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1.
Mol Vis ; 24: 801-817, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30713420

RESUMO

Purpose: Pseudoexfoliation (PEX) syndrome is an age-related progressive disease of the extracellular matrix with ocular manifestations. PEX is clinically diagnosed by the presence of extracellular exfoliative deposits on the anterior surface of the ocular lens. PEX syndrome is a major risk factor for developing glaucoma, the leading cause of irreversible blindness in the world, and is often associated with the development of cataract. PEX reportedly coexists with Alzheimer disease and increases the risk of heart disease and stroke. PEX material deposited on the anterior surface of the ocular lens is highly proteinaceous, complex, and insoluble, making deciphering the protein composition of the material challenging. Thus, to date, only a small proportion of the protein composition of PEX material is known. The aim of this study was to decipher the protein composition of pathological PEX material deposited on the ocular lens in patients and advance the understanding of pathophysiology of PEX syndrome. Methods: Liquid-chromatography and tandem mass spectrometry (LC-MS/MS) was employed to discover novel proteins in extracts of neat PEX material surgically isolated from patients (n = 4) with PEX syndrome undergoing cataract surgery. A sub-set of the identified proteins was validated with immunohistochemistry using lens capsule specimens from independent patients (n=3); lens capsules from patients with cataract but without PEX syndrome were used as controls (n=4). Expression of transcripts of the validated proteins in the human lens epithelium was analyzed with reverse transcription PCR (RT-PCR). Functional relationships among the proteins identified in this study and genes and proteins previously implicated in the disease were bioinformatically determined using InnateDB. Results: Peptides corresponding to 66 proteins, including ten proteins previously known to be present in PEX material, were identified. Thirteen newly identified proteins were chosen for validation. Of those proteins, 12 were found to be genuine components of the material. The novel protein constituents include apolipoproteins (APOA1 and APOA4), stress response proteins (CRYAA and PRDX2), and blood-related proteins (fibrinogen and hemoglobin subunits), including iron-free hemoglobin. The gene expression data suggest that the identified stress-response proteins and hemoglobin are contributed by the lens epithelium and apolipoproteins and fibrinogen by the aqueous humor to the PEX material. Pathway analysis of the identified novel protein constituents and genes or proteins previously implicated in the disease reiterated the involvement of extracellular matrix organization and degradation, elastic fiber formation, and complement cascade in PEX syndrome. Network analysis suggested a central role of fibronectin in the pathophysiology of the disease. The identified novel protein constituents of PEX material also shed light on the molecular basis of the association of PEX syndrome with heart disease, stroke, and Alzheimer disease. Conclusions: This study expands the understanding of the protein composition of pathological PEX material deposited on the ocular lens in patients with PEX syndrome and provides useful insights into the pathophysiology of this disease. This study together with the previous study by our group (Sharma et al. Experimental Eye Research 2009;89(4):479-85) demonstrate that using neat PEX material, devoid of the underlying lens capsule, for proteomics analysis is an effective approach for deciphering the protein composition of complex and highly insoluble extracellular pathological ocular deposits present in patients with PEX syndrome.


Assuntos
Catarata/metabolismo , Síndrome de Exfoliação/metabolismo , Cápsula do Cristalino/química , Agregados Proteicos , Agregação Patológica de Proteínas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Apolipoproteína A-I/química , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Apolipoproteínas A/química , Apolipoproteínas A/genética , Apolipoproteínas A/metabolismo , Catarata/genética , Catarata/patologia , Cristalinas/química , Cristalinas/genética , Cristalinas/metabolismo , Tecido Elástico/química , Tecido Elástico/metabolismo , Tecido Elástico/patologia , Síndrome de Exfoliação/genética , Síndrome de Exfoliação/patologia , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Fibrinogênio/química , Fibrinogênio/genética , Fibrinogênio/metabolismo , Expressão Gênica , Hemoglobinas/química , Hemoglobinas/genética , Hemoglobinas/metabolismo , Humanos , Cápsula do Cristalino/metabolismo , Cápsula do Cristalino/patologia , Masculino , Peroxirredoxinas/química , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/patologia , Espectrometria de Massas em Tandem
2.
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
3.
Artigo em Inglês | MEDLINE | ID: mdl-26453798

