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1.
J Virol ; 98(1): e0084923, 2024 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-38174935

RESUMO

Hepatitis C virus (HCV) is a member of the Flaviviridae family; however, unlike other family members, the HCV virion has an unusually high lipid content. HCV has two envelope glycoproteins, E1 and E2. E2 contributes to receptor binding, cell membrane attachment, and immune evasion. In contrast, the functions of E1 are poorly characterized due, in part, to challenges in producing the protein. This manuscript describes the expression and purification of a soluble E1 ectodomain (eE1) that is recognized by conformational, human monoclonal antibodies. eE1 forms a complex with apolipoproteins AI and AII, cholesterol, and phospholipids by recruiting high-density lipoprotein (HDL) from the extracellular media. We show that HDL binding is a function specific to eE1 and HDL hinders recognition of E1 by a neutralizing monoclonal antibody. Either low-density lipoprotein or HDL increases the production and infectivity of cell culture-produced HCV, but E1 preferentially selects HDL, influencing both viral life cycle and antibody evasion.IMPORTANCEHepatitis C virus (HCV) infection is a significant burden on human health, but vaccine candidates have yet to provide broad protection against this infection. We have developed a method to produce high quantities of soluble E1 or E2, the viral proteins located on the surface of HCV. HCV has an unusually high lipid content due to the recruitment of apolipoproteins. We found that E1 (and not E2) preferentially recruits host high-density lipoprotein (HDL) extracellularly. This recruitment of HDL by E1 prevents binding of E1 by a neutralizing antibody and furthermore prevents antibody-mediated neutralization of the virus. By comparison, low-density lipoprotein does not protect the virus from antibody-mediated neutralization. Our findings provide mechanistic insight into apolipoprotein recruitment, which may be critical for vaccine development.


Assuntos
Hepacivirus , Hepatite C , Evasão da Resposta Imune , Lipoproteínas HDL , Proteínas do Envelope Viral , Humanos , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Apolipoproteínas/metabolismo , Hepacivirus/patogenicidade , Hepatite C/imunologia , Hepatite C/virologia , Anticorpos Anti-Hepatite C/imunologia , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Proteínas do Envelope Viral/metabolismo , Células HEK293
2.
J Allergy Clin Immunol ; 153(4): 1010-1024.e14, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38092139

RESUMO

RATIONALE: Serum amyloid A (SAA) is bound to high-density lipoproteins (HDL) in blood. Although SAA is increased in the blood of patients with asthma, it is not known whether this modifies asthma severity. OBJECTIVE: We sought to define the clinical characteristics of patients with asthma who have high SAA levels and assess whether HDL from SAA-high patients with asthma is proinflammatory. METHODS: SAA levels in serum from subjects with and without asthma were quantified by ELISA. HDLs isolated from subjects with asthma and high SAA levels were used to stimulate human monocytes and were intravenously administered to BALB/c mice. RESULTS: An SAA level greater than or equal to 108.8 µg/mL was defined as the threshold to identify 11% of an asthmatic cohort (n = 146) as being SAA-high. SAA-high patients with asthma were characterized by increased serum C-reactive protein, IL-6, and TNF-α; older age; and an increased prevalence of obesity and severe asthma. HDL isolated from SAA-high patients with asthma (SAA-high HDL) had an increased content of SAA as compared with HDL from SAA-low patients with asthma and induced the secretion of IL-6, IL-1ß, and TNF-α from human monocytes via a formyl peptide receptor 2/ATP/P2X purinoceptor 7 axis. Intravenous administration to mice of SAA-high HDL, but not normal HDL, induced systemic inflammation and amplified allergen-induced neutrophilic airway inflammation and goblet cell metaplasia. CONCLUSIONS: SAA-high patients with asthma are characterized by systemic inflammation, older age, and an increased prevalence of obesity and severe asthma. HDL from SAA-high patients with asthma is proinflammatory and, when intravenously administered to mice, induces systemic inflammation, and amplifies allergen-induced neutrophilic airway inflammation. This suggests that systemic inflammation induced by SAA-high HDL may augment disease severity in asthma.


Assuntos
Asma , Lipoproteínas HDL , Humanos , Animais , Camundongos , Lipoproteínas HDL/metabolismo , Lipoproteínas HDL/farmacologia , Proteína Amiloide A Sérica/análise , Proteína Amiloide A Sérica/metabolismo , Proteína Amiloide A Sérica/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Interleucina-6 , Inflamação/metabolismo , Obesidade , Alérgenos
3.
Front Cardiovasc Med ; 10: 1223920, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37547254

