RESUMEN
Scavenger receptor class B type 1 (SR-B1) and CD36 are both members of the class B scavenger receptor family that play important roles in lipoprotein metabolism and atherosclerotic disease. SR-B1 is the primary receptor for high-density lipoproteins, while CD36 is the receptor responsible for the internalization of oxidized low-density lipoproteins. Despite their importance, class B scavenger receptor structure has only been studied by functional domain or peptide fragments-there are currently no reports of utilizing purified full-length protein. Here we report the successful expression and purification of full-length human SR-B1 and CD36 using an Spodoptera frugiperda insect cell system. We demonstrate that both SR-B1 and CD36 retained their normal functions in Spodoptera frugiperda cells, including lipoprotein binding, lipid transport, and the formation of higher order oligomers in the plasma membrane. Purification schemes for both scavenger receptors were optimized and their purity was confirmed by SDS-PAGE. Both purified scavenger receptors were assessed for stability by thermal shift assay and shown to maintain stable melting temperatures up to 6 weeks post-purification. Microscale thermophoresis was used to demonstrate that purified SR-B1 and CD36 were able to bind their native lipoprotein ligands. Further, there was no difference in affinity of SR-B1 for high-density lipoprotein or CD36 for oxidized low-density lipoprotein, when comparing glycosylated and deglycosylated receptors. These studies mark a significant step forward in creating physiologically relevant tools to study scavenger receptor function and lay the groundwork for future functional studies and determination of receptor structure.
RESUMEN
During reverse cholesterol transport, high-density lipoprotein (HDL) carries excess cholesterol from peripheral cells to the liver for excretion in bile. The first and last steps of this pathway involve the HDL receptor, scavenger receptor BI (SR-BI). While the mechanism of SR-BI-mediated cholesterol transport has not yet been established, it has long been suspected that cholesterol traverses through a hydrophobic tunnel in SR-BI's extracellular domain. Confirmation of a hydrophobic tunnel is hindered by the lack of a full-length SR-BI structure. Part of SR-BI's structure has been resolved, encompassing residues 405 to 475, which includes the C-terminal transmembrane domain and its adjacent extracellular region. Within the extracellular segment is an amphipathic helix (residues 427-436, referred to as AH(427-436)) that showed increased protection from solvent in NMR-based studies. Homology models predict that hydrophobic residues in AH(427-436) line a core cavity in SR-BI's extracellular region that may facilitate cholesterol transport. Therefore, we hypothesized that hydrophobic residues in AH(427-436) are required for HDL cholesterol transport. Here, we tested this hypothesis by mutating individual residues along AH(427-436) to a charged residue (aspartic acid), transiently transfecting COS-7 cells with plasmids encoding wild-type and mutant SR-BI, and performing functional analyses. We found that mutating hydrophobic, but not hydrophilic, residues in AH(427-436) impaired SR-BI bidirectional cholesterol transport. Mutating phenylalanine-430 was particularly detrimental to SR-BI's functions, suggesting that this residue may facilitate important interactions for cholesterol delivery within the hydrophobic tunnel. Our results support the hypothesis that a hydrophobic tunnel within SR-BI mediates cholesterol transport.
Asunto(s)
HDL-Colesterol , Lipoproteínas HDL , Receptores de Lipoproteína , Receptores Depuradores de Clase B , Ácido Aspártico/química , Ácido Aspártico/genética , Transporte Biológico , Antígenos CD36/química , HDL-Colesterol/química , HDL-Colesterol/metabolismo , Lipoproteínas HDL/química , Lipoproteínas HDL/genética , Fenilalanina/química , Fenilalanina/genética , Conformación Proteica en Hélice alfa , Receptores de Lipoproteína/química , Receptores de Lipoproteína/genética , Receptores Depuradores de Clase B/química , Receptores Depuradores de Clase B/genética , SolventesRESUMEN
PURPOSE OF REVIEW: The binding of high-density lipoprotein (HDL) to its primary receptor, scavenger receptor class B type 1 (SR-B1), is critical for lowering plasma cholesterol levels and reducing cardiovascular disease risk. This review provides novel insights into how the structural elements of SR-B1 drive efficient function with an emphasis on bidirectional cholesterol transport. RECENT FINDINGS: We have generated a new homology model of full-length human SR-B1 based on the recent resolution of the partial structures of other class B scavenger receptors. Interrogating this model against previously published observations allows us to generate structurally informed hypotheses about SR-B1's ability to mediate HDL-cholesterol (HDL-C) transport. Furthermore, we provide a structural perspective as to why human variants of SR-B1 may result in impaired HDL-C clearance. A comprehensive understanding of SR-B1's structure-function relationships is critical to the development of therapeutic agents targeting SR-B1 and modulating cardiovascular disease risk.
