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1.
Molecules ; 26(5)2021 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-33652957

RESUMO

Multidrug resistance (MDR) remains a major problem in cancer therapy and is characterized by the overexpression of p-glycoprotein (P-gp) efflux pump, upregulation of anti-apoptotic proteins or downregulation of pro-apoptotic proteins. In this study, an Apolipoprotein A1 (ApoA1)-modified cationic liposome containing a synthetic cationic lipid and cholesterol was developed for the delivery of a small-molecule chemotherapeutic drug, doxorubicin (Dox) to treat MDR tumor. The liposome-modified by ApoA1 was found to promote drug uptake and elicit better therapeutic effects than free Dox and liposome in MCF-7/ADR cells. Further, loading Dox into the present ApoA1-liposome systems enabled a burst release at the tumor location, resulting in enhanced anti-tumor effects and reduced off-target effects. More importantly, ApoA1-lip/Dox caused fewer adverse effects on cardiac function and other organs in 4T1 subcutaneous xenograft models. These features indicate that the designed liposomes represent a promising strategy for the reversal of MDR in cancer treatment.


Assuntos
Apolipoproteína A-I/química , Neoplasias da Mama/tratamento farmacológico , Doxorrubicina/análogos & derivados , Apolipoproteína A-I/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/química , Doxorrubicina/farmacologia , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Células MCF-7 , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia
2.
Mol Pharmacol ; 99(3): 175-183, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33384285

RESUMO

Acyl-CoA:cholesterol acyltransferase (ACAT) mediates cellular cholesterol esterification. In atherosclerotic plaque macrophages, ACAT promotes cholesteryl ester accumulation, resulting in foam cell formation and atherosclerosis progression. Its complete inactivation in mice, however, showed toxic effects because of an excess of free cholesterol (FC) in macrophages, which can cause endoplasmic reticulum stress, cholesterol crystal formation, and inflammasome activation. Our previous studies showed that long-term partial ACAT inhibition, achieved by dietary supplementation with Fujirebio F1394, delays atherosclerosis progression in apoprotein E-deficient (Apoe -/-) mice by reducing plaque foam cell formation without inflammatory or toxic effects. Here, we determined whether short-term partial inhibition of ACAT, in combination with an enhanced systemic FC acceptor capacity, has synergistic benefits. Thus, we crossbred Apoe -/- with human apoprotein A1-transgenic (APOA1 tg/tg) mice, which have elevated cholesterol-effluxing high-density lipoprotein particles, and subjected Apoe -/- and APOA1 tg/tg/Apoe -/- mice to an atherogenic diet to develop advanced plaques. Then mice were either euthanized (baseline) or fed purified standard diet with or without F1394 for 4 more weeks. Plaques of APOA1 tg/tg/Apoe -/- mice fed F1394 showed a 60% reduction of macrophages accompanied by multiple other benefits, such as reduced inflammation and favorable changes in extracellular composition, in comparison with Apoe -/- baseline mice. In addition, there was no accumulation of cholesterol crystals or signs of toxicity. Overall, these results show that short-term partial ACAT inhibition, coupled to increased cholesterol efflux capacity, favorably remodels atherosclerosis lesions, supporting the potential of these combined therapies in the treatment of advanced atherosclerosis. SIGNIFICANCE STATEMENT: Short-term pharmacological inhibition of acyl-CoA:cholesterol acyltransferase-mediated cholesterol esterification, in combination with increased free cholesterol efflux acceptors, has positive effects in mice by 1) reducing the inflammatory state of the plaque macrophages and 2) favoring compositional changes associated with plaque stabilization. These effects occur without toxicity, showing the potential of these combined therapies in the treatment of advanced atherosclerosis.


Assuntos
Acetil-CoA C-Acetiltransferase/antagonistas & inibidores , Apolipoproteína A-I/genética , Apolipoproteínas E/genética , Aterosclerose/terapia , Cicloexanos/administração & dosagem , Dioxanos/administração & dosagem , Animais , Aterosclerose/genética , Cruzamento , Cicloexanos/farmacologia , Suplementos Nutricionais , Dioxanos/farmacologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Marcadores Genéticos/efeitos dos fármacos , Humanos , Lipoproteínas HDL/sangue , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Resultado do Tratamento
3.
Arch Biochem Biophys ; 699: 108748, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33444627

RESUMO

ApoA-I is the main protein of HDL which has anti-atherogenic properties attributed to reverse cholesterol transport. It shares with other exchangeable apolipoproteins a high level of structural plasticity. In the lipid-free state, the apolipoprotein amphipathic α-helices interact intra- and inter-molecularly, providing structural stabilization by a complex self-association mechanism. In this study, we employed a multi-parametric fluorescent probe to study the self-association of apoA-I. We constructed six single cysteine mutants spanning positions along three helices: F104C, K107C (H4), K133C, L137C (H5), F225C and K226C (H10); and labelled them with N-Maleimide Pyrene. Taking advantage of its spectral properties, namely formation of an excited dimer (excimer) and polarity-dependent changes in its fluorescence fine structure (P-value), we monitored the apoA-I self-association in its lipid-free form as a function of its concentration. Interactions in helices H5 (K133C) and H10 (F225C and K226C) were highlighted by excimer emission; while polarity changes were reported in helix H4 (K107C), as well as in helices H5 and H10. Mathematical models were developed to enrich data analysis and estimate association constants (KA) and oligomeric species distribution. Furthermore, we briefly discuss the usefulness of the multi-parametric fluorescent probe to monitor different equilibria, even at a single labelling position. Results suggest that apoA-I self-association must be considered to fully understand its physiological roles. Particularly, some contacts that stabilize discoidal HDL particles seem to be already present in the lipid-free apoA-I oligomers.