RESUMO

The apolipoprotein A-IV (ApoA-IV) plays a key role in lipid transport and feed intake regulation. In this work, four cDNA sequences encoding ApoA-IV paralogs were identified. Sequence analysis revealed conserved structural features including the common 33-codon block and nine repeated motifs. Gene structure analysis identified four exons and three introns except for apoA-IVAa1 (with only 3 exons). Synteny analysis showed that the four paralogs were structured into two clusters (cluster A containing apoA-IVAa1 and apoA-IVAa2 and cluster B with apoA-IVBa3 and apoA-IVBa4) linked to an apolipoprotein E. Phylogenetic analysis clearly separated the paralogs according to their cluster organization as well as revealed four subclades highly conserved in Acanthopterygii. Whole-mount analyses (WISH) in early larvae (0 and 1day post-hatch (dph)) showed that the four paralogs were mainly expressed in yolk syncytial layer surrounding the oil globules. Later, at 3 and 5dph, the four paralogs were mainly expressed in liver and intestine although with differences in their relative abundance and temporal expression patterns. Diet supply triggered the intensity of WISH signals in the intestine of the four paralogs. Quantification of mRNA abundance by qPCR using whole larvae only detected the induction by diet at 5dph. Moreover, transcript levels increased progressively with age except for apoA-IVAa2, which appeared as a low-expressed isoform. Expression analysis in juvenile tissues confirmed that the four paralogs were mainly expressed in liver and intestine and secondary in other tissues. The role of these ApoA-IV genes in lipid transport and the possible role of apoA-IVAa2 as a regulatory form are discussed.


Assuntos
Apolipoproteínas A/genética , Linguados/genética , Regulação da Expressão Gênica no Desenvolvimento , Genômica , Filogenia , Homologia de Sequência do Ácido Nucleico , Sequência de Aminoácidos , Animais , Apolipoproteínas A/química , Apolipoproteínas A/metabolismo , Dieta , Linguados/crescimento & desenvolvimento , Larva/genética , Larva/crescimento & desenvolvimento , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sintenia
4.
Atherosclerosis ; 241(2): 649-56, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26117402

RESUMO

OBJECTIVE: Previous studies have indicated that supplementation with probiotics might improve lipid metabolism. The objective of the study was to evaluate the effect of supplementation with probiotic strains Lactobacillus curvatus (L. curvatus) HY7601 and Lactobacillus plantarum (L. plantarum) KY1032 on triglyceride (TG) and apolipoprotein A-V (apo A-V) levels. METHODS: A randomized, double-blinded, placebo-controlled study was conducted with 128 non-diabetic subjects with hypertriglyceridemia. Over a 12-week test period, the probiotic group consumed 2 g/day of a powdered supplement containing L. curvatus HY7601 and L. plantarum KY1032, whereas the placebo group consumed a powder lacking probiotics. RESULTS: After the treatment, the probiotic group showed an 18.3% (P < 0.001) reduction in TGs and increases of 21.1% (P = 0.001) and 15.6% (P < 0.001) in the apo A-V and LDL particle size, respectively. The probiotic group had a significant reduction in TGs (P = 0.040) and increases in the plasma apo A-V (P = 0.003) and LDL particle size (P < 0.001) compared with the placebo group. In the probiotic group, the reduction in the TG levels was negatively correlated with changes in the apo A-V and baseline TGs, regardless of the APOA5 -1131T > C genotype. CONCLUSION: The consumption of two probiotic strains for 12 weeks reduced TGs and increased the apo A-V and LDL particle size in hypertriglyceridemic subjects. This effect was more pronounced in subjects with higher levels of fasting TGs regardless of their APOA5 -1131T > C genotype.


Assuntos
Apolipoproteínas A/química , Hipertrigliceridemia/terapia , Lactobacillus plantarum , Lactobacillus , Probióticos/uso terapêutico , Antropometria , Apolipoproteína A-V , Apolipoproteínas A/genética , Glicemia/análise , Pressão Sanguínea , Proteína C-Reativa/química , LDL-Colesterol/sangue , Estudos de Coortes , Suplementos Nutricionais , Método Duplo-Cego , Jejum , Ácidos Graxos não Esterificados/sangue , Feminino , Genótipo , Humanos , Hipertrigliceridemia/microbiologia , Lipoproteínas LDL/química , Masculino , Pessoa de Meia-Idade , Tamanho da Partícula , Triglicerídeos/sangue
5.
Mol Cells ; 38(6): 573-9, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25997739

RESUMO

Apolipoprotein A-I and A-IV are protein constituents of high-density lipoproteins although their functional difference in lipoprotein metabolism is still unclear. To compare anti-atherogenic properties between apoA-I and apoA-4, we characterized both proteins in lipid-free and lipid-bound state. In lipid-free state, apoA4 showed two distinct bands, around 78 and 67 Å on native gel electrophoresis, while apoA-I showed scattered band pattern less than 71 Å. In reconstituted HDL (rHDL) state, apoA-4 showed three major bands around 101 Å and 113 Å, while apoA-I-rHDL showed almost single band around 98 Å size. Lipid-free apoA-I showed 2.9-fold higher phospholipid binding ability than apoA-4. In lipid-free state, BS3-crosslinking revealed that apoA-4 showed less multimerization tendency upto dimer, while apoA-I showed pentamerization. In rHDL state (95:1), apoA-4 was existed as dimer as like as apoA-I. With higher phospholipid content (255:1), five apoA-I and three apoA-4 were required to the bigger rHDL formation. Regardless of particle size, apoA-I-rHDL showed superior LCAT activation ability than apoA-4-rHDL. Uptake of acetylated LDL was inhibited by apoA-I in both lipid-free and lipid-bound state, while apoA-4 inhibited it only lipid-free state. ApoA-4 showed less anti-atherogenic activity with more sensitivity to glycation. In conclusion, apoA-4 showed inferior physiological functions in lipid-bound state, compared with those of apoA-I, to induce more pro-atherosclerotic properties.


