propensity for ß-aggregation.


Assuntos
Amiloidose/sangue , Apolipoproteína A-II/sangue , Sequência de Aminoácidos , Animais , Apolipoproteína A-II/química , Humanos , Lipoproteínas HDL/sangue , Camundongos , Dobramento de Proteína , Estrutura Secundária de Proteína
10.
J Biol Chem ; 289(10): 7121-7130, 2014 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-24492607

RESUMO

Human TDP-43 represents the main component of neuronal inclusions found in patients with neurodegenerative diseases, especially frontotemporal lobar degeneration and amyotrophic lateral sclerosis. In vitro and in vivo studies have shown that the TAR DNA-binding protein 43 (TDP-43) Drosophila ortholog (TBPH) can biochemically and functionally overlap the properties of the human factor. The recent direct implication of the human heterogeneous nuclear ribonucleoproteins (hnRNPs) A2B1 and A1, known TDP-43 partners, in the pathogenesis of multisystem proteinopathy and amyotrophic lateral sclerosis supports the hypothesis that the physical and functional interplay between TDP-43 and hnRNP A/B orthologs might play a crucial role in the pathogenesis of neurodegenerative diseases. To test this hypothesis and further validate the fly system as a useful model to study this type of diseases, we have now characterized human TDP-43 and Drosophila TBPH similarity in terms of protein-protein interaction pathways. In this work we show that TDP-43 and TBPH share the ability to associate in vitro with Hrp38/Hrb98DE/CG9983, the fruit fly ortholog of the human hnRNP A1/A2 factors. Interestingly, the protein regions of TDP-43 and Hrp38 responsible for reciprocal interactions are conserved through evolution. Functionally, experiments in HeLa cells demonstrate that TDP-43 is necessary for the inhibitory activity of Hrp38 on splicing. Finally, Drosophila in vivo studies show that Hrp38 deficiency produces locomotive defects and life span shortening in TDP-43 with and without animals. These results suggest that hnRNP protein levels can play a modulatory role on TDP-43 functions.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Ribonucleoproteínas/metabolismo , Sequência de Aminoácidos , Animais , Apolipoproteína A-II/química , Sequência Conservada , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Evolução Molecular , Éxons/genética , Humanos , Imunoprecipitação , Locomoção/genética , Longevidade/genética , Dados de Sequência Molecular , Splicing de RNA
11.
Biochemistry ; 52(25): 4324-30, 2013 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-23721456

RESUMO

Reassembled high-density lipoproteins (rHDL) of various sizes and compositions containing apo A-I or apo A-II as their sole protein, dimyristoylphosphatidylcholine (DMPC), and various amounts of free cholesterol (FC) have been isolated and analyzed by differential scanning calorimetry (DSC) and by circular dichroism to determine their stability and the temperature dependence of their helical content. Our data show that the multiple rHDL species obtained at each FC mole percent usually do not have the same FC mole percent as the starting mixture and that the size of the multiple species increases in a quantized way with their respective FC mole percent. DSC studies reveal multiple phases or domains that can be classified as virtual DMPC, which contains a small amount of DMPC that slightly reduces the melting temperature (Tm), a boundary phase that is adjacent to the apo A-I or apo A-II that circumscribes the discoidal rHDL, and a mixed FC/DMPC phase that has a Tm that increases with FC mole percent. Only the large rHDL contain virtual DMPC, whereas all contain boundary phase and various amounts of the mixed FC/DMPC phase according to increasing size and FC mole percent. As reported by others, FC stabilizes the rHDL. For rHDL (apo A-II) compared to rHDL (apo A-I), this occurs in spite of the reduced number of helical regions that mediate binding to the DMPC surface. This effect is attributed to the very high lipophilicity of apo A-II and the reduction in the polarity of the interface between DMPC and the aqueous phase with an increasing FC mole percent, an effect that is expected to increase the strength of the hydrophobic associations with the nonpolar face of the amphipathic helices of apo A-II. These data are relevant to the differential effects of FC and apolipoprotein species on intracellular and plasma membrane nascent HDL assembly and subsequent remodeling by plasma proteins.