RESUMO

Introduction: Defects in lipolysis can lead to hypertriglyceridemia, which can trigger acute pancreatitis and is also associated with cardiovascular disease. Decreasing plasma triglycerides (TGs) by activating lipoprotein lipase (LPL) with ApoC2 mimetic peptides is a new treatment strategy for hypertriglyceridemia. We recently described a dual ApoC2 mimetic/ApoC3 antagonist peptide called D6PV that effectively lowered TG in several mouse models but has limitations in terms of drug development. The aim of this study was to create the next generation of ApoC2 mimetic peptides. Methods: We employed hydrocarbon staples, as well as select amino acid substitutions, to make short single helical mimetic peptides based on the last helix of ApoC2. Peptides were first tested for their ability to activate LPL and then in hypertriglyceridemia mouse models. All-atom simulations of peptides were performed in a lipid-trilayer model of TG-rich lipoproteins to discern their possible mechanism of action. Results: We designed a single stapled peptide called SP1 (21 residues), and a double stapled (stitched) peptide called SP2 (21 residues) and its N-terminal acylated analogue, SP2a. The hydrocarbon staples increased the amphipathicity of the peptides and their ability to bind lipids without interfering with LPL activation. Indeed, from all-atom simulations, the conformations of SP1 and SP2a are restrained by the staples and maintains the proper orientation of the LPL activation motif, while still allowing their deeper insertion into the lipid-trilayer model. Intraperitoneal injection of stapled peptides (1-5 umoles/kg) into ApoC2-hypomorphic mice or human ApoC3-transgenic resulted in an 80%-90% reduction in plasma TG within 3 h, similar to the much longer D6PV peptide (41 residues). Other modifications (replacement L-Glu20, L-Glu21 with their D-isomers, N-methylation of Gly19, Met2NorLeu and Ala1alpha-methylAla substitutions, N-terminal octanoylation) were introduced into the SP2a peptide. These changes made SP2a highly resistant to proteolysis against trypsin, pepsin, and Proteinase K, while maintaining similar efficacy in lowering plasma TG in mice. Conclusion: We describe a new generation of ApoC2 mimetic peptides based on hydron carbon stapling that are at least equally potent to earlier peptides but are much shorter and resistant to proteolysis and could be further developed into a new therapy for hypertriglyceridemia.

4.
Nanomedicine ; 48: 102646, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36549559

RESUMO

Synthetic high-density lipoproteins nanomedicine (sHDL) composed of apolipoprotein A-I (ApoA-I) mimetic peptides and lipids have shown very promising results for the treatment of various cardiovascular diseases. Numerous efforts have also been made to design different ApoA-I mimetic peptides to improve the potency of sHDL, especially the efficiency of reverse cholesterol transport. However, the way in which ApoA-I mimetic peptides affect the properties of sHDL, including stability, cholesterol efflux, cholesterol esterification, elimination in vivo, and the relationship of these properties, is still poorly understood. Revealing the effect of these factors on the potency of sHDL is important for the design of better ApoA-I mimetic peptides. In this study, three widely used ApoA-I mimetic peptides with different sequences, lengths, LCAT activation and lipid binding affinities were used for the preparation of sHDL and were evaluated in terms of physical/chemical properties, cholesterol efflux, cholesterol esterification, remodeling, and pharmacokinetics/pharmacodynamics. Our results showed that ApoA-I mimetic peptides with the highest cholesterol efflux and cholesterol esterification in vitro did not exhibit the highest cholesterol mobilization in vivo. Further analysis indicated that other factors, such as pharmacokinetics and remodeling of sHDL, need to be considered in order to predict the efficiency of cholesterol mobilization in vivo. Thus, our study highlights the importance of using the overall performance, rather than in vitro results alone, as the blueprint for the design and optimization of ApoA-I mimetic peptides.


Assuntos
Apolipoproteína A-I , Lipoproteínas HDL , Lipoproteínas HDL/química , Apolipoproteína A-I/farmacologia , Apolipoproteína A-I/química , Peptídeos/farmacologia , Peptídeos/química , Colesterol/química , Transporte Biológico
5.
Blood Adv ; 7(8): 1366-1378, 2023 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-36219587

RESUMO

Low-density lipoprotein (LDL) contributes to atherogenesis and cardiovascular disease through interactions with peripheral blood cells, especially platelets. However, mechanisms by which LDL affects platelet activation and atherothrombosis, and how to best therapeutically target and safely prevent such responses remain unclear. Here, we investigate how oxidized low-density lipoprotein (oxLDL) enhances glycoprotein VI (GPVI)-mediated platelet hemostatic and procoagulant responses, and how traditional and emerging antiplatelet therapies affect oxLDL-enhanced platelet procoagulant activity ex vivo. Human platelets were treated with oxLDL and the GPVI-specific agonist, crosslinked collagen-related peptide, and assayed for hemostatic and procoagulant responses in the presence of inhibitors of purinergic receptors (P2YR), cyclooxygenase (COX), and tyrosine kinases. Ex vivo, oxLDL enhanced GPVI-mediated platelet dense granule secretion, α-granule secretion, integrin activation, thromboxane generation and aggregation, as well as procoagulant phosphatidylserine exposure and fibrin generation. Studies of washed human platelets, as well as platelets from mouse and nonhuman primate models of hyperlipidemia, further determined that P2YR antagonists (eg, ticagrelor) and Bruton tyrosine kinase inhibitors (eg, ibrutinib) reduced oxLDL-mediated platelet responses and procoagulant activity, whereas COX inhibitors (eg, aspirin) had no significant effect. Together, our results demonstrate that oxLDL enhances GPVI-mediated platelet procoagulant activity in a manner that may be more effectively reduced by P2YR antagonists and tyrosine kinase inhibitors compared with COX inhibitors.