Assuntos
Apolipoproteína A-I/química , Corantes Fluorescentes/química , Sondas Moleculares/química , Multimerização Proteica , Pirenos/química , Apolipoproteína A-I/genética , Cisteína/química , Humanos , Mutação , Espectrometria de Fluorescência
4.
Biochim Biophys Acta Gen Subj ; 1865(1): 129732, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32946930

RESUMO

BACKGROUND: The identification of dysfunctional human apolipoprotein A-I (apoA-I) in atherosclerotic plaques suggests that protein structure and function may be hampered under a chronic pro inflammatory scenario. Moreover, the fact that natural mutants of this protein elicit severe cardiovascular diseases (CVD) strongly indicates that the native folding could shift due to the mutation, yielding a structure more prone to misfold or misfunction. To understand the events that determine the failure of apoA-I structural flexibility to fulfill its protective role, we took advantage of the study of a natural variant with a deletion of the residue lysine 107 (K107del) associated with atherosclerosis. METHODS: Biophysical approaches, such as electrophoresis, fluorescence and spectroscopy were used to characterize proteins structure and function, either in native conformation or under oxidation or intramolecular crosslinking. RESULTS: K107del structure was more flexible than the protein with the native sequence (Wt) but interactions with artificial membranes were preserved. Instead, structural restrictions by intramolecular crosslinking impaired the Wt and K107del lipid solubilization function. In addition, controlled oxidation decreased the yield of the native dimer conformation for both variants. CONCLUSIONS: We conclude that even though mutations may alter protein structure and spatial arrangement, the highly flexible conformation compensates the mild shift from the native folding. Instead, post translational apoA-I modifications (probably chronic and progressive) are required to raise a protein conformation with significant loss of function and increased aggregation tendency. GENERAL SIGNIFICANCE: The results learnt from this variant strength a close association between amyloidosis and atherosclerosis.


Assuntos
Apolipoproteína A-I/metabolismo , Aterosclerose/metabolismo , Processamento de Proteína Pós-Traducional , Apolipoproteína A-I/química , Apolipoproteína A-I/genética , Aterosclerose/genética , Humanos , Lipídeos de Membrana/metabolismo , Mutação , Oxirredução , Conformação Proteica
5.
Nutr Metab Cardiovasc Dis ; 30(5): 788-795, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32127339

RESUMO

BACKGROUND AND AIMS: Type 2 diabetes mellitus (T2DM) is a condition defined by hyperglycaemia, but also often presents with dyslipidaemia and suppressed HDL cholesterol. Mendelian randomization studies have suggested a causal link between low HDL cholesterol and T2DM. However, influences of gender, polymorphisms and lifestyle, all known to influence HDL cholesterol, have not been fully explored in a prospective cohort. METHODS AND RESULTS: In 2001-2002, a random sample of 1514 males (18-87 years old) and 1528 females (18-89 years old) were recruited in the ATTICA study. The 10-year follow-up (2011-2012) included 1485 participants. Lipids and lipoproteins levels, glucose and insulin levels were measured together with apolipoprotein A1 (apoA1) 75 G/A genotype, which is known to influence HDL-cholesterol. In total, 12.9% of the study sample developed T2DM within the 10-year follow-up period. In multivariable models, for each mg/dL increase in apoA1 levels in males, 10-year T2DM risk decreased 1.02%; while every unit increase in apoB/LDL-cholesterol ratio increased risk 4-fold. Finally, for every unit increase in triglycerides/apoA1 ratio, the risk increased 85%. HOMA-IR independently predicted T2DM 10-year incidence only for carriers of GG polymorphism (all, p < 0.05), but not in carriers of the GA polymorphism (all, p > 0.05). CONCLUSION: ApoA1 was associated with decreased T2DM risk and TG/ApoA1 and apoB/LDL were associated with increased risk of T2DM, only in males. ApoA1 polymorphism, which is associated with lower HDL cholesterol, influenced the predictive effects of HOMA-IR on T2DM incidence, which appeared to be moderated by physical activity, suggesting potential scope for more targeted preventative strategies.


Assuntos
Apolipoproteína A-I/sangue , Apolipoproteína A-I/genética , Diabetes Mellitus Tipo 2/sangue , Dislipidemias/sangue , Lipídeos/sangue , Polimorfismo Genético , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Apolipoproteína B-100/sangue , Biomarcadores/sangue , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/genética , Dislipidemias/diagnóstico , Dislipidemias/epidemiologia , Dislipidemias/genética , Feminino , Predisposição Genética para Doença , Grécia/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Inquéritos Nutricionais , Prognóstico , Estudos Prospectivos , Medição de Risco , Fatores de Risco , Fatores Sexuais , Fatores de Tempo , Triglicerídeos/sangue , Adulto Jovem
6.
Arch Biochem Biophys ; 685: 108347, 2020 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-32194045