Asunto(s)
Enfermedades Cardiovasculares , HDL-Colesterol , Humanos , Lipoproteínas HDL/metabolismo , Receptores de Lipoproteína , Receptores Depuradores de Clase B/genética , Receptores Depuradores de Clase B/metabolismoRESUMEN
Objective: To investigate the role of adipocyte Pcpe2 (procollagen C-endopeptidase enhancer 2) in SR-BI (scavenger receptor class BI)-mediated HDL-C (high-density lipoprotein cholesterol) uptake and contributions to adipose lipid storage. Approach and Results: Pcpe2, a glycoprotein devoid of intrinsic proteolytic activity, is believed to participate in extracellular protein-protein interactions, supporting SR-BI- mediated HDL-C uptake. In published studies, Pcpe2 deficiency increased the development of atherosclerosis by reducing SR-BI-mediated HDL-C catabolism, but the biological impact of this deficiency on adipocyte SR-BI-mediated HDL-C uptake is unknown. Differentiated cells from Ldlr-/-/Pcpe2-/- (Pcpe2-/-) mouse adipose tissue showed elevated SR-BI protein levels, but significantly reduced HDL-C uptake compared to Ldlr-/- (control) adipose tissue. SR-BI-mediated HDL-C uptake was restored by preincubation of cells with exogenous Pcpe2. In diet-fed mice lacking Pcpe2, significant reductions in visceral, subcutaneous, and brown adipose tissue mass were observed, despite elevations in plasma triglyceride and cholesterol concentrations. Significant positive correlations exist between adipose mass and Pcpe2 expression in both mice and humans. Conclusions: Overall, these findings reveal a novel and unexpected function for Pcpe2 in modulating SR-BI expression and function as it relates to adipose tissue expansion and cholesterol balance in both mice and humans.
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Adipocitos/metabolismo , Aterosclerosis/metabolismo , HDL-Colesterol/metabolismo , Glicoproteínas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Microdominios de Membrana/metabolismo , Obesidad/metabolismo , Receptores Depuradores de Clase B/metabolismo , Grasa Subcutánea/metabolismo , Adipocitos/patología , Adipogénesis , Adiposidad , Adulto , Animales , Aterosclerosis/genética , Aterosclerosis/patología , Células CHO , Caveolina 1/metabolismo , Cricetulus , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Metabolismo Energético , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Glicoproteínas/genética , Humanos , Mediadores de Inflamación/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Microdominios de Membrana/genética , Microdominios de Membrana/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Obesidad/genética , Obesidad/patología , Receptores de LDL/genética , Receptores de LDL/metabolismo , Receptores Depuradores de Clase B/genética , Grasa Subcutánea/patologíaRESUMEN
HDL and its primary receptor, scavenger receptor class B type I (SR-BI), work together to promote the clearance of excess plasma cholesterol, thereby protecting against atherosclerosis. Human variants of SR-BI have been identified in patients with high HDL-cholesterol levels, and at least one variant has been linked to cardiovascular disease. Therefore, while often regarded as beneficial, very high levels of HDL-cholesterol may result from impaired cholesterol clearance through SR-BI and contribute to cardiovascular risk. In this study, we characterized the function of a rare human variant of SR-BI, resulting in the substitution of arginine-174 with cysteine (R174C), which was previously identified in a heterozygous individual with high levels of HDL-cholesterol. We hypothesized that the R174C-SR-BI variant has impaired cholesterol transport functions, which were assessed in COS-7 cells after transient transfection with full-length WT or R174C-SR-BI. Although R174C-SR-BI was expressed at levels comparable to the WT receptor, HDL binding, cholesteryl hexadecyl ether uptake, free cholesterol efflux, and modulation of membrane cholesterol were disrupted in the presence of R174C-SR-BI. We further examined the role of salt bridges as a potential mechanism for R174C-SR-BI dysfunction. If translatable, this human variant could lead to increased plasma HDL-cholesterol levels, impaired cholesterol clearance, and increased cardiovascular disease risk.