Assuntos
Hemostáticos , Inibidores da Agregação Plaquetária , Humanos , Camundongos , Animais , Inibidores da Agregação Plaquetária/farmacologia , Lipoproteínas LDL/farmacologia
6.
Int J Mol Sci ; 23(19)2022 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-36232786

RESUMO

ApoB-100 is a member of a large lipid transfer protein superfamily and is one of the main apolipoproteins found on low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) particles. Despite its clinical significance for the development of cardiovascular disease, there is limited information on apoB-100 structure. We have developed a novel method based on the "divide and conquer" algorithm, using PSIPRED software, by dividing apoB-100 into five subunits and 11 domains. Models of each domain were prepared using I-TASSER, DEMO, RoseTTAFold, Phyre2, and MODELLER. Subsequently, we used disuccinimidyl sulfoxide (DSSO), a new mass spectrometry cleavable cross-linker, and the known position of disulfide bonds to experimentally validate each model. We obtained 65 unique DSSO cross-links, of which 87.5% were within a 26 Å threshold in the final model. We also evaluated the positions of cysteine residues involved in the eight known disulfide bonds in apoB-100, and each pair was measured within the expected 5.6 Å constraint. Finally, multiple domains were combined by applying constraints based on detected long-range DSSO cross-links to generate five subunits, which were subsequently merged to achieve an uninterrupted architecture for apoB-100 around a lipoprotein particle. Moreover, the dynamics of apoB-100 during particle size transitions was examined by comparing VLDL and LDL computational models and using experimental cross-linking data. In addition, the proposed model of receptor ligand binding of apoB-100 provides new insights into some of its functions.


Assuntos
Apolipoproteínas B , Cisteína , Apolipoproteína B-100 , Apolipoproteínas B/metabolismo , Simulação por Computador , Dissulfetos , Ligantes , Lipoproteínas LDL/química , Lipoproteínas VLDL , Modelos Estruturais , Sulfóxidos
7.
Cells ; 10(3)2021 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-33800446

RESUMO

Since the seminal breakthrough of treating diabetic patients with insulin in the 1920s, there has been great interest in developing other proteins and their peptide mimetics as therapies for a wide variety of other medical disorders. Currently, there are at least 60 different peptides that have been approved for human use and over 150 peptides that are in various stages of clinical development. Peptides mimetic of the major proteins on lipoproteins, namely apolipoproteins, have also been developed first as tools for understanding apolipoprotein structure and more recently as potential therapeutics. In this review, we discuss the biochemistry, peptide mimetics design and clinical trials for peptides based on apoA-I, apoE and apoC-II. We primarily focus on applications of peptide mimetics related to cardiovascular diseases. We conclude with a discussion on the limitations of peptides as therapeutic agents and the challenges that need to be overcome before apolipoprotein mimetic peptides can be developed into new drugs.


Assuntos
Apolipoproteína A-I/uso terapêutico , Apolipoproteínas/metabolismo , Doenças Cardiovasculares/terapia , Peptídeos/metabolismo , Humanos
8.
PLoS One ; 15(10): e0240659, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33057430

RESUMO

SR-BI binds various lipoproteins, including HDL, LDL as well as VLDL, and mediates selective cholesteryl ester (CE) uptake. HDL derived CE accumulates in cellular lipid droplets (LDs), which also store triacylglycerol (TAG). We hypothesized that SR-BI could significantly facilitate LD formation, in part, by directly transporting LDL derived neutral lipids (NL) such as CE and TAG into LDs without lipolysis and de novo lipid synthesis. SR-BI overexpression greatly increased LDL uptake and LD formation in stably transfected HeLa cells (SR-BI-HeLa). LDs isolated from SR-BI-HeLa contained 4- and 7-times more CE and TAG, respectively, than mock-transfected HeLa (Mock-HeLa). In contrast, LDL receptor overexpression in HeLa (LDLr-HeLa) greatly increased LDL uptake, degradation with moderate 1.5- and 2-fold increases of CE and TAG, respectively. Utilizing CE and TAG analogs, BODIPY-TAG (BP-TAG) and BODIPY-CE (BP-CE), for tracking LDL NL, we found that after initial binding of LDL to SR-BI-HeLa, apoB remained at the cell surface, while BP-CE and BP-TAG were sorted and simultaneously transported together to LDs. Both lipids demonstrated limited internalization to lysosomes or endoplasmic reticulum in SR-BI-HeLa. In LDLr-HeLa, NLs demonstrated clear lysosomal sequestration without their sorting to LDs. An inhibition of TAG and CE de novo synthesis by 90-95% only reduced TAG and CE LD content by 45-50%, and had little effect on BP-CE and BP-TAG transport to LDs in SR-BI HeLa. Furthermore, intravenous infusion of 1-2 mg of LDL increased liver LDs in normal (WT) but not in SR-BI KO mice. Mice transgenic for human SR-BI demonstrated higher liver LD accumulation than WT mice. Finally, Electro Spray Infusion Mass Spectrometry (ESI-MS) using deuterated d-CE found that LDs accumulated up to 40% of unmodified d-CE LDL. We conclude that SR-BI mediates LDL-induced LD formation in vitro and in vivo. In addition to cytosolic NL hydrolysis and de novo lipid synthesis, this process includes selective sorting and transport of LDL NL to LDs with limited lysosomal NL sequestration and the transport of LDL CE, and TAG directly to LDs independently of de novo synthesis.