RESUMO

The reason that determines the pathological deposition of human apolipoprotein A-I variants inducing organ failure has been under research since the early description of natural mutations in patients. To shed light into the events associated with protein aggregation, we studied the structural perturbations that may occur in the natural variant that shows a substitution of a Leucine by an Arginine in position 60 (L60R). Circular dichroism, intrinsic fluorescence measurements, and proteolysis analysis indicated that L60R was more unstable, more sensitive to cleavage and the N-terminus was more disorganized than the protein with the native sequence (Wt). A higher tendency to aggregate was also detected when L60R was incubated at physiological pH. In addition, the small structural rearrangement observed for the freshly folded variant led to the release of tumor necrosis factor-α and interleukin-1ß from a model of macrophages. However, the mutant preserved both its dimeric conformation and its lipid-binding capacity. Our results strongly suggest that the chronic disease may be a consequence of the native conformation loss which elicits the release of protein conformations that could be either cytotoxic or precursors of amyloid conformations.


Assuntos
Proteínas Amiloidogênicas/metabolismo , Apolipoproteína A-I/metabolismo , Proteínas Amiloidogênicas/química , Proteínas Amiloidogênicas/genética , Amiloidose/etiologia , Amiloidose/genética , Apolipoproteína A-I/química , Apolipoproteína A-I/genética , Humanos , Mutação Puntual , Multimerização Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína
7.
PLoS Med ; 17(3): e1003062, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32203549

RESUMO

BACKGROUND: Circulating lipoprotein lipids cause coronary heart disease (CHD). However, the precise way in which one or more lipoprotein lipid-related entities account for this relationship remains unclear. Using genetic instruments for lipoprotein lipid traits implemented through multivariable Mendelian randomisation (MR), we sought to compare their causal roles in the aetiology of CHD. METHODS AND FINDINGS: We conducted a genome-wide association study (GWAS) of circulating non-fasted lipoprotein lipid traits in the UK Biobank (UKBB) for low-density lipoprotein (LDL) cholesterol, triglycerides, and apolipoprotein B to identify lipid-associated single nucleotide polymorphisms (SNPs). Using data from CARDIoGRAMplusC4D for CHD (consisting of 60,801 cases and 123,504 controls), we performed univariable and multivariable MR analyses. Similar GWAS and MR analyses were conducted for high-density lipoprotein (HDL) cholesterol and apolipoprotein A-I. The GWAS of lipids and apolipoproteins in the UKBB included between 393,193 and 441,016 individuals in whom the mean age was 56.9 y (range 39-73 y) and of whom 54.2% were women. The mean (standard deviation) lipid concentrations were LDL cholesterol 3.57 (0.87) mmol/L and HDL cholesterol 1.45 (0.38) mmol/L, and the median triglycerides was 1.50 (IQR = 1.11) mmol/L. The mean (standard deviation) values for apolipoproteins B and A-I were 1.03 (0.24) g/L and 1.54 (0.27) g/L, respectively. The GWAS identified multiple independent SNPs associated at P < 5 × 10-8 for LDL cholesterol (220), apolipoprotein B (n = 255), triglycerides (440), HDL cholesterol (534), and apolipoprotein A-I (440). Between 56%-93% of SNPs identified for each lipid trait had not been previously reported in large-scale GWASs. Almost half (46%) of these SNPs were associated at P < 5 × 10-8 with more than one lipid-related trait. Assessed individually using MR, LDL cholesterol (odds ratio [OR] 1.66 per 1-standard-deviation-higher trait; 95% CI: 1.49-1.86; P < 0.001), triglycerides (OR 1.34; 95% CI: 1.25-1.44; P < 0.001) and apolipoprotein B (OR 1.73; 95% CI: 1.56-1.91; P < 0.001) had effect estimates consistent with a higher risk of CHD. In multivariable MR, only apolipoprotein B (OR 1.92; 95% CI: 1.31-2.81; P < 0.001) retained a robust effect, with the estimate for LDL cholesterol (OR 0.85; 95% CI: 0.57-1.27; P = 0.44) reversing and that of triglycerides (OR 1.12; 95% CI: 1.02-1.23; P = 0.01) becoming weaker. Individual MR analyses showed a 1-standard-deviation-higher HDL cholesterol (OR 0.80; 95% CI: 0.75-0.86; P < 0.001) and apolipoprotein A-I (OR 0.83; 95% CI: 0.77-0.89; P < 0.001) to lower the risk of CHD, but these effect estimates attenuated substantially to the null on accounting for apolipoprotein B. A limitation is that, owing to the nature of lipoprotein metabolism, measures related to the composition of lipoprotein particles are highly correlated, creating a challenge in making exclusive interpretations on causation of individual components. CONCLUSIONS: These findings suggest that apolipoprotein B is the predominant trait that accounts for the aetiological relationship of lipoprotein lipids with risk of CHD.