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Receptores Depuradores , AnimalesRESUMEN
RATIONALE: A hallmark of chronic inflammatory disorders is persistence of proinflammatory macrophages in diseased tissues. In atherosclerosis, this is associated with dyslipidemia and oxidative stress, but mechanisms linking these phenomena to macrophage activation remain incompletely understood. OBJECTIVE: To investigate mechanisms linking dyslipidemia, oxidative stress, and macrophage activation through modulation of immunometabolism and to explore therapeutic potential targeting specific metabolic pathways. METHODS AND RESULTS: Using a combination of biochemical, immunologic, and ex vivo cell metabolic studies, we report that CD36 mediates a mitochondrial metabolic switch from oxidative phosphorylation to superoxide production in response to its ligand, oxidized LDL (low-density lipoprotein). Mitochondrial-specific inhibition of superoxide inhibited oxidized LDL-induced NF-κB (nuclear factor-κB) activation and inflammatory cytokine generation. RNA sequencing, flow cytometry, 3H-labeled palmitic acid uptake, lipidomic analysis, confocal and electron microscopy imaging, and functional energetics revealed that oxidized LDL upregulated effectors of long-chain fatty acid uptake and mitochondrial import, while downregulating fatty acid oxidation and inhibiting ATP5A (ATP synthase F1 subunit alpha)-an electron transport chain component. The combined effect is long-chain fatty acid accumulation, alteration of mitochondrial structure and function, repurposing of the electron transport chain to superoxide production, and NF-κB activation. Apoe null mice challenged with high-fat diet showed similar metabolic changes in circulating Ly6C+ monocytes and peritoneal macrophages, along with increased CD36 expression. Moreover, mitochondrial reactive oxygen species were positively correlated with CD36 expression in aortic lesional macrophages. CONCLUSIONS: These findings reveal that oxidized LDL/CD36 signaling in macrophages links dysregulated fatty acid metabolism to oxidative stress from the mitochondria, which drives chronic inflammation. Thus, targeting to CD36 and its downstream effectors may serve as potential new strategies against chronic inflammatory diseases such as atherosclerosis.
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Antígenos CD36/deficiencia , Reprogramación Celular/fisiología , Macrófagos/metabolismo , Mitocondrias/metabolismo , Estrés Oxidativo/fisiología , Transducción de Señal/fisiología , Animales , Antígenos CD36/genética , Células Cultivadas , Femenino , Humanos , Inflamación/genética , Inflamación/metabolismo , Masculino , Metabolismo/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/genéticaRESUMEN
The HDL (high-density lipoprotein) Workshop was established in 2009 as a forum for candid discussions among academic basic scientists, clinical investigators, and industry researchers about the role of HDL in cardiovascular disease. This ninth HDL Workshop was held on May 16 to 17, 2019 in Boston, MA, and included outstanding oral presentations from established and emerging investigators. The Workshop featured 5 sessions with topics that tackled the role of HDL in the vasculature, its structural complexity, its role in health and disease states, and its interaction with the intestinal microbiome. The highlight of the program was awarding the Jack Oram Award to the distinguished professor emeritus G.S. Getz from the University of Chicago. The tenth HDL Workshop will be held on May 2020 in Chicago and will continue the focus on intellectually stimulating presentations by established and emerging investigators on novel roles of HDL in cardiovascular and noncardiovascular health and disease states.
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Investigación Biomédica/métodos , Vasos Sanguíneos/metabolismo , Cardiología , Enfermedades Cardiovasculares/metabolismo , HDL-Colesterol/metabolismo , Hipolipemiantes/uso terapéutico , Sociedades Médicas , Animales , Enfermedades Cardiovasculares/prevención & control , Congresos como Asunto , HumanosRESUMEN
High-density lipoproteins (HDLs) facilitate reverse cholesterol transport, a process in which HDL removes cholesterol from circulation and carries it to the liver for biliary excretion. Reverse cholesterol transport is also facilitated by HDL's high-affinity receptor, scavenger receptor-BI (SR-BI), by mechanisms that are not fully understood. To improve our understanding of SR-BI function, we previously solved the NMR (nuclear magnetic resonance) structure of a peptide encompassing amino acids 405-475 of SR-BI. This segment of SR-BI, that includes the functionally critical C-terminal transmembrane domain and part of the extracellular domain, also contains four conserved proline (Pro) residues. We hypothesized that these proline residues support SR-BI in a conformation that allows for efficient cholesterol transport. To test this, we generated individual Pro-to-alanine mutations in full-length SR-BI and transiently expressed the mutant receptors in COS-7 cells to measure the effects on SR-BI-mediated cholesterol transport functions. Our findings reveal that HDL cell association and uptake of HDL-cholesteryl esters are impaired by mutation of Pro-412, Pro-438, or the transmembrane proline kink residue (Pro-459). In addition, SR-BI-mediated cholesterol efflux and membrane cholesterol distribution are impaired by mutation of Pro-412 or Pro-438, indicating that these residues are essential for a fully functional SR-BI receptor. Furthermore, we demonstrate that Pro-408 is necessary for proper SR-BI expression, but mutation of Pro-408 does not cause SR-BI to become misfolded or rapidly degraded by the proteasome or the lysosome. We conclude that key proline residues play an important role in SR-BI function by allowing for the efficient transport of cholesterol between cells and HDL.