Assuntos
Gotículas Lipídicas/metabolismo , Lipídeos/química , Lipoproteínas LDL/metabolismo , Receptores Depuradores Classe B/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Compostos de Boro/metabolismo , Ésteres do Colesterol/metabolismo , Coenzima A Ligases/antagonistas & inibidores , Coenzima A Ligases/metabolismo , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Gotículas Lipídicas/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de LDL/metabolismo , Triazenos/farmacologia , Triglicerídeos/metabolismo
9.
J Lipid Res ; 61(12): 1577-1588, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32907987

RESUMO

Lipoproteins play a key role in transport of cholesterol to and from tissues. Recent studies have also demonstrated that red blood cells (RBCs), which carry large quantities of free cholesterol in their membrane, play an important role in reverse cholesterol transport. However, the exact role of RBCs in systemic cholesterol metabolism is poorly understood. RBCs were incubated with autologous plasma or isolated lipoproteins resulting in a significant net amount of cholesterol moved from RBCs to HDL, while cholesterol from LDL moved in the opposite direction. Furthermore, the bi-directional cholesterol transport between RBCs and plasma lipoproteins was saturable and temperature-, energy-, and time-dependent, consistent with an active process. We did not find LDLR, ABCG1, or scavenger receptor class B type 1 in RBCs but found a substantial amount of ABCA1 mRNA and protein. However, specific cholesterol efflux from RBCs to isolated apoA-I was negligible, and ABCA1 silencing with siRNA or inhibition with vanadate and Probucol did not inhibit the efflux to apoA-I, HDL, or plasma. Cholesterol efflux from and cholesterol uptake by RBCs from Abca1+/+ and Abca1-/- mice were similar, arguing against the role of ABCA1 in cholesterol flux between RBCs and lipoproteins. Bioinformatics analysis identified ABCA7, ABCG5, lipoprotein lipase, and mitochondrial translocator protein as possible candidates that may mediate the cholesterol flux. Together, these results suggest that RBCs actively participate in cholesterol transport in the blood, but the role of cholesterol transporters in RBCs remains uncertain.


Assuntos
Colesterol/metabolismo , Eritrócitos/metabolismo , Lipoproteínas/metabolismo , Transporte Biológico , Biologia Computacional , Humanos
10.
Biochem Biophys Res Commun ; 526(2): 349-354, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32222278

RESUMO

Apolipoprotein A-I (ApoA-I) mimetic peptides are potential therapeutic agents for promoting the efflux of excess cellular cholesterol, which is dependent upon the presence of an amphipathic helix. Since α-methylated Ala enhances peptide helicity, we hypothesized that incorporating other types of α-methylated amino acids into ApoA-I mimetic peptides may also increase their helicity and cholesterol efflux potential. The last helix of apoA-I, peptide 'A' (VLESFKVSFLSALEEYTKKLNT), was used to design peptides containing a single type of α-methylated amino acid substitution (Ala/Aα, Glu/Dα, Lys/Kα, Leu/Lα), as well as a peptide containing both α-methylated Lys and Leu (6α). Depending on the specific residue, the α-helical content as measured by CD-spectroscopy and calculated hydrophobic moments were sometimes higher for peptides containing other types of α-methylated amino acids than those with α-methylated Ala. In ABCA1-transfected cells, cholesterol efflux to the peptides showed the following order of potency: 6α>Kα≈Lα≈Aα≫Dα≈A. In general, α-methylated peptides were resistant to proteolysis, but this varied depending on the type of protease and specific amino acid substitution. In summary, increased helicity and amphilicity due to α-methylated amino acid substitutions in ApoA-I mimetic peptides resulted in improved cholesterol efflux capacity and resistance to proteolysis, indicating that this modification may be useful in the future design of therapeutic ApoA-I mimetic peptides.