Assuntos
Apolipoproteína A-I/química , Apolipoproteína A-I/fisiologia , Apolipoproteínas A/química , Apolipoproteínas A/fisiologia , Antioxidantes/química , Antioxidantes/fisiologia , Apolipoproteína A-I/isolamento & purificação , Apolipoproteína A-I/farmacologia , Apolipoproteínas A/isolamento & purificação , Apolipoproteínas A/farmacologia , Aterosclerose/tratamento farmacológico , Aterosclerose/metabolismo , Linhagem Celular , Dicroísmo Circular , Humanos , Lipoproteínas HDL/química , Masculino , Fosfolipídeos/química , Relação Estrutura-Atividade
6.
Biomacromolecules ; 16(4): 1311-21, 2015 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-25794196

RESUMO

Understanding nanoparticle-protein interactions is a crucial issue in the development of targeted nanomaterial delivery. Besides unraveling the composition of the nanoparticle's protein coronas, distinct proteins thereof could control nanoparticle uptake into specific cell types. Here we differentially analyzed the protein corona composition on four polymeric differently functionalized nanoparticles by label-free quantitative mass spectrometry. Next, we correlated the relative abundance of identified proteins in the corona with enhanced or decreased cellular uptake of nanoparticles into human cancer and bone marrow stem cells to identify key candidates. Finally, we verified these candidate proteins by artificially decorating nanoparticles with individual proteins showing that nanoparticles precoated with the apolipoproteins ApoA4 or ApoC3 significantly decreased the cellular uptake, whereas precoating with ApoH increased the cellular uptake.


Assuntos
Apolipoproteína C-III/metabolismo , Apolipoproteínas A/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Nanopartículas/química , Apolipoproteína C-III/química , Apolipoproteínas A/química , Transporte Biológico , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/metabolismo , Nanopartículas/metabolismo
7.
J Biol Chem ; 290(17): 10689-702, 2015 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-25733664

RESUMO

Apolipoprotein (apo)A-IV is a lipid emulsifying protein linked to a range of protective roles in obesity, diabetes, and cardiovascular disease. It exists in several states in plasma including lipid-bound in HDL and chylomicrons and as monomeric and dimeric lipid-free/poor forms. Our recent x-ray crystal structure of the central domain of apoA-IV shows that it adopts an elongated helical structure that dimerizes via two long reciprocating helices. A striking feature is the alignment of conserved proline residues across the dimer interface. We speculated that this plays important roles in the structure of the lipid-free protein and its ability to bind lipid. Here we show that the systematic conversion of these prolines to alanine increased the thermodynamic stability of apoA-IV and its propensity to oligomerize. Despite the structural stabilization, we noted an increase in the ability to bind and reorganize lipids and to promote cholesterol efflux from cells. The novel properties of these mutants allowed us to isolate the first trimeric form of an exchangeable apolipoprotein and characterize it by small-angle x-ray scattering and chemical cross-linking. The results suggest that the reciprocating helix interaction is a common feature of all apoA-IV oligomers. We propose a model of how self-association of apoA-IV can result in spherical lipoprotein particles, a model that may have broader applications to other exchangeable apolipoprotein family members.


Assuntos
Apolipoproteínas A/química , Apolipoproteínas A/metabolismo , Alanina/química , Substituição de Aminoácidos , Apolipoproteínas A/genética , Colesterol/metabolismo , Sequência Conservada , Humanos , Lipossomos/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Prolina/química , Ligação Proteica , Multimerização Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espalhamento a Baixo Ângulo , Termodinâmica , Difração de Raios X
8.
J Biomol Struct Dyn ; 33(9): 1973-88, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25372987

RESUMO

Human transthyretin (hTTR) is a multifunctional protein involved in several amyloidogenic diseases. Besides transportation of thyroxin and vitamin-A, its role towards the catalysis of apolipoprotein-A1 and Aß-peptide are also drawing interest. The role of water molecules in the catalytic mechanism is still unknown. Extensive analyses of 14 high-resolution X-ray structures of human transthyretin and MD simulation studies have revealed the presence of eight conserved hydrophilic centres near its catalytic zone which may be indispensable for the function, dynamics and stability of the protein. Three water molecules (W1, W2 and W3) form a cluster and play an important role in the recognition of the catalytic and RBP-binding residues. They also induce the reorganisation of the His88 for coupling with other catalytic residues (His90, Glu92). Another water molecule (W5) participate in inter-monomer recognition between the catalytic and thyroxin binding sites. The rest four water molecules (W6, W*, W(#) and W(†)) form a distorted tetrahedral cluster and impart stability to the catalytic core of hTTR. The conserved water mediated recognition dynamics of the different functional sites may provide some rational clues towards the understanding of the activity and mechanism of hTTR.