Assuntos
HDL-Colesterol/química , Dimiristoilfosfatidilcolina/química , Fosfolipídeos/química , Apolipoproteína A-I/química , Apolipoproteína A-II/química , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Humanos , Transição de Fase , Espectrofotometria Ultravioleta , Termodinâmica
12.
J Lipid Res ; 54(7): 1939-48, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23620136

RESUMO

Many of the apolipoproteins in HDL can elicit cholesterol efflux via ABCA1, a critical initial step in HDL formation. Recent work has indicated that omnipresent amphipathic helices play a critical role, and these have been studied intensively in the most common HDL protein, apolipoprotein (apo)A-I. However, little information exists about helical domain arrangement in other apolipoproteins. We studied two of the smallest apolipoproteins known to interact with ABCA1, human apoA-II and apoC-I, in terms of ability to reorganize phospholipid (PL) bilayers and to promote ABCA1-mediated cholesterol. We found that both proteins contained helical domains that were fast and slow with respect to solubilizing PL. ABCA1-medated efflux required a minimum of a bihelical polypeptide comprised of at least one each of a slow and fast lipid reorganizing domain. In both proteins, the fast helix was located at the C terminus preceded by a slow helix. Helical placement in apoC-I was not critical for ABCA1 activity, but helix swaps in apoA-II dramatically disrupted cholesterol efflux, indicating that the tertiary structure of the longer apolipoprotein is important for the pathway. This work has implications for a more complete molecular understanding of apolipoprotein-mediated cholesterol efflux.


Assuntos
Transportador 1 de Cassete de Ligação de ATP/química , Apolipoproteína A-II/química , Apolipoproteína C-I/química , Colesterol/química , Fosfolipídeos/química , Apolipoproteína C-I/genética , Humanos , Bicamadas Lipídicas/química , Mutação Puntual , Solubilidade
13.
Biochemistry ; 51(23): 4633-41, 2012 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-22631438

RESUMO

High-density lipoproteins (HDL, or "good cholesterol") are heterogeneous nanoparticles that remove excess cell cholesterol and protect against atherosclerosis. The cardioprotective action of HDL and its major protein, apolipoprotein A-I (apoA-I), is well-established, yet the function of the second major protein, apolipoprotein A-II (apoA-II), is less clear. In this review, we postulate an ensemble of apolipoprotein conformations on various HDL. This ensemble is based on the crystal structure of Δ(185-243)apoA-I determined by Mei and Atkinson combined with the "double-hairpin" conformation of apoA-II(dimer) proposed in the cross-linking studies by Silva's team, and is supported by the wide array of low-resolution structural, biophysical, and biochemical data obtained by many teams over decades. The proposed conformational ensemble helps integrate and improve several existing HDL models, including the "buckle-belt" conformation of apoA-I on the midsize disks and the "trefoil/tetrafoil" arrangement on spherical HDL. This ensemble prompts us to hypothesize that endogenous apoA-II (i) helps confer lipid surface curvature during conversion of nascent discoidal HDL(A-I) and HDL(A-II) containing either apoA-I or apoA-II to mature spherical HDL(A-I/A-II) containing both proteins, and (ii) hinders remodeling of HDL(A-I/A-II) by hindering the expansion of the apoA-I conformation. Also, we report that, although endogenous apoA-II circulates mainly on the midsize spherical HDL(A-I/A-II), exogenous apoA-II can bind to HDL of any size, thereby slightly increasing this size and stabilizing the HDL assembly. This suggests distinctly different effects of the endogenous and exogenous apoA-II on HDL. Taken together, the existing results and models prompt us to postulate a new structural and functional role of apoA-II on human HDL.


Assuntos
Apolipoproteína A-II/química , Lipoproteínas HDL/química , Transporte Biológico , Colesterol/metabolismo , Humanos , Modelos Químicos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas , Estabilidade Proteica
14.
J Lipid Res ; 53(8): 1708-15, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22636422

RESUMO

Recombinant expression systems have become powerful tools for understanding the structure and function of proteins, including the apolipoproteins that comprise human HDL. However, human apolipoprotein (apo)A-II has proven difficult to produce by recombinant techniques, likely contributing to our lack of knowledge about its structure, specific biological function, and role in cardiovascular disease. Here we present a novel Escherichia coli-based recombinant expression system that produces highly pure mature human apoA-II at substantial yields. A Mxe GyrA intein containing a chitin binding domain was fused at the C terminus of apoA-II. A 6× histidine-tag was also added at the fusion protein's C terminus. After rapid purification on a chitin column, intein auto-cleavage was induced under reducing conditions, releasing a peptide with only one extra N-terminal Met compared with the sequence of human mature apoA-II. A pass through a nickel chelating column removed any histidine-tagged residual fusion protein, leaving highly pure apoA-II. A variety of electrophoretic, mass spectrometric, and spectrophotometric analyses demonstrated that the recombinant form is comparable in structure to human plasma apoA-II. Similarly, recombinant apoA-II is comparable to the plasma form in its ability to bind and reorganize lipid and promote cholesterol efflux from macrophages via the ATP binding cassette transporter A1. This system is ideal for producing large quantities of recombinant wild-type or mutant apoA-II for structural or functional studies.