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Colesterol/química , Colesterol/metabolismo , Receptores Depuradores de Clase B/química , Receptores Depuradores de Clase B/metabolismo , Sustitución de Aminoácidos , Animales , Transporte Biológico Activo/fisiología , Células COS , Chlorocebus aethiops , Colesterol/genética , Humanos , Lipoproteínas HDL/química , Lipoproteínas HDL/genética , Lipoproteínas HDL/metabolismo , Mutación Missense , Resonancia Magnética Nuclear Biomolecular , Dominios Proteicos , Receptores Depuradores de Clase B/genéticaRESUMEN
Stress-inducible heat shock protein 70 (hsp70) interacts with superoxide dismutase 2 (SOD2) in the cytosol after synthesis to transfer the enzyme to the mitochondria for subsequent activation. However, the structural basis for this interaction remains to be defined. To map the SOD2-binding site in hsp70, mutants of hsp70 were made and tested for their ability to bind SOD2. These studies showed that SOD2 binds in the amino acid 393-537 region of the chaperone. To map the hsp70-binding site in SOD2, we used a series of pulldown assays and showed that hsp70 binds to the amino-terminal domain of SOD2. To better define the binding site, we used a series of decoy peptides derived from the primary amino acid sequence in the SOD2-binding site in hsp70. This study shows that SOD2 specifically binds to hsp70 at 445GERAMT450 Small peptides containing GERAMT inhibited the transfer of SOD2 to the mitochondria and decreased SOD2 activity in vitro and in vivo To determine the amino acid residues in hsp70 that are critical for SOD2 interactions, we substituted each amino acid residue for alanine or more conservative residues, glutamine or asparagine, in the GERAMT-binding site. Substitutions of E446A/Q and R447A/Q inhibited the ability of the GERAMT peptide to bind SOD2 and preserved SOD2 function more than other substitutions. Together, these findings indicate that the GERAMT sequence is critical for hsp70-mediated regulation of SOD2 and that Glu446 and Arg447 cooperate with other amino acid residues in the GERAMT-binding site for proper chaperone-dependent regulation of SOD2 antioxidant function.
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Arginina/metabolismo , Ácido Glutámico/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Superóxido Dismutasa/metabolismo , Sustitución de Aminoácidos , Animales , Sitios de Unión , Células Cultivadas , Proteínas HSP70 de Choque Térmico/química , Mitocondrias/metabolismo , Ratas , Ovinos , Superóxidos/metabolismoRESUMEN
High density lipoproteins (HDL) are anti-atherogenic particles, primarily due to their role in the reverse cholesterol transport pathway whereby HDL delivers cholesteryl esters (CE) to the liver for excretion upon interaction with its receptor, scavenger receptor BI (SR-BI). We designed experiments to test the hypothesis that one or more of the eight highly conserved tryptophan (Trp; W) residues in SR-BI are critical for mediating function. We created a series of Trp-to-phenylalanine (Phe, F) mutant receptors, as well as Trp-less SR-BI (ΔW-SR-BI), and assessed their ability to mediate cholesterol transport. Wild-type (WT) or mutant SR-BI receptors were transiently expressed in COS-7 cells, and cell surface expression was confirmed. Next, we showed that Trp-less- and W415F-SR-BI had significantly decreased abilities to bind HDL and promote selective uptake of HDL-CE, albeit with higher selective uptake efficiency as compared to WT-SR-BI. Interestingly, only Trp-less-, but not W415F-SR-BI, showed an impaired ability to mediate efflux of free cholesterol (FC). Furthermore, both W415F- and Trp-less-SR-BI were unable to reorganize plasma membrane pools of FC based on lack of sensitivity to exogenous cholesterol oxidase. Restoration of Trp 415 into the Trp-less-SR-BI background was unable to rescue Trp-less-SR-BI's impaired functions, suggesting that Trp 415 is critical, but not sufficient for full receptor function. Furthermore, with the exception of Trp 262, restoration of individual extracellular Trp residues, in combination with Trp 415, into the Trp-less-SR-BI background partially rescued SR-BI function, indicating that Trp 415 must be present in combination with other Trp residues for proper cholesterol transport functions.