Assuntos
Histidina/química , Pré-Albumina/química , Tiroxina/química , Água/química , Apolipoproteínas A/química , Sítios de Ligação , Catálise , Cristalografia por Raios X , Histidina/genética , Humanos , Ligações de Hidrogênio , Simulação de Dinâmica Molecular , Pré-Albumina/metabolismo , Estrutura Terciária de Proteína , Vitamina A/química , Vitamina A/metabolismo
9.
Arq Bras Cardiol ; 103(1): 76-84, 2014 Jul.
Artigo em Inglês, Português | MEDLINE | ID: mdl-25120086

RESUMO

The chemical structure of lipoprotein (a) is similar to that of LDL, from which it differs due to the presence of apolipoprotein (a) bound to apo B100 via one disulfide bridge. Lipoprotein (a) is synthesized in the liver and its plasma concentration, which can be determined by use of monoclonal antibody-based methods, ranges from < 1 mg to > 1,000 mg/dL. Lipoprotein (a) levels over 20-30 mg/dL are associated with a two-fold risk of developing coronary artery disease. Usually, black subjects have higher lipoprotein (a) levels that, differently from Caucasians and Orientals, are not related to coronary artery disease. However, the risk of black subjects must be considered. Sex and age have little influence on lipoprotein (a) levels. Lipoprotein (a) homology with plasminogen might lead to interference with the fibrinolytic cascade, accounting for an atherogenic mechanism of that lipoprotein. Nevertheless, direct deposition of lipoprotein (a) on arterial wall is also a possible mechanism, lipoprotein (a) being more prone to oxidation than LDL. Most prospective studies have confirmed lipoprotein (a) as a predisposing factor to atherosclerosis. Statin treatment does not lower lipoprotein (a) levels, differently from niacin and ezetimibe, which tend to reduce lipoprotein (a), although confirmation of ezetimibe effects is pending. The reduction in lipoprotein (a) concentrations has not been demonstrated to reduce the risk for coronary artery disease. Whenever higher lipoprotein (a) concentrations are found, and in the absence of more effective and well-tolerated drugs, a more strict and vigorous control of the other coronary artery disease risk factors should be sought.


Assuntos
Lipoproteína(a)/fisiologia , Apolipoproteínas A/química , Apolipoproteínas A/genética , Humanos , Lipoproteína(a)/análise , Lipoproteína(a)/metabolismo , Fatores de Risco
10.
Arq. bras. cardiol ; 103(1): 76-84, 07/2014. tab
Artigo em Inglês | LILACS | ID: lil-718102

RESUMO

The chemical structure of lipoprotein (a) is similar to that of LDL, from which it differs due to the presence of apolipoprotein (a) bound to apo B100 via one disulfide bridge. Lipoprotein (a) is synthesized in the liver and its plasma concentration, which can be determined by use of monoclonal antibody-based methods, ranges from < 1 mg to > 1,000 mg/dL. Lipoprotein (a) levels over 20-30 mg/dL are associated with a two-fold risk of developing coronary artery disease. Usually, black subjects have higher lipoprotein (a) levels that, differently from Caucasians and Orientals, are not related to coronary artery disease. However, the risk of black subjects must be considered. Sex and age have little influence on lipoprotein (a) levels. Lipoprotein (a) homology with plasminogen might lead to interference with the fibrinolytic cascade, accounting for an atherogenic mechanism of that lipoprotein. Nevertheless, direct deposition of lipoprotein (a) on arterial wall is also a possible mechanism, lipoprotein (a) being more prone to oxidation than LDL. Most prospective studies have confirmed lipoprotein (a) as a predisposing factor to atherosclerosis. Statin treatment does not lower lipoprotein (a) levels, differently from niacin and ezetimibe, which tend to reduce lipoprotein (a), although confirmation of ezetimibe effects is pending. The reduction in lipoprotein (a) concentrations has not been demonstrated to reduce the risk for coronary artery disease. Whenever higher lipoprotein (a) concentrations are found, and in the absence of more effective and well-tolerated drugs, a more strict and vigorous control of the other coronary artery disease risk factors should be sought.