Assuntos
Apolipoproteína A-II/genética , Apolipoproteína A-II/isolamento & purificação , Escherichia coli/metabolismo , Engenharia Genética/métodos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Sequência de Aminoácidos , Apolipoproteína A-II/química , Apolipoproteína A-II/metabolismo , Dicroísmo Circular , Técnicas de Cultura , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Expressão Gênica , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo
15.
Biochim Biophys Acta ; 1818(11): 2732-41, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22627110

RESUMO

Exchangeable apolipoproteins A-I and A-II play distinct roles in reverse cholesterol transport. ApoA-I interacts with phospholipids and cholesterol of the cell membrane to make high density lipoprotein particles whereas apolipoprotein A-II interacts with high density lipoprotein particles to release apolipoprotein A-I. The two proteins show a high activity at the aqueous solution/lipid interface and are characterized by a high content of amphipathic α-helices built upon repetition of the same structural motif. We set out to investigate to what extent the number of α-helix repeats of this structural motif modulates the affinity of the protein for lipids and the sensitivity to lipid packing. To this aim we have compared the insertion of apolipoproteins A-I and A-II in phospholipid monolayers formed on a Langmuir trough in conditions where lipid packing, surface pressure and charge were controlled. We also used atomic force microscopy to obtain high resolution topographic images of the surface at a resolution of several nanometers and performed statistical image analysis to calculate the spatial distribution and geometrical shape of apolipoproteins A-I and A-II clusters. Our data indicate that apolipoprotein A-I is sensitive to packing of zwitterionic lipids but insensitive to the packing of negatively charged lipids. Interestingly, apolipoprotein A-II proved to be insensitive to the packing of zwitterionic lipids. The different sensitivity to lipid packing provides clues as to why apolipoprotein A-II barely forms nascent high density lipoprotein particles while apolipoprotein A-I promotes their formation. We conclude that the different interfacial behaviors of apolipoprotein A-I and apolipoprotein A-II in lipidic monolayers are important determinants of their distinctive roles in lipid metabolism.


Assuntos
Apolipoproteína A-II/metabolismo , Apolipoproteína A-I/metabolismo , Fosfolipídeos/química , Apolipoproteína A-I/química , Apolipoproteína A-I/isolamento & purificação , Apolipoproteína A-II/química , Dicroísmo Circular , Microscopia de Força Atômica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
16.
Artigo em Inglês | MEDLINE | ID: mdl-22377539

RESUMO

As a continuation of our proteogenomic studies of equine apolipoproteins, we have obtained molecular masses for several of the apolipoproteins associated with the HDL in horse cerebrospinal fluid (CSF). Using electrospray-ionization mass spectrometry (ESI-MS), we report on values for apolipoproteins, A-I and A-II, as well as acylated apoA-I. In comparison with our previously published data on equine plasma apolipoproteins, there appears to be a higher percentage of acylated apoA-I in the CSF than in plasma. As was the case in plasma, apoA-II circulates as a homodimer. These studies also revealed a protein with a mass of 34,468Da that we are speculating is the value for horse apoE.


Assuntos
Apolipoproteína A-II/líquido cefalorraquidiano , Apolipoproteína A-I/líquido cefalorraquidiano , Cavalos/líquido cefalorraquidiano , Animais , Apolipoproteína A-I/química , Apolipoproteína A-II/química , Masculino , Peso Molecular , Espectrometria de Massas por Ionização por Electrospray
17.
J Biol Chem ; 287(10): 7615-25, 2012 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-22235130

RESUMO

It is well accepted that HDL has the ability to reduce risks for several chronic diseases. To gain insights into the functional properties of HDL, it is critical to understand the HDL structure in detail. To understand interactions between the two major apolipoproteins (apos), apoA-I and apoA-II in HDL, we generated highly defined benchmark discoidal HDL particles. These particles were reconstituted using a physiologically relevant phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) incorporating two molecules of apoA-I and one homodimer of apoA-II per particle. We utilized two independent mass spectrometry techniques to study these particles. The techniques are both sensitive to protein conformation and interactions and are namely: 1) hydrogen deuterium exchange combined with mass spectrometry and 2) partial acetylation of lysine residues combined with MS. Comparison of mixed particles with apoA-I only particles of similar diameter revealed that the changes in apoA-I conformation in the presence of apoA-II are confined to apoA-I helices 3-4 and 7-9. We discuss these findings with respect to the relative reactivity of these two particle types toward a major plasma enzyme, lecithin:cholesterol acyltransferase responsible for the HDL maturation process.