Assuntos
Apolipoproteína B-100/genética , Doença das Coronárias/genética , Polimorfismo de Nucleotídeo Único , Adulto , Idoso , Apolipoproteína A-I/sangue , Apolipoproteína A-I/genética , Apolipoproteína B-100/sangue , Biomarcadores/sangue , HDL-Colesterol/sangue , HDL-Colesterol/genética , LDL-Colesterol/sangue , LDL-Colesterol/genética , Doença das Coronárias/sangue , Doença das Coronárias/diagnóstico , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Masculino , Análise da Randomização Mendeliana , Pessoa de Meia-Idade , Análise Multivariada , Fenótipo , Medição de Risco , Fatores de Risco , Triglicerídeos/sangue
8.
Clin Chim Acta ; 505: 55-59, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32092318

RESUMO

ApoA-I is a major protein component of high-density lipoprotein (HDL) that is widely known for regulating cholesterol trafficking and inflammatory and immune responses and for protecting against atherosclerosis. ApoA-I is generally considered to be synthesized in the liver (hepatocytes) and small intestine (enterocytes). However, computer analysis of ApoA-I has shown that the ApoA-I gene may be expressed in not only hepatocytes and enterocytes but also monocyte-macrophage cells, dendritic cells (DCs) and T cells. ApoA-I expression has been detected in THP-1 monocytes and macrophages, peripheral blood mononuclear cells (PBMCs) from postmenopausal women, human PBMC-derived monocytes and macrophages, mouse peritoneal macrophages, etc. Endogenous ApoA-I in macrophages has anti-inflammatory and cholesterol efflux effects. However, our understanding of the detailed roles of macrophage-synthesized ApoA-I is still at an early stage and very limited. More experiments are needed to elucidate the exact roles of endogenous ApoA-I in macrophages. Several lines of evidence indicate that recombinant exogenous human ApoA-I in mouse macrophages increases cholesterol efflux and thus reduces atherosclerosis development. Considering the antiatherogenic effect of exogenous ApoA-I overexpression in mouse macrophages, better understanding the role and mechanisms underlying macrophage-synthesized ApoA-I in atherosclerosis will enable macrophage-synthesized ApoA-I therapy to open new avenues for reducing the risk of atherosclerosis.


Assuntos
Apolipoproteína A-I/biossíntese , Apolipoproteína A-I/genética , Aterosclerose/prevenção & controle , Macrófagos/metabolismo , Animais , Colesterol/metabolismo , Humanos
9.
Metabolism ; 105: 154186, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32084429

RESUMO

BACKGROUND: Apolipoprotein A-I (ApoA-I) is involved in reverse cholesterol transport as a major component of HDL, but also conveys anti-thrombotic, anti-oxidative, anti-inflammatory and immune-regulatory properties that are pertinent to its protective roles in cardiovascular, inflammatory and malignant pathologies. Despite the pleiotropy in ApoA-I functions, the regulation of intracellular ApoA-I levels remains poorly explored. METHODS: HepG2 hepatoma cells and primary mouse hepatocytes were used as in vitro models to study the impact of genetic and chemical inhibitors of autophagy and the proteasome on ApoA-I by immunoblot, immunofluorescence and electron microscopy. Different growth conditions were implemented in conjunction with mTORC inhibitors to model the influence of nutrient scarcity versus sufficiency on ApoA-I regulation. Hepatic ApoA-I expression was also evaluated in high fat diet-fed mice displaying blockade in autophagy. RESULTS: Under nutrient-rich conditions, basal ApoA-I levels in liver cells are sustained by the balancing act of autophagy and of mTORC1-dependent de novo protein synthesis. ApoA-I proteolysis occurs through a canonical autophagic pathway involving Beclin1 and ULK1 and the receptor protein p62/SQSTM1 that targets ApoA-I to autophagosomes. However, upon aminoacid insufficiency, suppression of ApoA-I synthesis prevails, rendering mTORC1 inactivation dispensable for autophagy-mediated ApoA-I proteolysis. CONCLUSION: These data underscore the major contribution of post-transcriptional mechanisms to ApoA-I levels which differentially involve mTORC1-dependent signaling to protein synthesis and autophagy, depending on nutrient availability. Given the established role of ApoA-I in HDL-mediated reverse cholesterol transport, this mode of ApoA-I regulation may reflect a hepatocellular response to the organismal requirement for maintenance of cholesterol and lipid reserves under conditions of nutrient scarcity.


Assuntos
Apolipoproteína A-I/biossíntese , Autofagia/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Animais , Apolipoproteína A-I/genética , Colesterol/metabolismo , Dieta Hiperlipídica , Regulação da Expressão Gênica , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Metabolismo dos Lipídeos , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Camundongos , Hepatopatia Gordurosa não Alcoólica/genética , Estado Nutricional , Complexo de Endopeptidases do Proteassoma/genética
10.
J Biol Chem ; 295(7): 1973-1984, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31907281

RESUMO

Apolipoprotein A-I (apoA-I) is cross-linked and dysfunctional in human atheroma. Although multiple mechanisms of apoA-I cross-linking have been demonstrated in vitro, the in vivo mechanisms of cross-linking are not well-established. We have recently demonstrated the highly selective and efficient modification of high-density lipoprotein (HDL) apoproteins by endogenous oxidized phospholipids (oxPLs), including γ-ketoalkenal phospholipids. In the current study, we report that γ-ketoalkenal phospholipids effectively cross-link apoproteins in HDL. We further demonstrate that cross-linking impairs the cholesterol efflux mediated by apoA-I or HDL3 in vitro and in vivo Using LC-MS/MS analysis, we analyzed the pattern of apoprotein cross-linking in isolated human HDL either by synthetic γ-ketoalkenal phospholipids or by oxPLs generated during HDL oxidation in plasma by the physiologically relevant MPO-H2O2-NO2 - system. We found that five histidine residues in helices 5-8 of apoA-I are preferably cross-linked by oxPLs, forming stable pyrrole adducts with lysine residues in the helices 3-4 of another apoA-I or in the central domain of apoA-II. We also identified cross-links of apoA-I and apoA-II with two minor HDL apoproteins, apoA-IV and apoE. We detected a similar pattern of apoprotein cross-linking in oxidized murine HDL. We further detected oxPL cross-link adducts of HDL apoproteins in plasma and aorta of hyperlipidemic LDLR-/- mice, including cross-link adducts of apoA-I His-165-apoA-I Lys-93, apoA-I His-154-apoA-I Lys-105, apoA-I His-154-apoA-IV Lys-149, and apoA-II Lys-30-apoE His-227. These findings suggest an important mechanism that contributes to the loss of HDL's atheroprotective function in vivo.