A partícula de lipoproteína (a) apresenta estrutura semelhante à da LDL, diferenciando-se pela presença da apolipoproteína (a) ligada por uma ponte dissulfeto à apolipoproteína B. Sua síntese ocorre no fígado e sua concentração plasmática varia de < 1 mg a > 1.000 mg/dL, podendo ser dosada de rotina em laboratório clínico por método baseado em anticorpos monoclonais. Acima de 20 a 30 mg/dL o risco de desenvolvimento de doença cardiovascular aumenta em cerca de duas vezes, o que não é válido para os afrodescendentes, que já apresentam normalmente níveis mais altos dessa lipoproteína, do que caucasianos e orientais. Entretanto, o risco para indivíduos negros também deve ser levado em conta. Gênero e idade exercem pouca influência na concentração de lipoproteína (a). A homologia com o plasminogênio, que interfere na cascata fibrinolítica, pode ser um mecanismo da aterogenicidade da lipoproteína (a). Entretanto, a deposição direta na parede da artéria também é um dos mecanismos possíveis, sendo a lipoprotrína (a) mais oxidável do que a LDL. De forma geral estudos prospectivos confirmam a lipoproteína (a) como fator predisponente à aterosclerose. O uso de estatinas não interfere no nível da lipoproteína (a), diferentemente da niacina e da ezetimiba, que promovem sua diminuição, embora essa última dependa de confirmação. Não está demonstrado que a redução de lipoproteína (a) resulte em diminuição de risco de doença arterial coronária. Diante de concentrações mais elevadas de lipoproteína (a) e na falta de medicações mais efetivas e de boa tolerabilidade, deve-se, pelo menos, procurar controlar, de forma mais rigorosa, os outros fatores de risco de doença arterial coronária.


Assuntos
Humanos , Lipoproteína(a)/fisiologia , Apolipoproteínas A/química , Apolipoproteínas A/genética , Lipoproteína(a)/análise , Lipoproteína(a)/metabolismo , Fatores de Risco
11.
Glycoconj J ; 31(4): 289-98, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24723206

RESUMO

Lipoprotein(a) immune complexes [Lp(a) IC] of varying particle density obtained by ultracentrifugation of plasma from normal healthy donors were markedly dominated by IgG. Lp(a) and immunoglobulins were liberated from plasma Lp(a) IC by treatment with melibiose, a sugar specific for circulating anti-α-galactoside antibody (anti-Gal). Upon incubation with plasma lipoprotein fraction anti-Gal but not the α-glucoside-specific antibody from human plasma formed de novo IC with Lp(a). Binding of Lp(a) sugar-reversibly enhanced the fluorescence of FITC-labeled anti-Gal as did binding of α-galactoside-containing glycoproteins. This effect apparently due to conformational shift in the Fc region of the antibody was also produced by apo(a) subunit separated from Lp(a) and de-O-glycosylated apo(a) but not by any other plasma lipoproteins or by Lp(a) pre-incubated with the O-glycan-specific lectin jacalin. O-Glycans and their terminal sialic acid moieties in apo(a) of circulating Lp(a)-anti-Gal IC, in contrast to those in pure Lp(a), were inaccessible to jacalin and anion exchange resin, respectively. Unlike other plasma lipoproteins, Lp(a) inhibited Griffonia simplicifolia isolectin B4 which also accommodates serine- and threonine-rich peptide sequence (STPS) as surrogate ligand to α-galactosides at its binding site. Results suggest that anti-Gal recognizes STPS in the O-glycan-rich regions of apo(a) subunit in Lp(a) which contains no α-linked galactose.


Assuntos
Complexo Antígeno-Anticorpo/imunologia , Apolipoproteínas A/imunologia , Galactosídeos/imunologia , Imunoglobulina G/imunologia , Motivos de Aminoácidos , Complexo Antígeno-Anticorpo/sangue , Apolipoproteínas A/química , Sítios de Ligação , Humanos , Imunoglobulina G/sangue , Serina/química , Treonina/química
12.
J Biol Chem ; 289(9): 5596-608, 2014 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-24425874

RESUMO

Apolipoprotein (apo)A-IV plays important roles in dietary lipid and glucose metabolism, and knowledge of its structure is required to fully understand the molecular basis of these functions. However, typical of the entire class of exchangeable apolipoproteins, its dynamic nature and affinity for lipid has posed challenges to traditional high resolution structural approaches. We previously reported an x-ray crystal structure of a dimeric truncation mutant of apoA-IV, which showed a unique helix-swapping molecular interface. Unfortunately, the structures of the N and C termini that are important for lipid binding were not visualized. To build a more complete model, we used chemical cross-linking to derive distance constraints across the full-length protein. The approach was enhanced with stable isotope labeling to overcome ambiguities in determining molecular span of the cross-links given the remarkable similarities in the monomeric and dimeric apoA-IV structures. Using 51 distance constraints, we created a starting model for full-length monomeric apoA-IV and then subjected it to two modeling approaches: (i) molecular dynamics simulations and (ii) fitting to small angle x-ray scattering data. This resulted in the most detailed models yet for lipid-free monomeric or dimeric apoA-IV. Importantly, these models were of sufficient detail to direct the experimental identification of new functional residues that participate in a "clasp" mechanism to modulate apoA-IV lipid affinity. The isotope-assisted cross-linking approach should prove useful for further study of this family of apolipoproteins in both the lipid-free and -bound states.