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Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Receptores Depuradores de Clase B/metabolismo , Triptófano/metabolismo , Animales , Transporte Biológico , Células COS , Chlorocebus aethiops , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Multimerización de Proteína , Receptores Depuradores de Clase B/química , Receptores Depuradores de Clase B/genética , Triptófano/química , Triptófano/genéticaRESUMEN
Studies in human populations have shown a significant correlation between procollagen C-endopeptidase enhancer protein 2 (PCPE2) single nucleotide polymorphisms and plasma HDL cholesterol concentrations. PCPE2, a 52-kDa glycoprotein located in the extracellular matrix, enhances the cleavage of C-terminal procollagen by bone morphogenetic protein 1 (BMP1). Our studies here focused on investigating the basis for the elevated concentration of enlarged plasma HDL in PCPE2-deficient mice to determine whether they protected against diet-induced atherosclerosis. PCPE2-deficient mice were crossed with LDL receptor-deficient mice to obtain LDLr(-/-), PCPE2(-/-) mice, which had elevated HDL levels compared with LDLr(-/-) mice with similar LDL concentrations. We found that LDLr(-/-), PCPE2(-/-) mice had significantly more neutral lipid and CD68+ infiltration in the aortic root than LDLr(-/-) mice. Surprisingly, in light of their elevated HDL levels, the extent of aortic lipid deposition in LDLr(-/-), PCPE2(-/-) mice was similar to that reported for LDLr(-/-), apoA-I(-/-) mice, which lack any apoA-I/HDL. Furthermore, LDLr(-/-), PCPE2(-/-) mice had reduced HDL apoA-I fractional clearance and macrophage to fecal reverse cholesterol transport rates compared with LDLr(-/-) mice, despite a 2-fold increase in liver SR-BI expression. PCPE2 was shown to enhance SR-BI function by increasing the rate of HDL-associated cholesteryl ester uptake, possibly by optimizing SR-BI localization and/or conformation. We conclude that PCPE2 is atheroprotective and an important component of the reverse cholesterol transport HDL system.
Asunto(s)
Aterosclerosis/metabolismo , Aterosclerosis/prevención & control , Ésteres del Colesterol/metabolismo , Glicoproteínas/metabolismo , Lipoproteínas HDL/metabolismo , Receptores Depuradores de Clase B/metabolismo , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos de Diferenciación Mielomonocítica/metabolismo , Aorta/metabolismo , Aorta/patología , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Aterosclerosis/inducido químicamente , Aterosclerosis/genética , Aterosclerosis/patología , Transporte Biológico Activo/genética , Células CHO , Ésteres del Colesterol/genética , Cricetulus , Glicoproteínas/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Lipoproteínas HDL/genética , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Ratones Noqueados , Receptores de LDL/genética , Receptores de LDL/metabolismo , Receptores Depuradores de Clase B/genéticaRESUMEN
OBJECTIVE: Oxidative stress associated with cardiovascular disease can produce various oxidized lipids, including cholesterol oxides, such as 7-hydroperoxide (7-OOH), 7-hydroxide (7-OH), and 7-ketone (7=O). Unlike 7=O and 7-OH, 7-OOH is redox active, giving rise to the others via potentially toxic-free radical reactions. We tested the novel hypothesis that under oxidative stress conditions, steroidogenic acute regulatory (StAR) family proteins not only deliver cholesterol to/into mitochondria of vascular macrophages, but also 7-OOH, which induces peroxidative damage that impairs early stage reverse cholesterol transport. APPROACH AND RESULTS: Stimulation of human monocyte-derived THP-1 macrophages with dibutyryl-cAMP resulted in substantial upregulation of StarD1 and ATP-binding cassette (ABC) transporter, ABCA1. Small interfering RNA-induced StarD1 knockdown before stimulation had no effect on StarD4, but reduced ABCA1 upregulation, linking the latter to StarD1 functionality. Mitochondria in stimulated StarD1-knockdown cells internalized 7-OOH slower than nonstimulated controls and underwent less 7-OOH-induced lipid peroxidation and membrane depolarization, as probed with C11-BODIPY (4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-inda-cene-3-undecanoic acid) and JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine iodide), respectively. Major functional consequences of 7-OOH exposure were (1) loss of mitochondrial CYP27A1 activity, (2) reduced 27-hydroxycholesterol (27-OH) output, and (3) downregulation of cholesterol-exporting ABCA1 and ABCG1. Consistently, 7-OOH-challenged macrophages exported less cholesterol to apoA-I or high-density lipoprotein than did nonchallenged controls. StarD1-mediated 7-OOH transport was also found to be highly cytotoxic, whereas 7=O and 7-OH were minimally toxic. CONCLUSIONS: This study describes a previously unrecognized mechanism by which macrophage cholesterol efflux can be incapacitated under oxidative stress-linked disorders, such as chronic obesity and hypertension. Our findings provide new insights into the role of macrophage redox damage/dysfunction in atherogenesis.
