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1.
Proc Natl Acad Sci U S A ; 116(13): 6319-6328, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30850549

RESUMO

Lipoprotein lipase (LPL), the enzyme that hydrolyzes triglycerides in plasma lipoproteins, is assumed to be active only as a homodimer. In support of this idea, several groups have reported that the size of LPL, as measured by density gradient ultracentrifugation, is ∼110 kDa, twice the size of LPL monomers (∼55 kDa). Of note, however, in those studies the LPL had been incubated with heparin, a polyanionic substance that binds and stabilizes LPL. Here we revisited the assumption that LPL is active only as a homodimer. When freshly secreted human LPL (or purified preparations of LPL) was subjected to density gradient ultracentrifugation (in the absence of heparin), LPL mass and activity peaks exhibited the size expected of monomers (near the 66-kDa albumin standard). GPIHBP1-bound LPL also exhibited the size expected for a monomer. In the presence of heparin, LPL size increased, overlapping with a 97.2-kDa standard. We also used density gradient ultracentrifugation to characterize the LPL within the high-salt and low-salt peaks from a heparin-Sepharose column. The catalytically active LPL within the high-salt peak exhibited the size of monomers, whereas most of the inactive LPL in the low-salt peak was at the bottom of the tube (in aggregates). Consistent with those findings, the LPL in the low-salt peak, but not that in the high-salt peak, was easily detectable with single mAb sandwich ELISAs, in which LPL is captured and detected with the same antibody. We conclude that catalytically active LPL can exist in a monomeric state.


Assuntos
Lipase Lipoproteica/química , Lipase Lipoproteica/isolamento & purificação , Animais , Células CHO , Bovinos , Centrifugação com Gradiente de Concentração/métodos , Cromatografia de Afinidade , Cromatografia em Agarose , Cricetulus , Epitopos , Heparina , Humanos , Lipase Lipoproteica/sangue , Receptores de Lipoproteínas/sangue , Receptores de Lipoproteínas/química , Receptores de Lipoproteínas/isolamento & purificação , Sefarose/análogos & derivados , Triglicerídeos/metabolismo , Ultracentrifugação
2.
Biochem Biophys Res Commun ; 504(1): 54-60, 2018 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-30170729

RESUMO

The ß-isoform of diacylglycerol kinase (DGK) localizes predominantly to neurons and induces neurite outgrowth and spine formation. However, the detailed molecular mechanisms underlying the functions of DGKß remain elusive. During the course of studies on other DGK isozymes, we unexpectedly found that the overexpression of wild-type DGKß in COS-7 cells markedly induced filopodium formation. Because filopodium formation is closely related to neurite outgrowth and spine formation, we constructed various DGKß mutants and compared their abilities to induce filopodium formation in order to elucidate the structure-function relationships of DGKß. We found that the C-terminal, C1 and catalytic domains and catalytic activity were indispensable for filopodium formation, but the recoverin homology domain and EF-hand motifs were not. Moreover, the extent of plasma membrane localization and F-actin colocalization were positively correlated with filopodium formation. Intriguingly, DGKß selectively interacted and colocalized at the plasma membrane with a Rac1-GTPase-activating protein, ß2-chimaerin, which is an inducer of filopodia; it also interacted, to lesser extent, with α2-chimaerin, but not with α1- or ß1-chimaerin. Moreover, DGKß enhanced the plasma membrane localization of ß2-chimaerin. These results suggest that DGKß plays an important role in neurite outgrowth and spine formation in neurons via its ability to induce filopodium formation.


Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Lipase Lipoproteica/metabolismo , Proteínas de Neoplasias/metabolismo , Pseudópodes/fisiologia , Animais , Células COS , Domínio Catalítico , Lipase Lipoproteica/química , Lipase Lipoproteica/genética , Mutação , Domínios Proteicos , Pseudópodes/ultraestrutura
3.
Bioorg Chem ; 80: 347-360, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29986183