Assuntos
Aminoácidos/química , Apolipoproteína A-I/química , Colesterol/metabolismo , Peptidomiméticos/química , Peptidomiméticos/farmacologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Desenho de Fármacos , Humanos , Metilação
11.
Sci Transl Med ; 12(528)2020 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-31996466

RESUMO

Recent genetic studies have established that hypertriglyceridemia (HTG) is causally related to cardiovascular disease, making it an active area for drug development. We describe a strategy for lowering triglycerides (TGs) with an apolipoprotein C-II (apoC-II) mimetic peptide called D6PV that activates lipoprotein lipase (LPL), the main plasma TG-hydrolyzing enzyme, and antagonizes the TG-raising effect of apoC-III. The design of D6PV was motivated by a combination of all-atom molecular dynamics simulation of apoC-II on the Anton 2 supercomputer, structural prediction programs, and biophysical techniques. Efficacy of D6PV was assessed ex vivo in human HTG plasma and was found to be more potent than full-length apoC-II in activating LPL. D6PV markedly lowered TG by more than 80% within a few hours in both apoC-II-deficient mice and hAPOC3-transgenic (Tg) mice. In hAPOC3-Tg mice, D6PV treatment reduced plasma apoC-III by 80% and apoB by 65%. Furthermore, low-density lipoprotein (LDL) cholesterol did not accumulate but rather was decreased by 10% when hAPOC3-Tg mice lacking the LDL-receptor (hAPOC3-Tg × Ldlr-/- ) were treated with the peptide. D6PV lowered TG by 50% in whole-body inducible Lpl knockout (iLpl-/- ) mice, confirming that it can also act independently of LPL. D6PV displayed good subcutaneous bioavailability of about 80% in nonhuman primates. Because it binds to high-density lipoproteins, which serve as a long-term reservoir, it also has an extended terminal half-life (42 to 50 hours) in nonhuman primates. In summary, D6PV decreases plasma TG by acting as a dual apoC-II mimetic and apoC-III antagonist, thereby demonstrating its potential as a treatment for HTG.


Assuntos
Apolipoproteína C-III/antagonistas & inibidores , Apolipoproteína C-II/agonistas , Peptídeos/farmacologia , Triglicerídeos/sangue , Animais , Modelos Animais de Doenças , Feminino , Meia-Vida , Humanos , Hipertrigliceridemia/sangue , Hipertrigliceridemia/tratamento farmacológico , Lipólise , Lipase Lipoproteica/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Simulação de Dinâmica Molecular , Peptídeos/química , Peptídeos/farmacocinética , Peptídeos/uso terapêutico , Primatas
12.
Biochem Biophys Res Commun ; 519(1): 67-72, 2019 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-31477272

RESUMO

Elevated plasma triglyceride (TG) levels are associated with higher risk of atherosclerotic cardiovascular disease. One way to reduce plasma TG is to increase the activity of lipoprotein lipase (LPL), the rate limiting enzyme in plasma TG metabolism. An apolipoprotein (apo) C-II mimetic peptide (18A-CII-a) has been recently developed that stimulated LPL activity in vitro and decreased plasma TG concentration in animal models for hypertriglyceridemia. Since this peptide can serve as a new therapeutic approach for treatment of hypertriglyceridemia, we investigated how 18A-CII-a peptide influences LPL activity in human plasma. We used recently described isothermal titration calorimetry based approach to assess the peptide, which enables the analysis in nearly undiluted human plasma. The 18A-CII-a peptide was 3.5-fold more efficient in stimulating LPL activity than full-length apoC-II in plasma sample from normolipidemic individual. Furthermore, 18A-CII-a also increased LPL activity in hypertriglyceridemic plasma samples. Unlike apoC-II, high concentrations of the 18A-CII-a peptide did not inhibit LPL activity. The increase in LPL activity after addition of 18A-CII-a or apoC-II to plasma was due to the increase of the amount of available substrate for LPL. Measurements with isolated lipoproteins revealed that the relative activation effects of 18A-CII-a and apoC-II on LPL activity were greater in smaller size lipoprotein fractions, such as remnant lipoproteins, low-density lipoproteins and high-density lipoproteins. In summary, this report describes a novel mechanism of action for stimulation of LPL activity by apoC-II mimetic peptides.


Assuntos
Apolipoproteína C-II/metabolismo , Calorimetria/métodos , Lipase Lipoproteica/sangue , Peptídeos/metabolismo , Animais , Bovinos , Ácidos Graxos/metabolismo , Humanos , Hidrólise , Especificidade por Substrato
13.
Biology (Basel) ; 8(3)2019 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-31336888

RESUMO

We describe simple, sensitive and robust methods to monitor lipoprotein remodeling and cholesterol and apolipoprotein exchange, using fluorescent Lissamine Rhodamine B head-group tagged phosphatidylethanolamine (*PE) as a lipoprotein reference marker. Fluorescent Bodipy cholesterol (*Chol) and *PE directly incorporated into whole plasma lipoproteins in proportion to lipoprotein cholesterol and phospholipid mass, respectively. *Chol, but not *PE, passively exchanged between isolated plasma lipoproteins. Fluorescent apoA-I (*apoA-I) specifically bound to high-density lipoprotein (HDL) and remodeled *PE- and *Chol-labeled synthetic lipoprotein-X multilamellar vesicles (MLV) into a pre-ß HDL-like particle containing *PE, *Chol, and *apoA-I. Fluorescent MLV-derived *PE specifically incorporated into plasma HDL, whereas MLV-derived *Chol incorporation into plasma lipoproteins was similar to direct *Chol incorporation, consistent with apoA-I-mediated remodeling of fluorescent MLV to HDL with concomitant exchange of *Chol between lipoproteins. Based on these findings, we developed a model system to study lipid transfer by depositing fluorescent *PE and *Chol-labeled on calcium silicate hydrate crystals, forming dense lipid-coated donor particles that are readily separated from acceptor lipoprotein particles by low-speed centrifugation. Transfer of *PE from donor particles to mouse plasma lipoproteins was shown to be HDL-specific and apoA-I-dependent. Transfer of donor particle *PE and *Chol to HDL in whole human plasma was highly correlated. Taken together, these studies suggest that cell-free *PE efflux monitors apoA-I functionality.