Assuntos
Apolipoproteína A-I/genética , Lipoproteínas HDL3/genética , Fosfolipídeos/genética , Receptores de LDL/genética , Animais , Aorta/metabolismo , Cromatografia Líquida , Humanos , Peróxido de Hidrogênio/metabolismo , Lipoproteínas HDL/genética , Macrófagos/metabolismo , Camundongos , Óxido Nítrico/genética , Óxido Nítrico/metabolismo , Oxirredução , Fosforilação Oxidativa , Fosfolipídeos/metabolismo , Espectrometria de Massas em Tandem
11.
Int J Mol Sci ; 21(3)2020 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-31979310

RESUMO

Epidemiological results revealed that there is an inverse correlation between high-density lipoprotein (HDL) cholesterol levels and risks of atherosclerotic cardiovascular disease (ASCVD). Mounting evidence supports that HDLs are atheroprotective, therefore, many therapeutic approaches have been developed to increase HDL cholesterol (HDL-C) levels. Nevertheless, HDL-raising therapies, such as cholesteryl ester transfer protein (CETP) inhibitors, failed to ameliorate cardiovascular outcomes in clinical trials, thereby casting doubt on the treatment of cardiovascular disease (CVD) by increasing HDL-C levels. Therefore, HDL-targeted interventional studies were shifted to increasing the number of HDL particles capable of promoting ATP-binding cassette transporter A1 (ABCA1)-mediated cholesterol efflux. One such approach was the development of reconstituted HDL (rHDL) particles that promote ABCA1-mediated cholesterol efflux from lipid-enriched macrophages. Here, we explore the manipulation of rHDL nanoparticles as a strategy for the treatment of CVD. In addition, we discuss technological capabilities and the challenge of relating preclinical in vivo mice research to clinical studies. Finally, by drawing lessons from developing rHDL nanoparticles, we also incorporate the viabilities and advantages of the development of a molecular imaging probe with HDL nanoparticles when applied to ASCVD, as well as gaps in technology and knowledge required for putting the HDL-targeted therapeutics into full gear.


Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Aterosclerose/tratamento farmacológico , Descoberta de Drogas , Lipoproteínas HDL/metabolismo , Nanopartículas , Animais , Apolipoproteína A-I/química , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Aterosclerose/diagnóstico por imagem , Aterosclerose/metabolismo , Biomimética , Humanos , Macrófagos/metabolismo , Camundongos , Imagem Molecular , Nanopartículas/química , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo
12.
Sci Rep ; 10(1): 1159, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980684

RESUMO

Apolipoprotein A-Ib (ApoA-Ib) is a high molecular weight form of Apolipoprotein A-I (ApoA-I) found specifically in the urine of kidney-transplanted patients with recurrent idiopathic focal segmental glomerulosclerosis (FSGS). To determine the nature of the modification present in ApoA-Ib, we sequenced the whole APOA1 gene in ApoA-Ib positive and negative patients, and we also studied the protein primary structure using mass spectrometry. No genetic variations in the APOA1 gene were found in the ApoA-Ib positive patients that could explain the increase in its molecular mass. The mass spectrometry analysis revealed three extra amino acids at the N-Terminal end of ApoA-Ib that were not present in the standard plasmatic form of ApoA-I. These amino acids corresponded to half of the propeptide sequence of the immature form of ApoA-I (proApoA-I) indicating that ApoA-Ib is a misprocessed form of proApoA-I. The description of ApoA-Ib could be relevant not only because it can allow the automated analysis of this biomarker in the clinical practice but also because it has the potential to shed light into the molecular mechanisms that cause idiopathic FSGS, which is currently unknown.


Assuntos
Apolipoproteína A-I/metabolismo , Glomerulosclerose Segmentar e Focal/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Especificidade de Anticorpos , Apolipoproteína A-I/genética , Apolipoproteína A-I/imunologia , Apolipoproteína A-I/urina , Biomarcadores , Western Blotting , Eletroforese em Gel Bidimensional , Humanos , Espectrometria de Massas , Peso Molecular , Polimorfismo de Nucleotídeo Único , Precursores de Proteínas/metabolismo , Recidiva
13.
PLoS One ; 15(1): e0221915, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31945064