Assuntos
Apolipoproteínas A/química , Simulação de Dinâmica Molecular , Apolipoproteínas A/genética , Cristalografia por Raios X , Humanos , Espalhamento a Baixo Ângulo , Difração de Raios X
13.
J Lipid Res ; 55(4): 625-34, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24478033

RESUMO

Apo(a), the distinguishing protein component of lipoprotein(a) [Lp(a)], exhibits sequence similarity to plasminogen and can inhibit binding of plasminogen to cell surfaces. Plasmin generated on the surface of vascular cells plays a role in cell migration and proliferation, two of the fibroproliferative inflammatory events that underlie atherosclerosis. The ability of apo(a) to inhibit pericellular plasminogen activation on vascular cells was therefore evaluated. Two isoforms of apo(a), 12K and 17K, were found to significantly decrease tissue-type plasminogen activator-mediated plasminogen activation on human umbilical vein endothelial cells (HUVECs) and THP-1 monocytes and macrophages. Lp(a) purified from human plasma decreased plasminogen activation on THP-1 monocytes and HUVECs but not on THP-1 macrophages. Removal of kringle V or the strong lysine binding site in kringle IV10 completely abolished the inhibitory effect of apo(a). Treatment with carboxypeptidase B to assess the roles of carboxyl-terminal lysines in cellular receptors leads in most cases to decreases in plasminogen activation as well as plasminogen and apo(a) binding; however, inhibition of plasminogen activation by apo(a) was unaffected. Our findings directly demonstrate that apo(a) inhibits pericellular plasminogen activation in all three cell types, although binding of apo(a) to cell-surface receptors containing carboxyl-terminal lysines does not appear to play a major role in the inhibition mechanism.


Assuntos
Apolipoproteínas A/fisiologia , Plasminogênio/fisiologia , Apolipoproteínas A/química , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana , Humanos , Leucócitos Mononucleares/metabolismo , Lisina/fisiologia , Macrófagos/metabolismo , Ativadores de Plasminogênio/química , Ativadores de Plasminogênio/farmacologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas
14.
Physiol Behav ; 120: 136-42, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23911688

RESUMO

Rodent apoA-IV is expressed predominantly in small intestine and also expressed to a small extent in liver and hypothalamus. ApoA-IV has been shown to inhibit food intake in rats when injected centrally. In the current study, we hypothesize that a specific sequence within rat apoA-IV is responsible for mediating the anorectic effect. We use a bacterial expression system to generate truncation mutants (Δ249-371, Δ117-371 and Δ1-61) of rat apoA-IV and assess the ability of various regions of the molecule to inhibit food intake. The results indicate that a responsible sequence exists within the N-terminal 61 amino acids of rat apoA-IV. Synthetic peptides (1-30 EVTSDQVANVMWDYFTQLSNNAKEAVEQLQ, 1-15 EVTSDQVANVMWDYF and 17-30 QLSNNAKEAVEQLQ) were used to specify the region in between residues 1 and 30. A 14-mer peptide (17-30) encompassing this sequence was capable of reducing food intake in a dose-dependent manner whereas a peptide designed on a more C-terminal region (211-232) of apoA-IV (QEKLNHQMEGLAFQMKKNAEEL) failed to exhibit the dose-dependent anorectic effect. The isolation of this sequence provides a valuable tool for future work directed at identifying apoA-IV binding proteins and is a key step for exploring the potential of therapeutic manipulation of food intake via this pathway.


Assuntos
Apolipoproteínas A/química , Apolipoproteínas A/farmacologia , Depressores do Apetite , Sequência de Aminoácidos , Animais , Relação Dose-Resposta a Droga , Ingestão de Alimentos/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Masculino , Dados de Sequência Molecular , Mutagênese , Peptídeos/síntese química , Peptídeos/farmacologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , Relação Estrutura-Atividade
15.
J Lipid Res ; 54(10): 2815-30, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23828779