14.
J Nutr Metab ; 2019: 7078241, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30863636

RESUMO

AIM: Plasma apolipoprotein C-II (apoC-II) activates lipoprotein lipase (LPL) and thus lowers plasma triglycerides (TG). We previously reported that a human apoC-II mimetic peptide (C-II-a) decreased plasma TG in apoC-II mutant mice, as well as in apoE-knockout mice. Because it is unknown what tissues take up free fatty acids (FFAs) released from TG after C-II-a peptide administration, we investigated in mice TG plasma clearance and tissue incorporation, using 3H-triolein as a tracer, with and without C-II-a treatment. METHODS AND RESULTS: Intralipid® fat emulsion was labeled with 3H-triolein and then mixed with or without C-II-a. Addition of the peptide did not alter mean particle size of the lipid emulsion particles (298 nm) but accelerated their plasma clearance. After intravenous injection into C57BL/6N mice, the plasma half-life of the 3H-triolein for control and C-II-a treated emulsions was 18.3 ± 2.2 min and 14.8 ± 0.1 min, respectively. In apoC-II mutant mice, the plasma half-life of 3H-triolein for injected control and C-II-a treated emulsions was 30.1 ± 0.1 min and 14.8 ± 0.1 min, respectively. C57BL/6N and apoC-II mutant mice at 120 minutes after the injection showed increased tissue incorporation of radioactivity in white adipose tissue when C-II-a treated emulsion was used. Higher radiolabeled uptake of lipids from C-II-a treated emulsion was also observed in the skeletal muscle of C57BL/6N mice only. In case of apoC-II mutant mice, decreased uptake of radioactive lipids was observed in the liver and kidney after addition of C-II-a to the lipid emulsion. CONCLUSIONS: C-II-a peptide promotes the plasma clearance of TG-rich lipid emulsions in wild type and apoC-II mutant mice and promotes the incorporation of fatty acids from TG in the lipid emulsions into specific peripheral tissues.

15.
Atherosclerosis ; 278: 278-285, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30347343

RESUMO

BACKGROUND AND AIMS: High density lipoprotein cholesterol (HDL-C) is associated with risk of cardiovascular disease (CVD); however, therapeutic manipulations of HDL-C have failed to reduce CVD events. This suggests that HDL-C and the atheroprotective capacity of HDL are not directly linked. The goal of this study was to evaluate the relationships between HDL-bound proteins and measures of atherosclerosis burden and HDL function. METHODS: The HDL proteome was analyzed using mass spectrometry in 126 human subjects, who had undergone coronary computed tomography angiography (CCTA) to quantify calcified (CB) and non-calcified (NCB) atherosclerosis burden. Partial least squares regression analysis was used to evaluate associations between HDL-bound proteins and CB, NCB, or cholesterol efflux capacity (CEC). RESULTS: Significant overlap was found among proteins associated with NCB and CEC. Proteins that were associated with NCB displayed an inverse relationship with CEC, supporting a link between this protective function of HDL and clinical plaque burden. CB was associated with a set of proteins mostly distinct from NCB and CEC. When CVD risk factors were evaluated, BMI had a stronger influence on important HDL proteins than gender, age, or HDL-C. Most HDL proteins associated with function or atherosclerosis burden were not significantly correlated with HDL-C. CONCLUSIONS: These findings indicate that the HDL proteome contains information not captured by HDL- C and, therefore, has potential for future development as a biomarker for CVD risk. Additionally, the proteome effects detected in this study may provide HDL compositional goals for evaluating new and existing HDL-modification therapies.