RESUMO

Studies on the lipid-regulating effects of alisol compounds are reported that include alisol B, alisol A 24-acetate (24A), alisol A and an alisol B - 24A - alisol A mixture (content ratio = 1:1:1). The effects on the activity of lipoprotein lipase (LPL), a key lipid-modulating enzyme, were studied to investigate the molecular mechanism of lipid-regulating activity of alisols. The effects of alisols on regulating blood lipids and the activities of LPL were determined using a reagent kit method. The structure of LPL was obtained by homology modeling and the interactive mechanism of alisol monomers and the mixture with LPL was investigated by molecular simulation. The alisol monomer and mixture were shown to regulate blood lipids, suggesting that alisols may decrease the level of triglyceride (TG) by improving the activity of LPL. The order of intensity was: mixture > alisol A > alisol B > 24A, indicating that alisols of alismatis rhizoma feature a synergistic effect on LPL. The N- and C-terminus of LPL both represented the catalytic active domains of this lipid-regulating effect. Cys306, Gln129 and Ser166 were the key amino acid residues resulting in the lipid-regulating effect of the alisol monomer while Ser166 and Arg18 were found to be responsible for the lipid-regulating effect of the mixture. The C-terminus of LPL was indirectly involved in the enzymatic process. A folded side chain of alisols or the parent ring was found to bind somewhat weaker to LPL than an open side chain or parent ring. The hydroxyl groups on the C14-, C22-, C28-, C30- and C31-terminus in the side chain, the ring ether structure in C23-position, and the acetyl group in C29-position represented the key sites for the lipid-regulating action of alisols. Meanwhile, the C30-site hydroxyl group played an important role in the synergistic effect of the alisol mixture.


Assuntos
Colestenonas/metabolismo , Lipase Lipoproteica/metabolismo , Animais , Sítios de Ligação , Colestenonas/química , Colestenonas/uso terapêutico , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Hiperlipidemias/tratamento farmacológico , Hiperlipidemias/metabolismo , Hiperlipidemias/veterinária , Lipídeos/sangue , Lipase Lipoproteica/química , Masculino , Camundongos , Camundongos Endogâmicos ICR , Simulação de Dinâmica Molecular , Eletricidade Estática
4.
Org Biomol Chem ; 16(29): 5250-5253, 2018 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-30004552

RESUMO

Diacylglycerol lipases (DAGL) produce the endocannabinoid 2-arachidonoylglycerol, a key modulator of neurotransmitter release. Chemical tools that visualize endogenous DAGL activity are desired. Here, we report the design, synthesis and application of a triazole urea probe for DAGL equipped with a norbornene as a biorthogonal handle. The activity and selectivity of the probe was assessed with activity-based protein profiling. This probe was potent against endogenous DAGLα (IC50 = 5 nM) and it was successfully applied as a two-step activity-based probe for labeling of DAGLα using an inverse electron-demand Diels-Alder ligation in living cells.


Assuntos
Lipase Lipoproteica/química , Lipase Lipoproteica/metabolismo , Animais , Encéfalo/metabolismo , Linhagem Celular Tumoral , Reação de Cicloadição , Teoria da Densidade Funcional , Endocanabinoides/química , Humanos , Lipase Lipoproteica/antagonistas & inibidores , Camundongos , Sondas Moleculares/química , Sondas Moleculares/toxicidade , Norbornanos/química , Proteoma , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Triazóis/química , Ureia/química
5.
Proc Natl Acad Sci U S A ; 115(26): E6020-E6029, 2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29899144

RESUMO

The intravascular processing of triglyceride-rich lipoproteins depends on lipoprotein lipase (LPL) and GPIHBP1, a membrane protein of endothelial cells that binds LPL within the subendothelial spaces and shuttles it to the capillary lumen. In the absence of GPIHBP1, LPL remains mislocalized within the subendothelial spaces, causing severe hypertriglyceridemia (chylomicronemia). The N-terminal domain of GPIHBP1, an intrinsically disordered region (IDR) rich in acidic residues, is important for stabilizing LPL's catalytic domain against spontaneous and ANGPTL4-catalyzed unfolding. Here, we define several important properties of GPIHBP1's IDR. First, a conserved tyrosine in the middle of the IDR is posttranslationally modified by O-sulfation; this modification increases both the affinity of GPIHBP1-LPL interactions and the ability of GPIHBP1 to protect LPL against ANGPTL4-catalyzed unfolding. Second, the acidic IDR of GPIHBP1 increases the probability of a GPIHBP1-LPL encounter via electrostatic steering, increasing the association rate constant (kon) for LPL binding by >250-fold. Third, we show that LPL accumulates near capillary endothelial cells even in the absence of GPIHBP1. In wild-type mice, we expect that the accumulation of LPL in close proximity to capillaries would increase interactions with GPIHBP1. Fourth, we found that GPIHBP1's IDR is not a key factor in the pathogenicity of chylomicronemia in patients with the GPIHBP1 autoimmune syndrome. Finally, based on biophysical studies, we propose that the negatively charged IDR of GPIHBP1 traverses a vast space, facilitating capture of LPL by capillary endothelial cells and simultaneously contributing to GPIHBP1's ability to preserve LPL structure and activity.