RESUMO

Oxidized phospholipids (OxPLs) are present on apolipoprotein (a) [apo(a)] and lipoprotein (a) [Lp(a)] but the determinants influencing their binding are not known. The presence of OxPLs on apo(a)/Lp(a) was evaluated in plasma from healthy humans, apes, monkeys, apo(a)/Lp(a) transgenic mice, lysine binding site (LBS) mutant apo(a)/Lp(a) mice with Asp(55/57)→Ala(55/57) substitution of kringle (K)IV10)], and a variety of recombinant apo(a) [r-apo(a)] constructs. Using antibody E06, which binds the phosphocholine (PC) headgroup of OxPLs, Western and ELISA formats revealed that OxPLs were only present in apo(a) with an intact KIV10 LBS. Lipid extracts of purified human Lp(a) contained both E06- and nonE06-detectable OxPLs by tandem liquid chromatography-mass spectrometry (LC-MS/MS). Trypsin digestion of 17K r-apo(a) showed PC-containing OxPLs covalently bound to apo(a) fragments by LC-MS/MS that could be saponified by ammonium hydroxide. Interestingly, PC-containing OxPLs were also present in 17K r-apo(a) with Asp(57)→Ala(57) substitution in KIV10 that lacked E06 immunoreactivity. In conclusion, E06- and nonE06-detectable OxPLs are present in the lipid phase of Lp(a) and covalently bound to apo(a). E06 immunoreactivity, reflecting pro-inflammatory OxPLs accessible to the immune system, is strongly influenced by KIV10 LBS and is unique to human apo(a), which may explain Lp(a)'s pro-atherogenic potential.


Assuntos
Apolipoproteínas A/química , Lipoproteína(a)/química , Fosfolipídeos/química , Animais , Apolipoproteínas A/sangue , Sítios de Ligação , Ensaio de Imunoadsorção Enzimática , Gorilla gorilla , Humanos , Lipoproteína(a)/sangue , Macaca fascicularis , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oxirredução , Pan paniscus , Pan troglodytes , Papio , Fosfolipídeos/sangue , Fosfolipídeos/isolamento & purificação , Ligação Proteica , Especificidade da Espécie , Espectrometria de Massas em Tandem
16.
Rapid Commun Mass Spectrom ; 27(12): 1294-302, 2013 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-23681806

RESUMO

RATIONALE: Apolipoprotein(a) [apo(a)] is the defining protein component of lipoprotein(a) [Lp(a)], an independent risk factor for cardiovascular disease. The regulation of Lp(a) levels in blood is poorly understood in part due to technical challenges in measuring Lp(a) kinetics. Improvements in the ability to readily and reliably measure the kinetics of apo(a) using a stable isotope labeled tracer is expected to facilitate studies of the role of Lp(a) in cardiovascular disease. Since investigators typically determine the isotopic labeling of protein-bound amino acids following acid-catalyzed hydrolysis of a protein of interest [e.g., apo(a)], studies of protein synthesis require extensive protein purification which limits throughput and often requires large sample volumes. We aimed to develop a rapid and efficient method for studying apo(a) kinetics that is suitable for use in studies involving human subjects. METHODS: Microfluidic device and tandem mass spectrometry were used to quantify the incorporation of [(2)H3]-leucine tracer into protein-derived peptides. RESULTS: We demonstrated that it is feasible to quantify the incorporation of [(2)H3]-leucine tracer into a proteolytic peptide from the non-kringle repeat region of apo(a) in human subjects. Specific attention was directed toward optimizing the multiple reaction monitoring (MRM) transitions, mass spectrometer settings, and chromatography (i.e., critical parameters that affect the sensitivity and reproducibility of isotopic enrichment measurements). The results demonstrated significant advantages with the use of a microfluidic device technology for studying apo(a) kinetics, including enhanced sensitivity relative to conventional micro-flow chromatography, a virtually drift-free elution profile, and a stable and robust electrospray. CONCLUSIONS: The technological advances described herein enabled the implementation of a novel method for studying the kinetics of apo(a) in human subjects infused with [(2)H3]-leucine.


Assuntos
Apolipoproteínas A/química , Técnicas Analíticas Microfluídicas/métodos , Espectrometria de Massas em Tandem/métodos , Humanos , Cinética
17.
Protein Eng Des Sel ; 26(6): 425-32, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23571426

RESUMO

Angiogenesis is crucial for tumor growth and metastasis. Blocking this process is, therefore, a potentially powerful approach for the treatment of cancer. Human apolipoprotein(a) kringle V (rhLK8) is an angiogenesis inhibitor and is currently under development as an anti-cancer therapeutic. However, a relatively short in vivo half-life limits its widespread clinical use. This study was performed to evaluate whether fusion of an Fc domain to rhLK8 can extend plasma half-life. RhLK8-Fc fusion protein was expressed in CHO DG44 cells as a dimer and was readily purified by protein G affinity chromatography. The anti-angiogenic activity of rhLK8-Fc was similar to that of rhLK8, as determined by migration and tube formation assays with endothelial cells in vitro and a chorioallantoic membrane assay in vivo. Pharmacokinetic profiles in mice after single intravenous administration of rhLK8 or rhLK8-Fc showed that Fc fusion significantly increased the elimination half-life (t(½)) and the systemic exposure (AUC(inf)) of the protein, in parallel with a significant decrease in total clearance (CL). These data suggest that Fc fusion to rhLK8 is a powerful strategy for extending the plasma half-life of rhLK8 without affecting its anti-angiogenic activity, and could thus improve the clinical applicability of rhLK8.