Assuntos
Aterosclerose/diagnóstico por imagem , Doenças Cardiovasculares/diagnóstico por imagem , Lipoproteínas HDL/metabolismo , Idoso , Animais , Aterosclerose/metabolismo , Biomarcadores/metabolismo , Índice de Massa Corporal , Doenças Cardiovasculares/metabolismo , Linhagem Celular , HDL-Colesterol , Angiografia por Tomografia Computadorizada , Angiografia Coronária , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Placa Aterosclerótica , Proteoma , Análise de Regressão , Fatores de Risco
16.
Sci Rep ; 8(1): 2956, 2018 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-29440748

RESUMO

Peptides mimicking the major protein of highdensity lipoprotein (HDL), apolipoprotein A-I (apoA-I), are promising therapeutics for cardiovascular diseases. Similar to apoA-I, their atheroprotective property is attributed to their ability to form discoidal HDL-like particles by extracting cellular cholesterol and phospholipids from lipid microdomains created by the ABCA1 transporter in a process called cholesterol efflux. The structural features of peptides that enable cholesterol efflux are not well understood. Herein, four synthetic amphipathic peptides denoted ELK, which only contain Glu, Leu, Lys, and sometimes Ala, and which have a wide range of net charges and hydrophobicities, were examined for cholesterol efflux. Experiments show that ELKs with a net neutral charge and a hydrophobic face that subtends an angle of at least 140° are optimal for cholesterol efflux. All-atom molecular dynamics simulations show that peptides that are effective in promoting cholesterol efflux stabilize HDL nanodiscs formed by these peptides by the orderly covering of the hydrophobic acyl chains on the edge of the disc. In contrast to apoA-I, which forms an anti-parallel double belt around the HDL, active peptides assemble in a mostly anti-parallel "picket fence" arrangement. These results shed light on the efflux ability of apoA-I mimetics and inform the future design of such therapeutics.


Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Apolipoproteína A-I/química , Colesterol/metabolismo , Peptidomiméticos/química , Peptidomiméticos/farmacologia , Sequência de Aminoácidos , Transporte Biológico/efeitos dos fármacos , Simulação de Dinâmica Molecular , Fosfolipídeos/metabolismo , Conformação Proteica
17.
Mol Pharm ; 15(1): 83-96, 2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29141139

RESUMO

Synthetic high density lipoprotein nanoparticles (sHDLs) capable of mobilizing excess cholesterol from atherosclerotic arteries and delivering it to the liver for elimination have been shown to reduce plaque burden in patients. Unfortunately, sHDLs have a narrow therapeutic index and relative to the endogenous HDL shorter circulation half-life. Surface modification with polyethylene glycol (PEG) was investigated for its potential to extend sHDL circulation in vivo. Various amounts (2.5, 5, and 10%) and different chain lengths (2 and 5 kDa) of PEG-modified lipids were incorporated in sHDL's lipid membrane. Incorporating PEG did not reduce the ability of sHDL to facilitate cholesterol efflux, nor did it inhibit cholesterol uptake by the liver cells. By either adding more PEG or using PEG of longer chain lengths, the circulation half-life was extended. Addition of PEG also increased the area under the curve for the phospholipid component of sHDL (p < 0.05), but not for the apolipoprotein A-I peptide component of sHDL, suggesting sHDL is remodeled by endogenous lipoproteins in vivo. The extended phospholipid circulation led to a higher mobilization of plasma free cholesterol, a biomarker for facilitation of reverse cholesterol transport. The area under the cholesterol mobilization increased about 2-4-fold (p < 0.05), with greater increases observed for longer PEG chains and higher molar percentages of incorporated PEGylated lipids. Mobilized cholesterol was associated primarily with the HDL fraction, led to a transient increase in VLDL cholesterol, and returned to baseline 24 h postdose. Overall, PEGylation of sHDL led to beneficial changes in sHDL particle pharmacokinetic and pharmacodynamic behaviors.


Assuntos
Lipoproteínas HDL/química , Nanopartículas/química , Polietilenoglicóis/química , Apolipoproteína A-I/química , Colesterol/química
18.
Atherosclerosis ; 267: 49-60, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29100061

RESUMO

Apolipoprotein C-II (apoC-II) is a small exchangeable apolipoprotein found on triglyceride-rich lipoproteins (TRL), such as chylomicrons (CM) and very low-density lipoproteins (VLDL), and on high-density lipoproteins (HDL), particularly during fasting. ApoC-II plays a critical role in TRL metabolism by acting as a cofactor of lipoprotein lipase (LPL), the main enzyme that hydrolyses plasma triglycerides (TG) on TRL. Here, we present an overview of the role of apoC-II in TG metabolism, emphasizing recent novel findings regarding its transcriptional regulation and biochemistry. We also review the 24 genetic mutations in the APOC2 gene reported to date that cause hypertriglyceridemia (HTG). Finally, we describe the clinical presentation of apoC-II deficiency and assess the current therapeutic approaches, as well as potential novel emerging therapies.