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Aterosclerosis/metabolismo , Colesterol/análogos & derivados , Peroxidación de Lípido/fisiología , Macrófagos/metabolismo , Estrés Oxidativo/fisiología , Transporte Biológico , Células Cultivadas , Colesterol/metabolismo , CMP Cíclico/análogos & derivados , CMP Cíclico/farmacología , Humanos , Macrófagos/citología , Mitocondrias/metabolismo , Transporte de Proteínas , Sensibilidad y EspecificidadRESUMEN
Scavenger receptor class B type I (SR-BI), the high density lipoprotein (HDL) receptor, is important for the delivery of HDL-cholesteryl esters to the liver for excretion via bile formation. The focus on therapeutic strategies aimed at reducing cholesterol levels highlights the critical need to understand the structural features of SR-BI that drive cholesterol removal. Yet, in the absence of a high-resolution structure of SR-BI, our understanding of how SR-BI interacts with HDL is limited. In this study, we have optimized the NMR solution conditions for the structural analysis of the C-terminal transmembrane domain of SR-BI that harbors putative domains required for receptor oligomerization. An isotopically-labeled SR-BI peptide encompassing residues 405-475 was bacterially-expressed and purified. [U-(15)N]-SR-BI(405-475) was incorporated into different detergent micelles and assessed by (1)H-(15)N-HSQC in order to determine which detergent micelle best maintained SR-BI(405-475) in a folded, native conformation for subsequent NMR analyses. We also determined the optimal detergent concentration used in micelles, as well as temperature, solution buffer and pH conditions. Based on (1)H-(15)N-HSQC peak dispersion, intensity, and uniformity, we determined that [U-(15)N]-SR-BI(405-475) should be incorporated into 5% detergent micelles consisting of 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-[1'-rac-glycerol] (LPPG) and data collected at 40°C in a non-buffered solution at pH 6.8. Furthermore, we demonstrate the ability of SR-BI(405-475) to form dimers upon chemical crosslinking. These studies represent the first steps in obtaining high-resolution structural information by NMR for the HDL receptor that plays a critical role in regulating whole body cholesterol removal.
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Receptores Depuradores de Clase B/química , Receptores Depuradores de Clase B/aislamiento & purificación , Animales , Detergentes/química , Dimerización , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Espectroscopía de Resonancia Magnética , Ratones , Micelas , Estructura Terciaria de Proteína , Receptores Depuradores de Clase B/genética , Receptores Depuradores de Clase B/metabolismoRESUMEN
High-density lipoproteins (HDLs) are athero-protective, primarily because of their ability to promote cholesterol flux from peripheral tissues to the liver by reverse cholesterol transport (RCT). The delivery of HDL-cholesteryl esters (CE) into cells is mediated by the HDL receptor, scavenger receptor class B type I (SR-BI), a promising target for enhancing whole body cholesterol disposal and preventing cardiovascular disease. A detailed understanding of the structural determinants underlying proper SR-BI/HDL alignment that supports the selective uptake of HDL-CE into cells remains lacking. To this end, we exploited CD36, a class B scavenger receptor with a predicted topology similar to that of SR-BI that binds HDL but is unable to mediate efficient selective uptake of HDL-CE. We generated a series of SR-BI/CD36 chimeric receptors that span the extracellular (EC) domain of SR-BI to delineate regions that are essential for SR-BI's cholesterol transport functions. All 16 SR-BI/CD36 chimeras were transiently expressed in COS-7 cells, and their plasma membrane localization was confirmed. The majority of SR-BI/CD36 chimeric receptors displayed significant reductions in their ability to (i) bind HDL, (ii) deliver HDL-CE to cells, (iii) mediate efflux of free cholesterol (FC) to HDL, and (iv) redistribute plasma membrane domains of FC. We also demonstrated that changes in SR-BI function were independent of receptor oligomerization. Altogether, we have identified discrete subdomains, particularly in the N-terminal and C-terminal regions of the EC domain of SR-BI, that are critical for productive receptor-ligand interactions and the various cholesterol transport functions of SR-BI.