Assuntos
Células Endoteliais/metabolismo , Lipase Lipoproteica/metabolismo , Receptores de Lipoproteínas/metabolismo , Proteína 4 Semelhante a Angiopoietina/química , Proteína 4 Semelhante a Angiopoietina/genética , Proteína 4 Semelhante a Angiopoietina/metabolismo , Animais , Células Endoteliais/patologia , Humanos , Hiperlipoproteinemia Tipo I/genética , Hiperlipoproteinemia Tipo I/metabolismo , Hiperlipoproteinemia Tipo I/patologia , Lipase Lipoproteica/química , Lipase Lipoproteica/genética , Camundongos , Ligação Proteica , Domínios Proteicos , Receptores de Lipoproteínas/química , Receptores de Lipoproteínas/genética , Tirosina/química , Tirosina/genética , Tirosina/metabolismo
6.
Chem Asian J ; 13(22): 3491-3500, 2018 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-29901868

RESUMO

Diacylglycerol lipases (DAGL) are responsible for the biosynthesis of the endocannabinoid 2-arachidonoylglycerol. The fluorescent activity-based probes DH379 and HT-01 have been previously shown to label DAGLs and to cross-react with the serine hydrolase ABHD6. Here, we report the synthesis and characterization of two new quenched activity-based probes 1 and 2, the design of which was based on the structures of DH379 and HT-01, respectively. Probe 1 contains a BODIPY-FL and a 2,4-dinitroaniline moiety as a fluorophore-quencher pair, whereas probe 2 employs a Cy5-fluorophore and a cAB40-quencher. The fluorescence of both probes was quenched with relative quantum yields of 0.34 and 0.0081, respectively. The probes showed target inhibition as characterized in activity-based protein profiling assays using human cell- and mouse brain lysates, but were unfortunately not active in living cells, presumably due to limited cell permeability.


Assuntos
Desenho de Fármacos , Corantes Fluorescentes/síntese química , Compostos Heterocíclicos com 3 Anéis/síntese química , Lipase Lipoproteica/metabolismo , Monoacilglicerol Lipases/metabolismo , Triazóis/síntese química , Compostos de Anilina/química , Animais , Encéfalo/metabolismo , Catálise , Linhagem Celular Tumoral , Cobre/química , Reação de Cicloadição , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Compostos Heterocíclicos com 3 Anéis/química , Compostos Heterocíclicos com 3 Anéis/metabolismo , Humanos , Lipase Lipoproteica/química , Camundongos , Monoacilglicerol Lipases/química , Triazóis/química , Triazóis/metabolismo
7.
Sci Rep ; 8(1): 6752, 2018 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-29713054

RESUMO

Coronary artery disease is the most common cause of death globally and is linked to a number of risk factors including serum low density lipoprotein, high density lipoprotein, triglycerides and lipoprotein(a). Recently two proteins, angiopoietin-like protein 3 and 4, have emerged from genetic studies as being factors that significantly modulate plasma triglyceride levels and coronary artery disease. The exact function and mechanism of action of both proteins remains to be elucidated, however, mutations in these proteins results in up to 34% reduction in coronary artery disease and inhibition of function results in reduced plasma triglyceride levels. Here we report the crystal structures of the fibrinogen-like domains of both proteins. These structures offer new insights into the reported loss of function mutations, the mechanisms of action of the proteins and open up the possibility for the rational design of low molecular weight inhibitors for intervention in coronary artery disease.


Assuntos
Proteína 4 Semelhante a Angiopoietina/química , Proteínas Semelhantes a Angiopoietina/química , Doença da Artéria Coronariana/genética , Conformação Proteica , Proteína 4 Semelhante a Angiopoietina/genética , Proteínas Semelhantes a Angiopoietina/genética , Angiopoietinas/química , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/patologia , Cristalografia por Raios X , Humanos , Lipase Lipoproteica/química , Lipase Lipoproteica/genética , Mutação com Perda de Função/genética , Domínios Proteicos/genética , Fatores de Risco , Triglicerídeos/sangue
8.
Biochemistry ; 57(2): 241-254, 2018 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-29303250