Assuntos
Inibidores da Angiogênese/farmacocinética , Apolipoproteínas A/farmacocinética , Fragmentos Fc das Imunoglobulinas/sangue , Fragmentos de Peptídeos/farmacocinética , Proteínas Recombinantes de Fusão/farmacocinética , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Animais , Apolipoproteínas A/química , Apolipoproteínas A/farmacologia , Células CHO , Movimento Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Meia-Vida , Células Endoteliais da Veia Umbilical Humana , Humanos , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Neovascularização Fisiológica/efeitos dos fármacos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia
18.
J Immunoassay Immunochem ; 34(2): 166-79, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23537301

RESUMO

Enzyme immunoassay for lipoprotein(a) [Lp(a)] using antibodies to both apoB and apo(a) subunits (a-B assay) is shown to be affected by differential masking of apoB by apo(a) and the presence of LDL-Lp(a) adducts. An apoB-independent immunoassay by capturing Lp(a) through its O-glycans on microplate-coated lectin jacalin and quantitation using peroxidase-labeled anti-apo(a) (J-a assay) is described. J-a assay response is linear, more than twice as sensitive as a-B assay, and is suppressed only 18 ± 5% by non-Lp(a) O-glycan-containing proteins of serum. Wide variations in IgA did not significantly affect Lp(a) binding to jacalin (CV = 6.4%).


Assuntos
Proteínas Imobilizadas/química , Técnicas Imunoenzimáticas , Lipoproteína(a)/sangue , Lectinas de Plantas/química , Adolescente , Adulto , Anticorpos/química , Apolipoproteínas A/química , Apolipoproteínas B/química , Humanos , Imunoglobulina A/sangue , Peroxidase/química
19.
J Lipid Res ; 54(3): 649-61, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23307945

RESUMO

During the diagnosis of three unrelated patients with severe hypertriglyceridemia, three APOA5 mutations [p.(Ser232_Leu235)del, p.Leu253Pro, and p.Asp332ValfsX4] were found without evidence of concomitant LPL, APOC2, or GPIHBP1 mutations. The molecular mechanisms by which APOA5 mutations result in severe hypertriglyceridemia remain poorly understood, and the functional impairment/s induced by these specific mutations was not obvious. Therefore, we performed a thorough structural and functional analysis that included follow-up of patients and their closest relatives, measurement of apoA-V serum concentrations, and sequencing of the APOA5 gene in 200 nonhyperlipidemic controls. Further, we cloned, overexpressed, and purified both wild-type and mutant apoA-V variants and characterized their capacity to activate LPL. The interactions of recombinant wild-type and mutated apoA-V variants with liposomes of different composition, heparin, LRP1, sortilin, and SorLA/LR11 were also analyzed. Finally, to explore the possible structural consequences of these mutations, we developed a three-dimensional model of full-length, lipid-free human apoA-V. A complex, wide array of impairments was found in each of the three mutants, suggesting that the specific residues affected are critical structural determinants for apoA-V function in lipoprotein metabolism and, therefore, that these APOA5 mutations are a direct cause of hypertriglyceridemia.


Assuntos
Apolipoproteínas A/química , Apolipoproteínas A/metabolismo , Hipertrigliceridemia/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Apolipoproteína A-V , Apolipoproteínas A/genética , Western Blotting , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Lipossomos/química , Lipossomos/metabolismo , Masculino , Pessoa de Meia-Idade , Mutagênese Sítio-Dirigida , Mutação , Adulto Jovem
20.
J Biol Chem ; 288(7): 4854-66, 2013 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-23288849

RESUMO

ApoA-IV is an amphipathic protein that can emulsify lipids and has been linked to protective roles against cardiovascular disease and obesity. We previously reported an x-ray crystal structure of apoA-IV that was truncated at its N and C termini. Here, we have extended this work by demonstrating that self-associated states of apoA-IV are stable and can be structurally studied using small-angle x-ray scattering. Both the full-length monomeric and dimeric forms of apoA-IV were examined, with the dimer showing an elongated rod core with two nodes at opposing ends. The monomer is roughly half the length of the dimer with a single node. Small-angle x-ray scattering visualization of several deletion mutants revealed that removal of both termini can have substantial conformational effects throughout the molecule. Additionally, the F334A point mutation, which we previously showed increases apoA-IV lipid binding, also exhibited large conformational effects on the entire dimer. Merging this study's low-resolution structural information with the crystal structure provides insight on the conformation of apoA-IV as a monomer and as a dimer and further defines that a clasp mechanism may control lipid binding and, ultimately, protein function.


Assuntos
Apolipoproteínas A/química , Apolipoproteínas A/metabolismo , Biofísica/métodos , Dimerização , Dimiristoilfosfatidilcolina/química , Humanos , Lipídeos/química , Conformação Molecular , Mutação Puntual , Conformação Proteica , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Ultracentrifugação , Raios X
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