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Antígenos CD36/metabolismo , Colesterol/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Transporte Biológico/genética , Antígenos CD36/química , Antígenos CD36/genética , Células COS , Chlorocebus aethiops , Colesterol/química , Electroforesis en Gel de Poliacrilamida , Lipoproteínas HDL/química , Lipoproteínas HDL/metabolismo , Ratones , Datos de Secuencia Molecular , Multimerización de Proteína , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Homología de Secuencia de AminoácidoRESUMEN
Obesity is a worldwide epidemic and places individuals at a higher risk for developing comorbidities that include cardiovascular disease and type 2 diabetes. Adipose tissue contains adipocytes that are responsible for lipid metabolism and reducing misdirected lipid storage. Adipocytes facilitate this process through insulin-mediated uptake of glucose and its subsequent metabolism into triglycerides for storage. During obesity, adipocytes become insulin resistant and have a reduced ability to mediate glucose import, thus resulting in whole-body metabolic dysfunction. Scavenger receptor class B type I (SR-BI) has been implicated in glucose uptake in skeletal muscle and adipocytes via its native ligands, apolipoprotein A-1 and high-density lipoproteins. Further, SR-BI translocation to the cell surface in adipocytes is sensitive to insulin stimulation. Using adipocytes differentiated from ear mesenchymal stem cells isolated from wild-type and SR-BI knockout (SR-BI -/- ) mice as our model system, we tested the hypothesis that SR-BI is required for insulin-mediated glucose uptake and regulation of energy balance in adipocytes. We demonstrated that loss of SR-BI in adipocytes resulted in inefficient glucose uptake regardless of cell surface expression levels of glucose transporter 4 compared to WT adipocytes. We also observed reduced glycolytic capacity, increased lipid biosynthesis, and dysregulated expression of lipid metabolism genes in SR-BI -/- adipocytes compared to WT adipocytes. These results partially support our hypothesis and suggest a novel role for SR-BI in glucose uptake and metabolic homeostasis in adipocytes.
RESUMEN
Introduction: Adipose tissue constantly secretes adipokines and extracellular vesicles including exosomes to crosstalk with distinct tissues and organs for whole-body homeostasis. However, dysfunctional adipose tissue under chronic inflammatory conditions such as obesity, atherosclerosis, and diabetes shows pro-inflammatory phenotypes accompanied by oxidative stress and abnormal secretion. Nevertheless, molecular mechanisms of how adipocytes are stimulated to secrete exosomes under those conditions remain poorly understood. Methods: Mouse and human in vitro cell culture models were used for performing various cellular and molecular studies on adipocytes and macrophages. Statistical analysis was performed using Student's t-test (two-tailed, unpaired, and equal variance) for comparisons between two groups or ANOVA followed by Bonferroni's multiple comparison test for comparison among more than two groups. Results and discussion: In this work, we report that CD36, a scavenger receptor for oxidized LDL, formed a signaling complex with another membrane signal transducer Na/K-ATPase in adipocytes. The atherogenic oxidized LDL induced a pro-inflammatory response in in vitro differentiated mouse and human adipocytes and also stimulated the cells to secrete more exosomes. This was largely blocked by either CD36 knockdown using siRNA or pNaKtide, a peptide inhibitor of Na/K-ATPase signaling. These results showed a critical role of the CD36/Na/K-ATPase signaling complex in oxidized LDL-induced adipocyte exosome secretion. Moreover, by co-incubation of adipocyte-derived exosomes with macrophages, we demonstrated that oxidized LDL-induced adipocyte-derived exosomes promoted pro-atherogenic phenotypes in macrophages, including CD36 upregulation, IL-6 secretion, metabolic switch to glycolysis, and mitochondrial ROS production. Altogether, we show here a novel mechanism through which adipocytes increase exosome secretion in response to oxidized LDL and that the secreted exosomes can crosstalk with macrophages, which may contribute to atherogenesis.
RESUMEN
Apolipoprotein B (apoB)-lipoproteins initiate and promote atherosclerotic cardiovascular disease. Plasma tissue plasminogen activator (tPA) activity is negatively associated with atherogenic apoB-lipoprotein cholesterol levels in humans, but the mechanisms are unknown. We found that tPA, partially through the lysine-binding site on its Kringle 2 domain, binds to the N terminus of apoB, blocking the interaction between apoB and microsomal triglyceride transfer protein (MTP) in hepatocytes, thereby reducing very-low-density lipoprotein (VLDL) assembly and plasma apoB-lipoprotein cholesterol levels. Plasminogen activator inhibitor 1 (PAI-1) sequesters tPA away from apoB and increases VLDL assembly. Humans with PAI-1 deficiency have smaller VLDL particles and lower plasma levels of apoB-lipoprotein cholesterol. These results suggest a mechanism that fine-tunes VLDL assembly by intracellular interactions among tPA, PAI-1, and apoB in hepatocytes.