RESUMO

Lipoprotein lipase (LPL) is a dimeric enzyme that is responsible for clearing triglyceride-rich lipoproteins from the blood. Although LPL plays a key role in cardiovascular health, an experimentally derived three-dimensional structure has not been determined. Such a structure would aid in understanding mutations in LPL that cause familial LPL deficiency in patients and help in the development of therapeutic strategies to target LPL. A major obstacle to structural studies of LPL is that LPL is an unstable protein that is difficult to produce in the quantities needed for nuclear magnetic resonance or crystallography. We present updated LPL structural models generated by combining disulfide mapping, computational modeling, and data derived from single-molecule Förster resonance energy transfer (smFRET). We pioneer the technique of smFRET for use with LPL by developing conditions for imaging active LPL and identifying positions in LPL for the attachment of fluorophores. Using this approach, we measure LPL-LPL intermolecular interactions to generate experimental constraints that inform new computational models of the LPL dimer structure. These models suggest that LPL may dimerize using an interface that is different from the dimerization interface suggested by crystal packing contacts seen in structures of pancreatic lipase.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Biotinilação , Biologia Computacional , Cisteína/química , Dimerização , Células HEK293 , Humanos , Lipase Lipoproteica/química , Lipase Lipoproteica/genética , Lipoproteínas/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Conformação Proteica , Proteínas Recombinantes/química , Triglicerídeos/metabolismo
9.
Biosens Bioelectron ; 102: 403-410, 2018 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-29175215

RESUMO

Single nucleotide polymorphism (SNP) in lipoprotein lipase (LPL) gene (rs1801177) is strongly associated with the increased progression of atherosclerosis, threatening global public health. In this work, a relatively simple, specific and ultrasensitive electrochemical DNA biosensor was constructed to detect rs1801177 for the first time. A glass carbon electrode was modified with fullerene (C60)/polyamidoamine (PAMAM)/gold (Au) nanoparticles nanocomposites film. In addition the nitrogen-doped graphene (N-G)/palladium platinum (PdPt) bimetallic nanoparticle/ polyaniline (PANI) nanohybrids were synthesised and used to label the signal probes. These nanohybrids have abundant active groups, and efficient redox and catalytic activity, allowing them to be used as the nanocarrier for a redox nanoprobe without the additional modification of electroactive substance and catalyst, which could effectively simplify the operation procedure and shorten the analysis time. With the catalysis of H2O2 by nanohybrids, the detection signal of N-G/PdPt/PANI itself could be significantly enhanced, lead to the improvement of the sensitivity. Under optimal conditions, the electrochemical DNA biosensor exhibited desirable performance for the determination of rs1801177 with a wide linearity ranging from 10 fM to 10nM and a relatively low detection limit of 3.33 fM (S/N=3). The proposed biosensor showed excellent selectivity to the target DNA compared to possible interfering substances. The results suggested that this method has potential applications in clinical research.


Assuntos
Técnicas Biossensoriais , DNA/genética , Lipase Lipoproteica/isolamento & purificação , Polimorfismo de Nucleotídeo Único/genética , Compostos de Anilina , Catálise , DNA/química , Dendrímeros/química , Técnicas Eletroquímicas , Fulerenos/química , Ouro/química , Humanos , Peróxido de Hidrogênio , Limite de Detecção , Lipase Lipoproteica/química , Lipase Lipoproteica/genética , Nanopartículas Metálicas/química
10.
Biomed Pharmacother ; 96: 1082-1093, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29174038

RESUMO

This study was undertaken to uncover the regulatory role of lycopene in targeting lipopolysaccharide (LPS) induced oxidative stress and inflammatory cascades and subsequent regulation of proprotein convertase subtilisin/kexin type-9 (PCSK-9) expression via sterol regulatory element binding protein-2 (SREBP-2) and hepatocyte nuclear factor-1α (HNF-1α). Further, protein-protein interaction (PPI) studies for Lycopene-Apo-CIII complex against lipoprotein lipase (LPL) were also performed to assess its regulatory role behind the enhanced circulatory TG/TRLs clearance. Lycopene treatment down-regulated hepatocyte PCSK-9 expression via down-regulation of HNF-1α, whereas, LDL-receptor (LDL-R) was up-regulated by subsequent up-regulation of SREBP-2. PPI studies showed that lycopene diminishes the affinity of Apo-CIII to complex with LPL (ΔG: -917.1 Kcal/mol) resulting in increased LPL functionality and TRLs clearance. Moreover, lycopene also ameliorated LPS stimulated oxidative-stress via enhanced total antioxidant and HDL associated PON-1 activity in addition to down-regulate the expression and plasma level of inflammatory mediators. Based on above findings, we concluded that lycopene exhibits dual role in targeting LPS induced oxidative stress and hypertriglyceridemia via down-regulation of PCSK-9, making greater no. of surface LDL-R available for LPS processing and clearance, as well as increased LPL activity through inhibition of Apo-CIII.