Assuntos
Apolipoproteína A-II/química , Apolipoproteína A-I/química , Lipoproteínas HDL/química , Lipoproteínas/química , Apolipoproteína A-I/metabolismo , Apolipoproteína A-II/metabolismo , Medição da Troca de Deutério , Humanos , Lipoproteínas/metabolismo , Lipoproteínas HDL/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Estrutura Secundária de Proteína
18.
Innate Immun ; 18(1): 171-8, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21382909

RESUMO

Lipid A, the active moiety of LPS, exerts its effects through interaction with TLR4, triggering a signalling cascade that results in the release of pro-inflammatory cytokines. Eritoran is a lipid A analogue that competes with LPS for binding to TLR4; however, after intravenous administration, it undergoes a time-dependent deactivation as a consequence of binding to high-density lipoproteins (HDLs). The site of eritoran association with HDL remains unknown. Therefore the aim of this study was to determine if HDL-associated apolipoproteins A1, A2, serum amyloid A (SAA) and C1, inhibit the ability of eritoran to block LPS-induced TNF-α release from whole blood. Eritoran activity after LPS stimulation in human whole blood was assessed in the presence of reconstituted HDL (rHDL) containing different apos. In rHDL, the major apolipoproteins in both the healthy and septic state, A1 and SAA, caused a significant reduction in eritoran antagonistic activity and had a greater effect than minor apolipoproteins A2 and C1. Apolipoproteins associated with HDL are likely to facilitate eritoran deactivation. Apolipoproteins A1 and SAA should be of particular focus as they are the major apos found on HDL in both the healthy and septic state. Further evaluation of the physical association between apolipoproteins and eritoran should be explored.


Assuntos
Células Sanguíneas/efeitos dos fármacos , Dissacarídeos/antagonistas & inibidores , Lipídeo A/antagonistas & inibidores , Lipoproteínas HDL/imunologia , Fosfatos Açúcares/antagonistas & inibidores , Receptor 4 Toll-Like/antagonistas & inibidores , Apolipoproteína A-I/química , Apolipoproteína A-I/metabolismo , Apolipoproteína A-II/química , Apolipoproteína A-II/metabolismo , Ligação Competitiva , Células Sanguíneas/imunologia , Células Sanguíneas/metabolismo , Células Sanguíneas/patologia , Dissacarídeos/farmacologia , Humanos , Estrutura Molecular , Proteína Amiloide A Sérica/metabolismo , Fosfatos Açúcares/farmacologia , Fator de Necrose Tumoral alfa/metabolismo
19.
PLoS One ; 6(8): e23589, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21887280

RESUMO

In a recent FIELD study the fenofibrate therapy surprisingly failed to achieve significant benefit over placebo in the primary endpoint of coronary heart disease events. Increased levels of atherogenic homocysteine were observed in some patients assigned to fenofibrate therapy but the molecular mechanisms behind this are poorly understood. Herein we investigated HDL lipidomic profiles associated with fenofibrate treatment and the drug-induced Hcy levels in the FIELD substudy. We found that fenofibrate leads to complex HDL compositional changes including increased apoA-II, diminishment of lysophosphatidylcholines and increase of sphingomyelins. Ethanolamine plasmalogens were diminished only in a subgroup of fenofibrate-treated patients with elevated homocysteine levels. Finally we performed molecular dynamics simulations to qualitatively reconstitute HDL particles in silico. We found that increased number of apoA-II excludes neutral lipids from HDL surface and apoA-II is more deeply buried in the lipid matrix than apoA-I. In conclusion, a detailed molecular characterization of HDL may provide surrogates for predictors of drug response and thus help identify the patients who might benefit from fenofibrate treatment.


Assuntos
Fenofibrato/uso terapêutico , Metabolismo dos Lipídeos , Lipoproteínas HDL/química , Apolipoproteína A-II/química , Fracionamento Químico , Análise por Conglomerados , Biologia Computacional , Simulação por Computador , Feminino , Humanos , Masculino , Análise Multivariada , Fatores de Tempo
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