RESUMO

The crystal structure of a C-terminal deletion of apolipoprotein A-I (apoA1) shows a large helical bundle structure in the amino half of the protein, from residues 8 to 115. Using site directed mutagenesis, guanidine or thermal denaturation, cell free liposome clearance, and cellular ABCA1-mediated cholesterol efflux assays, we demonstrate that apoA1 lipidation can occur when the thermodynamic barrier to this bundle unfolding is lowered. The absence of the C-terminus renders the bundle harder to unfold resulting in loss of apoA1 lipidation that can be reversed by point mutations, such as Trp8Ala, and by truncations as short as 8 residues in the amino terminus, both of which facilitate helical bundle unfolding. Locking the bundle via a disulfide bond leads to loss of apoA1 lipidation. We propose a model in which the C-terminus acts on the N-terminus to destabilize this helical bundle. Upon lipid binding to the C-terminus, Trp8 is displaced from its interaction with Phe57, Arg61, Leu64, Val67, Phe71, and Trp72 to destabilize the bundle. However, when the C-terminus is deleted, Trp8 cannot be displaced, the bundle cannot unfold, and apoA1 cannot be lipidated.


Assuntos
Transportador 1 de Cassete de Ligação de ATP/genética , Apolipoproteína A-I/genética , Metabolismo dos Lipídeos/genética , Lipídeos/química , Transportador 1 de Cassete de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Apolipoproteína A-I/química , Transporte Biológico/genética , Dicroísmo Circular , Cristalografia por Raios X , Humanos , Cinética , Lipídeos/genética , Camundongos , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Secundária de Proteína , Desdobramento de Proteína , Células RAW 264.7 , Deleção de Sequência/genética
14.
Biochim Biophys Acta Gen Subj ; 1864(4): 129515, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31904503

RESUMO

BACKGROUND: Different protein conformations may be involved in the development of clinical manifestations associated with human amyloidosis. Although a fibrillar conformation is usually the signature of damage in the tissues of patients, it is not clear whether this species is per se the cause or the consequence of the disease. Hereditary amyloidosis due to variants of apolipoprotein A-I (apoA-I) with a substitution of a single amino acid is characterized by the presence of fibrillar protein within the lesions. Thus mutations result in increased protein aggregation. Here we set up to characterize the folding of a natural variant with a mutation leading to a deletion at position 107 (apoA-I Lys107-0). Patients carrying this variant show amyloidosis and severe atherosclerosis. METHODS: We oxidized this variant under controlled concentrations of hydrogen peroxide and analyzed the structure obtained after 30-day incubation by fluorescence, circular dichroism and microscopy approaches. Neutrophils activation was characterized by confocal microscopy. RESULTS: We obtained a high yield of well-defined stable fibrillar structures of apoA-I Lys107-0. In an in vitro neutrophils system, we were able to detect the induction of Neutrophils Extracellular Traps (NETs) when we incubated with oxidized apoA-I variants. This effect was exacerbated by the fibrillar structure of oxidized Lys 107-0. CONCLUSIONS: We conclude that a pro-inflammatory microenvironment could result in the formation of aggregation-prone species, which, in addition may induce a positive feed-back in the activation of an inflammatory response. GENERAL SIGNIFICANCE: These events may explain a close association between amyloidosis due to apoA-I Lys107-0 and atherosclerosis.


Assuntos
Amiloidose Familiar/genética , Apolipoproteína A-I/química , Apolipoproteína A-I/genética , Aterosclerose/genética , Mutação , Amiloidose Familiar/metabolismo , Apolipoproteína A-I/metabolismo , Aterosclerose/metabolismo , Humanos , Conformação Proteica
15.
Artigo em Inglês | MEDLINE | ID: mdl-31863971

RESUMO

Several hereditary point mutations in human apolipoprotein A-I (apoA-I) have been associated with low HDL-cholesterol levels and/or increased coronary artery disease (CAD) risk. However, one apoA-I mutation, the V19L, recently identified in Icelanders, has been associated with increased HDL-cholesterol levels and decreased CAD risk. In an effort to gain mechanistic insight linking the presence of this mutation in apoA-I with the increase of HDL-cholesterol levels we evaluated the effect of V19L mutation on the conformational integrity and functional properties of apoA-I in lipid-free and lipidated form. ApoA-I[V19L] was found to be thermodynamically destabilized in lipid-free form and displays an increased capacity to associate with phospholipids compared to WT apoA-I. When associated to reconstituted HDL (rHDL), apoA-I[V19L] was more thermodynamically stabilized than WT apoA-I. ApoA-I[V19L] displayed normal capacity to promote ABCA1-mediated cholesterol efflux and to activate the enzyme LCAT, in lipid-free and rHDL-associated forms, respectively. Additionally, rHDL-associated apoA-I[V19L] showed normal capacity to promote ABCG1-mediated cholesterol efflux, but 45% increased capacity to promote SR-BI-mediated cholesterol efflux, while the SR-BI-mediated HDL-lipid uptake was normal. Overall, our findings show that the apoA-I V19L mutation does not affect the first steps of HDL biogenesis pathway. However, the increased capacity of apoA-I[V19L] to associate with phospholipids, in combination with the enhanced thermodynamic stability of lipoprotein-associated apoA-I[V19L] and increased capacity of apoA-I[V19L]-containing lipoprotein particles to accept additional cholesterol by SR-BI could account for the increased HDL-cholesterol levels observed in human carriers of the mutation.