Assuntos
Apolipoproteína C-III/metabolismo , Carotenoides/uso terapêutico , Hipertrigliceridemia/tratamento farmacológico , Lipase Lipoproteica/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Pró-Proteína Convertase 9/biossíntese , Animais , Apolipoproteína C-III/antagonistas & inibidores , Apolipoproteína C-III/química , Carotenoides/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Regulação Enzimológica da Expressão Gênica , Hipertrigliceridemia/induzido quimicamente , Hipertrigliceridemia/metabolismo , Lipopolissacarídeos/toxicidade , Lipase Lipoproteica/antagonistas & inibidores , Lipase Lipoproteica/química , Licopeno , Masculino , Estresse Oxidativo/fisiologia , Pró-Proteína Convertase 9/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley
11.
Eur J Pharm Sci ; 108: 101-110, 2017 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-28108360

RESUMO

Archaea contain membrane lipids that differ from those found in the other domains of life (Eukarya and Bacteria). These lipids consist of isoprenoid chains attached via ether bonds to the glycerol carbons at the sn-2,3 positions. Two types of ether lipids are known, polar diether lipids and bipolar tetraether lipids. The inherent chemical stability and unique membrane-spanning characteristics of tetraether lipids render them interesting for oral drug delivery purposes. Archaeal lipids form liposomes spontaneously (archaeosomes) and may be incorporated in conventional liposomes (mixed vesicles). Both types of liposomes are promising to protect their drug cargo, such as therapeutic peptides, against the acidic environment of the stomach and proteolytic degradation in the intestine. They appear to withstand lipolytic enzymes and bile salts and may thus deliver orally administered therapeutic peptides to distant sections of the intestine or to the colon, where they may be absorbed, eventually by the help of absorption enhancers. Archaeal lipids and their semisynthetic derivatives may thus serve as biological source for the next generation oral drug delivery systems. The aim of this review is to present a systematic overview over existing literature on archaea carrying diether and tetraether lipids, lipid diversity, means of lipid extraction and purification, preparation and in vitro stability studies of archaeal lipid-based liposomal drug carriers and in vivo proof-of concepts studies.


Assuntos
Archaea/química , Lipídeos/química , Peptídeos/administração & dosagem , Peptídeos/química , Administração Oral , Animais , Química Farmacêutica , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Estabilidade de Medicamentos , Excipientes/química , Humanos , Lipase Lipoproteica/química , Lipossomos , Solubilidade , Comprimidos
12.
Drug Discov Today ; 22(2): 352-365, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27771332

RESUMO

Although statins and other pharmacological approaches have improved the management of lipid abnormalities, there exists a need for newer treatment modalities especially for the management of hypertriglyceridemia. Lipoprotein lipase (LPL), by promoting hydrolytic cleavage of the triglyceride core of lipoproteins, is a crucial node in the management of plasma lipid levels. Although LPL expression and activity modulation is observed as a pleiotropic action of some the commonly used lipid lowering drugs, the deliberate development of drugs targeting LPL has not occurred yet. In this review, we present the biology of LPL, highlight the LPL modulation property of currently used drugs and review the novel emerging approaches to target LPL.


Assuntos
Doenças Cardiovasculares/enzimologia , Lipase Lipoproteica/metabolismo , Doenças Metabólicas/enzimologia , Animais , Doenças Cardiovasculares/tratamento farmacológico , Humanos , Lipase Lipoproteica/química , Doenças Metabólicas/tratamento farmacológico
13.
Biotechnol Appl Biochem ; 64(4): 464-470, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27097985

RESUMO

The human lipoprotein lipase (LPL) is a therapeutic target for obesity, and inhibition of LPL with the approved small molecule agent orlistat has been widely used in clinic to treat obesity-related health problems such as diabetes and cardiovascular diseases. However, a variety of missense mutations in LPL protein have been observed, which may cause resistance or sensitization for orlistat, largely limiting the clinical applications of orlistat in obesity therapy. Here, we integrated molecular dynamics simulations and enzyme inhibition to investigate orlistat response to 16 disorder-associated missense mutations in LPL catalytic domain. It was found that most mutations have a modest effect on orlistat binding, and only few can exert strong impact to the binding. Three unfavorable (Trp86Arg, Ile194Thr, and Glu242Lys) and two favorable (His136Arg and Gly188Glu) mutations were identified, which can alter the binding affinity and inhibitory activity of orlistat considerably. Structural and energetic analysis revealed that these potent mutations induce orlistat resistance and sensitization by directly influencing the intermolecular interaction between LPL and orlistat or by indirectly addressing allosteric effect on LPL structure.