Assuntos
Apolipoproteína C-II/genética , Apolipoproteína C-II/metabolismo , Triglicerídeos/metabolismo , Animais , Apolipoproteína C-II/deficiência , Quilomícrons/metabolismo , Regulação da Expressão Gênica , Humanos , Hidrólise , Mucosa Intestinal/metabolismo , Lipólise , Lipase Lipoproteica/metabolismo , Lipoproteínas/metabolismo , Lipoproteínas HDL/sangue , Lipoproteínas VLDL/metabolismo , Fígado/metabolismo , Macrófagos/metabolismo , Camundongos , Família Multigênica , Mutação , Ratos , Transcrição Gênica
19.
Atherosclerosis ; 262: 31-38, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28486149

RESUMO

BACKGROUND AND AIMS: Concentrated fish oils, containing a mixture of long-chain monounsaturated fatty acids (LCMUFA) with aliphatic chains longer than 18 C atoms (i.e., C20:1 and C22:1), have been shown to attenuate atherosclerosis development in mouse models. It is not clear, however, how individual LCMUFA isomers may act on atherosclerosis. METHODS: In the present study, we used saury fish oil-derived concentrates enriched in either C20:1 or C22:1 isomer fractions to investigate their individual effect on atherosclerosis and lipoprotein metabolism. LDLR-deficient (LDLr-/-) mice were fed a Western diet supplemented with 5% (w/w) of either C20:1 or C22:1 concentrate for 12 wk. RESULTS: Compared to the control Western diet with no supplement, both LCMUFA isomers increased hepatic levels of LCMUFA by 2∼3-fold (p < 0.05), and decreased atherosclerotic lesion areas by more than 40% (p < 0.05), although there were no major differences in plasma lipoproteins or hepatic lipid content. Both LCMUFA isomers significantly decreased plasma CRP levels, improved Abca1-dependent cholesterol efflux capacity of apoB-depleted plasma, and enhanced Ppar transcriptional activities in HepG2 cells. LC-MS/MS proteomic analysis of lipoproteins (HDL, LDL and VLDL) revealed that both LCMUFA isomer diets resulted in similar potentially beneficial alterations in proteins involved in complement activation, blood coagulation, and lipid metabolism. Several lipoprotein proteome changes were significantly correlated with atherosclerotic plaque reduction. CONCLUSIONS: Dietary supplementation with the LCMUFA isomers C20:1 or C22:1 was equally effective in reducing atherosclerosis in LDLr-/-mice and this may partly occur through activation of the Ppar signaling pathways and favorable alterations in the proteome of lipoproteins.


Assuntos
Doenças da Aorta/prevenção & controle , Aterosclerose/prevenção & controle , Suplementos Nutricionais , Ácidos Graxos Monoinsaturados/farmacologia , Óleos de Peixe/farmacologia , Hiperlipidemias/tratamento farmacológico , Lipoproteínas/sangue , Proteoma , Receptores de LDL/deficiência , Animais , Doenças da Aorta/sangue , Doenças da Aorta/genética , Aterosclerose/sangue , Aterosclerose/genética , Aterosclerose/patologia , Biomarcadores/sangue , Proteína C-Reativa/metabolismo , Cromatografia Líquida , Dieta Ocidental , Modelos Animais de Doenças , Predisposição Genética para Doença , Células Hep G2 , Humanos , Hiperlipidemias/sangue , Hiperlipidemias/genética , Hiperlipidemias/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos Knockout , Receptores Ativados por Proliferador de Peroxissomo/genética , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Fenótipo , Placa Aterosclerótica , Proteômica/métodos , Receptores de LDL/genética , Espectrometria de Massas em Tandem
20.
Sci Rep ; 7: 43410, 2017 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-28230201

RESUMO

Lipoproteins modulate innate and adaptive immune responses. In the chronic inflammatory disease multiple sclerosis (MS), reports on lipoprotein level alterations are inconsistent and it is unclear whether lipoprotein function is affected. Using nuclear magnetic resonance (NMR) spectroscopy, we analysed the lipoprotein profile of relapsing-remitting (RR) MS patients, progressive MS patients and healthy controls (HC). We observed smaller LDL in RRMS patients compared to healthy controls and to progressive MS patients. Furthermore, low-BMI (BMI ≤ 23 kg/m2) RRMS patients show increased levels of small HDL (sHDL), accompanied by larger, triglyceride (TG)-rich VLDL, and a higher lipoprotein insulin resistance (LP-IR) index. These alterations coincide with a reduced serum capacity to accept cholesterol via ATP-binding cassette (ABC) transporter G1, an impaired ability of HDL3 to suppress inflammatory activity of human monocytes, and modifications of HDL3's main protein component ApoA-I. In summary, lipoprotein levels and function are altered in RRMS patients, especially in low-BMI patients, which may contribute to disease progression in these patients.


Assuntos
Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/sangue , Apolipoproteína A-I/sangue , Lipoproteínas HDL/sangue , Monócitos/imunologia , Esclerose Múltipla Recidivante-Remitente/sangue , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Imunidade Adaptativa , Adulto , Apolipoproteína A-I/genética , Índice de Massa Corporal , Estudos de Casos e Controles , LDL-Colesterol/sangue , VLDL-Colesterol/sangue , Progressão da Doença , Feminino , Expressão Gênica , Humanos , Imunidade Inata , Resistência à Insulina , Lipoproteínas HDL/classificação , Lipoproteínas HDL/genética , Espectroscopia de Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Monócitos/patologia , Esclerose Múltipla Recidivante-Remitente/genética , Esclerose Múltipla Recidivante-Remitente/imunologia , Esclerose Múltipla Recidivante-Remitente/patologia , Triglicerídeos/sangue
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