Assuntos
Apolipoproteína A-I/química , HDL-Colesterol/metabolismo , Mutação de Sentido Incorreto , Fosfolipídeos/metabolismo , Animais , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Sítios de Ligação , Células HEK293 , Humanos , Camundongos , Simulação de Dinâmica Molecular , Ligação Proteica , Estabilidade Proteica
16.
Artigo em Inglês | MEDLINE | ID: mdl-31678510

RESUMO

Adipose organ is made of white (WAT) and brown (BAT) adipose tissue which are primarily responsible for lipid storage and energy production (heat and ATP) respectively. Metabolic activation of WAT may ascribe to this tissue characteristics of BAT, namely non-shivering thermogenesis and ATP production. Recent data indicate that apolipoproteins E (APOE) and A1 (APOA1) regulate WAT mitochondrial metabolic activation. Here, we investigated the functional cross-talk between natural human APOE2 and APOE4 isoforms with APOA1 in this process, using Apoe2knock-in and Apoe4knock-in mice. At baseline when Apoe2knock-in and Apoe4knock-in mice express both APOE and Apoa1, the Apoe2knock-in strain appears to have higher mitochondrial oxidative phosphorylation levels and non-shivering thermogenesis in WAT compared to Apoe4knock-in mice. When mice were switched to a high-fat diet for 18 weeks, circulating levels of endogenous Apoa1 in Apoe2knock-in mice became barely detectable though significant levels of APOE2 were still present. This change was accompanied by a significant reduction in WAT mitochondrial Ucp1 expression while BAT Ucp1 was unaffected. Ectopic APOA1 expression in Apoe2knock-in animals potently stimulated WAT but not BAT mitochondrial Ucp1 expression providing further evidence that APOA1 potently stimulates WAT non-shivering thermogenesis in the presence of APOE2. Ectopic expression of APOA1 in Apoe4knock-in mice stimulated BAT but no WAT mitochondrial Ucp1 levels, suggesting that in the presence of APOE4, APOA1 is a trigger of BAT non-shivering thermogenesis. Overall, our data identified a tissue-specific role of the natural human APOE2 and APOE4 isoforms in WAT- and BAT-metabolic activation respectively, that appears dependent on circulating APOA1 levels.


Assuntos
Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Apolipoproteína A-I/metabolismo , Apolipoproteína E2/metabolismo , Apolipoproteína E4/metabolismo , Animais , Apolipoproteína A-I/sangue , Apolipoproteína A-I/genética , Apolipoproteína E2/genética , Apolipoproteína E4/genética , Dieta Hiperlipídica/efeitos adversos , Técnicas de Introdução de Genes , Técnicas de Transferência de Genes , Masculino , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Modelos Animais , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/sangue , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termogênese/fisiologia , Proteína Desacopladora 1/metabolismo , Ganho de Peso/fisiologia
17.
Int J Mol Sci ; 20(24)2019 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-31842455

RESUMO

Apolipoprotein A-I (apoA-I) is the major protein component of high-density lipoproteins (HDL), mediating many of its atheroprotective properties. Increasing data reveal the pro-atherogenic effects of bisphenol A (BPA), one of the most prevalent environmental chemicals. In this study, we investigated the mechanisms by which BPA exerts pro-atherogenic effects. For this, LDLR-/- mice were fed with a high-fat diet and treated with 50 µg BPA/kg body weight by gavage. After two months of treatment, the area of atherosclerotic lesions in the aorta, triglycerides and total cholesterol levels were significantly increased, while HDL-cholesterol was decreased in BPA-treated LDLR-/- mice as compared to control mice. Real-Time PCR data showed that BPA treatment decreased hepatic apoA-I expression. BPA downregulated the activity of the apoA-I promoter in a dose-dependent manner. This inhibitory effect was mediated by MEKK1/NF-κB signaling pathways. Transfection experiments using apoA-I promoter deletion mutants, chromatin immunoprecipitation, and protein-DNA interaction assays demonstrated that treatment of hepatocytes with BPA induced NF-κB signaling and thus the recruitment of p65/50 proteins to the multiple NF-κB binding sites located in the apoA-I promoter. In conclusion, BPA exerts pro-atherogenic effects downregulating apoA-I by MEKK1 signaling and NF-κB activation in hepatocytes.


Assuntos
Poluentes Ocupacionais do Ar/efeitos adversos , Apolipoproteína A-I/genética , Aterosclerose/etiologia , Aterosclerose/metabolismo , Compostos Benzidrílicos/efeitos adversos , Regulação da Expressão Gênica , NF-kappa B/metabolismo , Fenóis/efeitos adversos , Animais , Apolipoproteína A-I/metabolismo , Aterosclerose/sangue , Aterosclerose/patologia , Biomarcadores , Modelos Animais de Doenças , Feminino , Hepatócitos/metabolismo , Lipídeos/sangue , Masculino , Camundongos , Camundongos Knockout , Receptores de LDL/deficiência
18.
Lipids ; 54(11-12): 687-695, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31814132

RESUMO

Although increasing apolipoprotein A-I (apoA-I) might lower the cardiovascular disease risk, knowledge on natural compounds that elevate apoA-I transcription is limited. Therefore, the aim of this study was to discover natural compounds that increase apoA-I transcription in HepG2 cells. Since BRD4 inhibition is known to elevate apoA-I transcription, we focused on natural BRD4 inhibitors. For this, the literature was screened for compounds that might increase apoA-I and or inhibit BRD4. This resulted in list A, (apoA-I increasers with unknown BRD4 inhibitor capacity), list B (known BRD4 inhibitors that increase apoA-I), and list C (BRD4 inhibitors with unknown effect on apoA-I). These compounds were compared with the compounds in two natural compound databases. This resulted in (1) a common substructure (ethyl-benzene) in 60% of selected BRD4-inhibitors, and (2) four compounds that increased ApoA-I: hesperetin, equilenin, 9(S)-HOTrE, and cymarin. Whether these increases are regulated via BRD4 inhibition and the ethyl-benzene structure inhibits BRD4 requires further study.