Assuntos
Lactonas/farmacologia , Lipase Lipoproteica/antagonistas & inibidores , Lipase Lipoproteica/química , Mutação de Sentido Incorreto/genética , Regulação Alostérica/efeitos dos fármacos , Humanos , Lactonas/química , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , Simulação de Dinâmica Molecular , Estrutura Molecular , Orlistate , Relação Estrutura-Atividade
14.
Biochemistry ; 56(3): 525-533, 2017 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-27984852

RESUMO

Lipoprotein lipase (LPL) is responsible for the hydrolysis of triglycerides from circulating lipoproteins. Whereas most identified mutations in the LPL gene are deleterious, one mutation, LPLS447X, causes a gain of function. This mutation truncates two amino acids from LPL's C-terminus. Carriers of LPLS447X have decreased VLDL levels and increased HDL levels, a cardioprotective phenotype. LPLS447X is used in Alipogene tiparvovec, the gene therapy product for individuals with familial LPL deficiency. It is unclear why LPLS447X results in a serum lipid profile more favorable than that of LPL. In vitro reports vary as to whether LPLS447X is more active than LPL. We report a comprehensive, biochemical comparison of purified LPLS447X and LPL dimers. We found no difference in specific activity on synthetic and natural substrates. We also did not observe a difference in the Ki for ANGPTL4 inhibition of LPLS447X relative to that of LPL. Finally, we analyzed LPL-mediated uptake of fluorescently labeled lipoprotein particles and found that LPLS447X enhanced lipoprotein uptake to a greater degree than LPL did. An LPL structural model suggests that the LPLS447X truncation exposes residues implicated in LPL binding to uptake receptors.


Assuntos
HDL-Colesterol/química , LDL-Colesterol/química , Lipase Lipoproteica/química , Mutação , Receptores de Lipoproteínas/química , Triglicerídeos/química , Proteína 4 Semelhante a Angiopoietina , Angiopoietinas/química , Angiopoietinas/genética , Angiopoietinas/metabolismo , Animais , Transporte Biológico , HDL-Colesterol/metabolismo , LDL-Colesterol/metabolismo , VLDL-Colesterol/química , VLDL-Colesterol/metabolismo , Expressão Gênica , Humanos , Hiperlipidemias/sangue , Hiperlipidemias/genética , Hiperlipidemias/patologia , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , Camundongos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Estrutura Secundária de Proteína , Receptores de Lipoproteínas/genética , Receptores de Lipoproteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/química , Serina/metabolismo , Especificidade por Substrato , Triglicerídeos/metabolismo
15.
Elife ; 52016 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-27929370

RESUMO

Lipoprotein lipase (LPL) undergoes spontaneous inactivation via global unfolding and this unfolding is prevented by GPIHBP1 (Mysling et al., 2016). We now show: (1) that ANGPTL4 inactivates LPL by catalyzing the unfolding of its hydrolase domain; (2) that binding to GPIHBP1 renders LPL largely refractory to this inhibition; and (3) that both the LU domain and the intrinsically disordered acidic domain of GPIHBP1 are required for this protective effect. Genetic studies have found that a common polymorphic variant in ANGPTL4 results in lower plasma triglyceride levels. We now report: (1) that this ANGPTL4 variant is less efficient in catalyzing the unfolding of LPL; and (2) that its Glu-to-Lys substitution destabilizes its N-terminal α-helix. Our work elucidates the molecular basis for regulation of LPL activity by ANGPTL4, highlights the physiological relevance of the inherent instability of LPL, and sheds light on the molecular defects in a clinically relevant variant of ANGPTL4.


Assuntos
Proteína 4 Semelhante a Angiopoietina/metabolismo , Lipase Lipoproteica/metabolismo , Dobramento de Proteína , Receptores de Lipoproteínas/metabolismo , Proteína 4 Semelhante a Angiopoietina/genética , Lipase Lipoproteica/química , Espectrometria de Massas , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Domínios Proteicos , Mapeamento de Interação de Proteínas
16.
Eur J Haematol ; 97(5): 409-415, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27504855

RESUMO

Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease characterized by the accumulation of a clonal population of B cells in peripheral blood, bone marrow, and lymphoid organs. More than 10 years ago, lipoprotein lipase (LPL) mRNA was identified as being strongly expressed in patients experiencing a more aggressive phenotype, while CLL patients with an indolent disease course lack expression of this marker. Since then, several reports confirmed the capability of LPL to predict CLL disease evolution at the moment of diagnosis. In contrast, data on the functional implications of LPL in CLL are scarce. LPL exerts a central role in overall lipid metabolism and transport, but plays additional, non-catalytic roles as well. Which of those is more important in the pathogenesis of CLL remains largely unclear. Here, we review the current knowledge on the prognostic and biological relevance of LPL in CLL.