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Apolipoproteínas B , Aterosclerosis , Hepatocitos , Lipoproteínas VLDL , Inhibidor 1 de Activador Plasminogénico , Activador de Tejido Plasminógeno , Humanos , Apolipoproteínas B/sangre , Aterosclerosis/sangre , Aterosclerosis/metabolismo , Hepatocitos/metabolismo , Lipoproteínas VLDL/metabolismo , Inhibidor 1 de Activador Plasminogénico/sangre , Inhibidor 1 de Activador Plasminogénico/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Animales , Ratones , Ratones Endogámicos C57BLRESUMEN
Na/K-ATPase (NKA), besides its ion transporter function, is a signal transducer by regulating Src family kinases (SFK). The signaling NKA contributes to oxidized LDL-induced macrophage foam cell formation and interacts with TLR4. However, its role in lipopolysaccharides (LPS)-induced signaling and glycolytic switch in macrophages remains unclear. Using peritoneal macrophages from NKA α1 haploinsufficient mice (NKA α1+/-), we found that NKA α1 haploinsufficiency led to enhanced LPS-stimulated NF-κB pathway, ROS signaling, and pro-inflammatory cytokines. Intraperitoneal injection of LPS resulted in more severe lung inflammation and injury with lower survival rate in NKA α1+/- mice. Additionally, LPS induced a higher extent of the metabolic switch from oxidative phosphorylation to glycolysis. Mechanistically, NKA α1 interacted with TLR4 and Lyn. The presence of NKA α1 in this complex attenuated Lyn activation by LPS, which subsequently restricted the downstream ROS and NF-κB signaling. In conclusion, we demonstrated that NKA α1 suppresses LPS-induced macrophage pro-inflammatory signaling through Lyn.
RESUMEN
Scavenger receptor class B type I (SR-BI) binds high-density lipoprotein (HDL) and mediates the selective uptake of cholesteryl esters (CE). Although the extracellular domain of SR-BI is critical for function, the structural characteristics of this region remain elusive. Using sulfhydryl labeling strategies, we report the novel finding that all six cysteine (Cys) residues in the extracellular domain of SR-BI are involved in disulfide bond formation that is intramolecular by nature. We hypothesized that an SR-BI conformation stabilized by extracellular disulfide bonds is a prerequisite for SR-BI-mediated cholesterol transport. Thus, single-Cys mutant SR-BI receptors (C251S-, C280S-, C321S-, C323S-, C334S-, and C384S-SR-BI), as well as Cys-less SR-BI, a mutant SR-BI receptor void of all Cys residues, were created, and plasma membrane localization was confirmed. Functional assays revealed that C280S-, C321S-, C323S-, and C334S-SR-BI and Cys-less SR-BI mutant receptors displayed weakened HDL binding and subsequent selective uptake of HDL-CE. However, only C323S-SR-BI and Cys-less SR-BI were unable to mediate wild-type levels of efflux of free cholesterol (FC) to HDL. None of the Cys mutations disrupted SR-BI's ability to redistribute plasma membrane FC. Taken together, the intramolecular disulfide bonds in the extracellular domain of SR-BI appear to maintain the receptor in a conformation integral to its cholesterol transport functions.
Asunto(s)
Antígenos CD36/química , HDL-Colesterol/metabolismo , Disulfuros/química , Líquido Extracelular/química , Animales , Transporte Biológico , Antígenos CD36/genética , Antígenos CD36/fisiología , Células COS , Chlorocebus aethiops , Colesterol/análogos & derivados , Colesterol/química , Colesterol/metabolismo , HDL-Colesterol/química , Disulfuros/metabolismo , Endocitosis , Líquido Extracelular/metabolismo , Hidrólisis , Ratones , Unión Proteica , Conformación ProteicaRESUMEN
Free fatty acids (FFAs) are implicated in the pathogenesis of metabolic diseases that includes obesity, type 2 diabetes mellitus, and cardiovascular disease (CVD). FFAs serve as ligands for free fatty acid receptors (FFARs) that belong to the family of rhodopsin-like G protein-coupled receptors (GPCRs) and are expressed throughout the body to maintain energy homeostasis under changing nutritional conditions. Free fatty acid receptor 4 (FFAR4), also known as G protein-coupled receptor 120, is a long-chain fatty acid receptor highly expressed in adipocytes, endothelial cells, and macrophages. Activation of FFAR4 helps maintain metabolic homeostasis by regulating adipogenesis, insulin sensitivity, and inflammation. Furthermore, dysfunction of FFAR4 is associated with insulin resistance, obesity, and eccentric remodeling in both humans and mice, making FFAR4 an attractive therapeutic target for treating or preventing metabolic diseases. While much of the previous literature on FFAR4 has focused on its role in obesity and diabetes, recent studies have demonstrated that FFAR4 may also play an important role in the development of atherosclerosis and CVD. Most notably, FFAR4 activation reduces monocyte-endothelial cell interaction, enhances cholesterol efflux from macrophages, reduces lesion size in atherogenic mouse models, and stimulates oxylipin production in myocytes that functions in a feed-forward cardioprotective mechanism. This review will focus on the role of FFAR4 in metabolic diseases and highlights an underappreciated role of FFAR4 in the development of atherosclerosis and CVD.