Assuntos
Apolipoproteína A-I/genética , Produtos Biológicos/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Ativação Transcricional/efeitos dos fármacos , Apolipoproteína A-I/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células Hep G2 , Humanos , Fatores de Transcrição/metabolismo , Ativação Transcricional/genética
19.
Int J Mol Sci ; 20(23)2019 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-31783518

RESUMO

In a recent human study, we observed that amoxicillin treatment decreased HDL-C concentration. We hypothesize that antibiotics lower the transcription and secretion of ApoA-I, the responsible protein for HDL production. HepG2 and Caco-2 cells were exposed to increasing dose of amoxicillin, penicillin, and streptomycin. Secreted ApoA-I protein and mRNA transcripts were analyzed using ELISA and qPCR, respectively. To unravel underlying mechanisms, KEAP1, CPT1, and CHOP mRNA expressions were determined as well as PPARα transactivation. In HepG2 and Caco-2, amoxicillin decreased ApoA-I transcription and secretion. Effects on ApoA-I expression were clearly there for amoxicillin while no effects were observed for penicillin or streptomycin. KEAP1, CPT1, and CHOP mRNA expressions were reduced by amoxicillin treatments. Moreover, a significant correlation between ApoA-I and CPT1 mRNA expressions was found. Furthermore, amoxicillin lowered PPARα transactivation. All together, these data suggest that inhibited PPARα transactivation is involved in the effects of amoxicillin on ApoA-I. In conclusion, the direct effect of amoxicillin in treated HepG2 and Caco-2 cells was a lower ApoA-I secretion and transcription. Based on evaluating alterations in KEAP1, CPT1, and CHOP mRNA expressions plus PPARα transactivation, we suggest that a reduced PPARα activation is a potential mechanism behind the observed amoxicillin effects on ApoA-I expression.


Assuntos
Amoxicilina/farmacologia , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , PPAR alfa/genética , Transcrição Genética/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Células CACO-2 , Carnitina O-Palmitoiltransferase/genética , Linhagem Celular Tumoral , Células Hep G2 , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , RNA Mensageiro/genética , Fator de Transcrição CHOP/genética
20.
Lipids Health Dis ; 18(1): 225, 2019 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-31861992

RESUMO

BACKGROUND: Hypocholesterolemia is the most frequently encountered lipid abnormality in sickle cell disease (SCD). We enrolled pediatric patients to determine the relationships between lipid profile and parameters of hemolysis, oxidative stress and chronic inflammation in SCD. METHODS: The study involved 35 pediatric SCD patients and 19 healthy controls. Patients were crisis-free and had not received transfusions for the last 3 months. Total cholesterol, triglyceride, HDL-C, LDL-C, VLDL-C, apolipoprotein A1, apolipoprotein B, LCAT, LDH, bilirubin, haptoglobin, iron, ferritin, hemin, serum amyloid A (SAA), myeloperoxidase (MPO), uric acid, ALT and GGT levels were evaluated in patients' blood. RESULTS: Patients had hypocholesterolemia depicted by lower levels of total cholesterol, HDL-C, LDL-C, as well as Apolipoprotein A1 and Apolipoprotein B compared to controls. The chronic hemolysis of SCD was evident in patients by higher LDH and bilirubin and almost undetectable haptoglobin levels. Hemin levels (as a measure of oxidized heme) were significantly increased in patients with SCD. Inflammation markers, SAA and MPO, were significantly increased in the patients as well. There were negative correlations between HDL-C and LDH, and Apo A1 and SAA. Hemin was positively correlated to MPO. CONCLUSION: Hemolysis was associated with decreased HDL -C, and Inflammation was linked to decreased apolipoprotein A1 levels in our SCD patients. Therefore, we suggest that the HDL particle is altered during the course of the disease. The altered HDL in SCD may become dysfunctional and result with a slowing down of the reverse cholesterol transport.


Assuntos
Anemia Falciforme/sangue , Apolipoproteína A-I/sangue , Colesterol/sangue , Inflamação/sangue , Adolescente , Adulto , Anemia Falciforme/metabolismo , Anemia Falciforme/patologia , Apolipoproteína A-I/genética , Apolipoproteínas B/sangue , Bilirrubina/sangue , Biomarcadores/sangue , Criança , Colesterol/metabolismo , HDL-Colesterol/sangue , LDL-Colesterol/sangue , VLDL-Colesterol/sangue , Feminino , Hemólise , Humanos , Inflamação/metabolismo , Inflamação/patologia , L-Lactato Desidrogenase/sangue , Lipídeos/sangue , Masculino , Peroxidase/sangue , Proteína Amiloide A Sérica/metabolismo , Triglicerídeos/sangue , Adulto Jovem
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