Assuntos
Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/mortalidade , Lipase Lipoproteica/metabolismo , Comunicação Celular , Ativação Enzimática , Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Lipase Lipoproteica/química , Lipase Lipoproteica/genética , Prognóstico , Transdução de Sinais , Relação Estrutura-Atividade , Microambiente Tumoral/genética
17.
J Lipid Res ; 57(10): 1889-1898, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27494936

RESUMO

LPL contains two principal domains: an amino-terminal catalytic domain (residues 1-297) and a carboxyl-terminal domain (residues 298-448) that is important for binding lipids and binding glycosylphosphatidylinositol-anchored high density lipoprotein binding protein 1 (GPIHBP1) (an endothelial cell protein that shuttles LPL to the capillary lumen). The LPL sequences required for GPIHBP1 binding have not been examined in detail, but one study suggested that sequences near LPL's carboxyl terminus (residues ∼403-438) were crucial. Here, we tested the ability of LPL-specific monoclonal antibodies (mAbs) to block the binding of LPL to GPIHBP1. One antibody, 88B8, abolished LPL binding to GPIHBP1. Consistent with those results, antibody 88B8 could not bind to GPIHBP1-bound LPL on cultured cells. Antibody 88B8 bound poorly to LPL proteins with amino acid substitutions that interfered with GPIHBP1 binding (e.g., C418Y, E421K). However, the sequences near LPL's carboxyl terminus (residues ∼403-438) were not sufficient for 88B8 binding; upstream sequences (residues 298-400) were also required. Additional studies showed that these same sequences are required for LPL binding to GPIHBP1. In conclusion, we identified an LPL mAb that binds to LPL's GPIHBP1-binding domain. The binding of both antibody 88B8 and GPIHBP1 to LPL depends on large segments of LPL's carboxyl-terminal domain.


Assuntos
Anticorpos Monoclonais Murinos/química , Lipase Lipoproteica/química , Receptores de Lipoproteínas/química , Substituição de Aminoácidos , Animais , Linhagem Celular , Drosophila melanogaster , Humanos , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , Mutação de Sentido Incorreto , Ligação Proteica , Domínios Proteicos , Receptores de Lipoproteínas/genética , Receptores de Lipoproteínas/metabolismo
18.
J Bioinform Comput Biol ; 14(5): 1650028, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27427383

RESUMO

Lipoprotein lipase (LPL) is the rate-limiting enzyme for the hydrolysis of the triglyceride (TG) core of circulating TG-rich lipoproteins, chylomicrons, and very low-density lipoproteins. The enzyme has been established as an efficacious and safe therapeutic target for the management of obesity. Here, a systematic profile of the lipase inhibitor response of three anti-obesity agents (Orlistat, Lipstatin, and Cetilistat) to clinical LPL missense mutations arising from disease single nucleotide polymorphisms (SNPs) was established by integrating complex structure modeling, virtual mutagenesis, molecular dynamics (MD) simulations, binding energy analysis, and radiolabeled TG hydrolysis assays. The profile was then used to characterize the resistance and sensitivity of systematic mutation-inhibitor pairs. It is suggested that the Orlistat and Lipstatin have a similar response profile to the investigated mutations due to their homologous chemical structures, but exhibit a distinct profile to that of Cetilistat. Most mutations were predicted to have a modest or moderate effect on inhibitor binding; they are located far away from the enzyme active site and thus can only influence the binding limitedly. A number of mutations were found to sensitize or cause resistance for lipase inhibitors by directly interacting with the inhibitor ligands or by indirectly addressing allosteric effect on enzyme active site. Long-term MD simulations revealed a different noncovalent interaction network at the complex interfaces of Orlistat with wild-type LPL as well as its sensitized mutant H163R and resistant mutant I221T.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Lipase Lipoproteica/antagonistas & inibidores , Lipase Lipoproteica/genética , Polimorfismo de Nucleotídeo Único , Fármacos Antiobesidade/farmacologia , Predisposição Genética para Doença , Humanos , Lactonas/química , Lactonas/farmacologia , Lipase Lipoproteica/química , Modelos Moleculares , Mutação de Sentido Incorreto , Obesidade/tratamento farmacológico , Obesidade/genética , Conformação Proteica
19.
J Biochem ; 160(5): 269-279, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27198176

RESUMO

DDHD2 has been reported to exhibit phospholipase A1, triacylglycerol (TG) lipase and diacylglycerol (DG) lipase activities. However, the detailed enzymatic properties of DDHD2 have not yet been elucidated. In the current study, the substrate specificity of DDHD2 towards DG, TG and phosphatidic acid (PA) has been examined using highly purified recombinant rat DDHD2 (rDDHD2) with a liquid chromatography mass spectrometer. The k cat/Km value for DG (18:0/20:4) was much higher than those for TG (18:1/18:1/18:1), and PA (18:0/20:4) in the presence of sodium deoxycholate. The enzyme activity of rDDHD2 towards DG (18:0/20:4) was highest among all of the substrates tested. In addition, rDDHD2 was highly specific to DG substrates with a polyunsaturated fatty acid at their sn-2 position. The levels of 2-arachidonoylglycerol (2-AG) in CHO cells were quantified by gas chromatography-tandem mass spectrometry, showing that CHO cells expressing recombinant rDDHD2 contained higher levels of 2-AG when cells were treated with a monoacylglycerol lipase inhibitor, URB602. These results therefore support the idea that DDHD2 functions as a DG lipase in vivo and produces 2-AG.


Assuntos
Ácidos Araquidônicos/química , Ácidos Araquidônicos/metabolismo , Endocanabinoides/química , Endocanabinoides/metabolismo , Glicerídeos/química , Glicerídeos/metabolismo , Lipase Lipoproteica/química , Lipase Lipoproteica/metabolismo , Animais , Compostos de Bifenilo/farmacologia , Células CHO , Cricetinae , Cricetulus , Lipase Lipoproteica/antagonistas & inibidores , Lipase Lipoproteica/genética , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/fisiologia
20.
J Lipid Res ; 57(6): 1074-85, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27040450

RESUMO

Reduced white adipose tissue (WAT) LPL activity delays plasma clearance of TG-rich lipoproteins (TRLs). We reported the secretion of apoC-I, an LPL inhibitor, from WAT ex vivo in women. Therefore we hypothesized that WAT-secreted apoC-I associates with reduced WAT LPL activity and TRL clearance. WAT apoC-I secretion averaged 86.9 ± 31.4 pmol/g/4 h and 74.1 ± 36.6 pmol/g/4 h in 28 women and 11 men with BMI ≥27 kg/m(2), respectively, with no sex differences. Following the ingestion of a (13)C-triolein-labeled high-fat meal, subjects with high WAT apoC-I secretion (above median) had delayed postprandial plasma clearance of dietary TRLs, assessed from plasma (13)C-triolein-labeled TGs and apoB48. They also had reduced hydrolysis and storage of synthetic (3)H-triolein-labeled ((3)H)-TRLs in WAT ex vivo (i.e., in situ LPL activity). Adjusting for WAT in situ LPL activity eliminated group differences in chylomicron clearance; while adjusting for plasma apoC-I, (3)H-NEFA uptake by WAT, or body composition did not. apoC-I inhibited in situ LPL activity in adipocytes in both a concentration- and time-dependent manner. There was no change in postprandial WAT apoC-I secretion. WAT apoC-I secretion may inhibit WAT LPL activity and promote delayed chylomicron clearance in overweight and obese subjects. We propose that reducing WAT apoC-I secretion ameliorates postprandial TRL clearance in humans.


Assuntos
Tecido Adiposo Branco/enzimologia , Apolipoproteína C-I/sangue , Lipase Lipoproteica/sangue , Obesidade/sangue , Tecido Adiposo Branco/química , Idoso , Animais , Apolipoproteína B-48/química , Apolipoproteína B-48/metabolismo , Apolipoproteínas E/química , Apolipoproteínas E/metabolismo , Índice de Massa Corporal , Isótopos de Carbono/química , Quilomícrons/sangue , Dieta Hiperlipídica , Feminino , Humanos , Lipase Lipoproteica/química , Lipase Lipoproteica/genética , Lipoproteínas HDL/sangue , Masculino , Camundongos , Pessoa de Meia-Idade , Obesidade/genética , Obesidade/patologia , Período Pós-Prandial , Triglicerídeos/sangue , Trioleína/química , Trioleína/metabolismo
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