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1.
Cell ; 175(2): 514-529.e20, 2018 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-30220461

RESUMO

The mechanisms underlying sterol transport in mammalian cells are poorly understood. In particular, how cholesterol internalized from HDL is made available to the cell for storage or modification is unknown. Here, we describe three ER-resident proteins (Aster-A, -B, -C) that bind cholesterol and facilitate its removal from the plasma membrane. The crystal structure of the central domain of Aster-A broadly resembles the sterol-binding fold of mammalian StARD proteins, but sequence differences in the Aster pocket result in a distinct mode of ligand binding. The Aster N-terminal GRAM domain binds phosphatidylserine and mediates Aster recruitment to plasma membrane-ER contact sites in response to cholesterol accumulation in the plasma membrane. Mice lacking Aster-B are deficient in adrenal cholesterol ester storage and steroidogenesis because of an inability to transport cholesterol from SR-BI to the ER. These findings identify a nonvesicular pathway for plasma membrane to ER sterol trafficking in mammals.


Assuntos
HDL-Colesterol/metabolismo , Proteínas de Membrana/fisiologia , Proteínas de Membrana/ultraestrutura , Células 3T3 , Animais , Transporte Biológico/fisiologia , Antígenos CD36/metabolismo , Células CHO , Proteínas de Transporte/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Colesterol/metabolismo , Cricetulus , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/fisiologia , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Membranas Mitocondriais/metabolismo , Alinhamento de Sequência , Esteróis/metabolismo
2.
Nat Immunol ; 21(7): 746-755, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32514064

RESUMO

Plasma membranes of animal cells are enriched for cholesterol. Cholesterol-dependent cytolysins (CDCs) are pore-forming toxins secreted by bacteria that target membrane cholesterol for their effector function. Phagocytes are essential for clearance of CDC-producing bacteria; however, the mechanisms by which these cells evade the deleterious effects of CDCs are largely unknown. Here, we report that interferon (IFN) signals convey resistance to CDC-induced pores on macrophages and neutrophils. We traced IFN-mediated resistance to CDCs to the rapid modulation of a specific pool of cholesterol in the plasma membrane of macrophages without changes to total cholesterol levels. Resistance to CDC-induced pore formation requires the production of the oxysterol 25-hydroxycholesterol (25HC), inhibition of cholesterol synthesis and redistribution of cholesterol to an esterified cholesterol pool. Accordingly, blocking the ability of IFN to reprogram cholesterol metabolism abrogates cellular protection and renders mice more susceptible to CDC-induced tissue damage. These studies illuminate targeted regulation of membrane cholesterol content as a host defense strategy.


Assuntos
Infecções Bacterianas/imunologia , Toxinas Bacterianas/imunologia , Hidroxicolesteróis/metabolismo , Interferons/isolamento & purificação , Fagócitos/imunologia , Estreptolisinas/imunologia , Animais , Bactérias/imunologia , Bactérias/metabolismo , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/imunologia , Células Cultivadas , Modelos Animais de Doenças , Suscetibilidade a Doenças/imunologia , Feminino , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Microscopia Intravital , Masculino , Camundongos , Camundongos Transgênicos , Fagócitos/citologia , Fagócitos/metabolismo , Cultura Primária de Células , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/metabolismo , Estreptolisinas/administração & dosagem , Estreptolisinas/metabolismo
3.
Nucleic Acids Res ; 49(1): 1-14, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33275144

RESUMO

Nucleic acid therapeutics (NATs) have proven useful in promoting the degradation of specific transcripts, modifying gene expression, and regulating mRNA splicing. In each situation, efficient delivery of nucleic acids to cells, tissues and intracellular compartments is crucial-both for optimizing efficacy and reducing side effects. Despite successes in NATs, our understanding of their cellular uptake and distribution in tissues is limited. Current methods have yielded insights into distribution of NATs within cells and tissues, but the sensitivity and resolution of these approaches are limited. Here, we show that nanoscale secondary ion mass spectrometry (NanoSIMS) imaging can be used to define the distribution of 5-bromo-2'-deoxythymidine (5-BrdT) modified antisense oligonucleotides (ASO) in cells and tissues with high sensitivity and spatial resolution. This approach makes it possible to define ASO uptake and distribution in different subcellular compartments and to quantify the impact of targeting ligands designed to promote ASO uptake by cells. Our studies showed that phosphorothioate ASOs are associated with filopodia and the inner nuclear membrane in cultured cells, and also revealed substantial cellular and subcellular heterogeneity of ASO uptake in mouse tissues. NanoSIMS imaging represents a significant advance in visualizing uptake and distribution of NATs; this approach will be useful in optimizing efficacy and delivery of NATs for treating human disease.


Assuntos
Oligonucleotídeos Antissenso/análise , Oligonucleotídeos Fosforotioatos/análise , Espectrometria de Massa de Íon Secundário/métodos , Células 3T3-L1 , Acetilgalactosamina/administração & dosagem , Acetilgalactosamina/análise , Animais , Receptor de Asialoglicoproteína/análise , Césio , Células HEK293 , Células HeLa , Humanos , Rim/química , Rim/ultraestrutura , Fígado/química , Fígado/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Miocárdio/química , Miocárdio/ultraestrutura , Oligonucleotídeos Antissenso/farmacocinética , Oligonucleotídeos Fosforotioatos/farmacocinética , Pseudópodes/química , Pseudópodes/ultraestrutura , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/genética , Frações Subcelulares/química , Enxofre/análise , Isótopos de Enxofre/análise , Distribuição Tecidual
4.
Proc Natl Acad Sci U S A ; 117(19): 10476-10483, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32354992

RESUMO

Cholesterol-laden macrophage foam cells are a hallmark of atherosclerosis. For that reason, cholesterol metabolism in macrophages has attracted considerable scrutiny, particularly the mechanisms by which macrophages unload surplus cholesterol (a process referred to as "cholesterol efflux"). Many studies of cholesterol efflux in macrophages have focused on the role of ABC transporters in moving cholesterol onto high-density lipoproteins (HDLs), but other mechanisms for cholesterol efflux likely exist. We hypothesized that macrophages have the capacity to unload cholesterol directly onto adjacent cells. To test this hypothesis, we used methyl-ß-cyclodextrin (MßCD) to load mouse peritoneal macrophages with [13C]cholesterol. We then plated the macrophages (in the absence of serum or HDL) onto smooth muscle cells (SMCs) that had been metabolically labeled with [15N]choline. After incubating the cells overnight in the absence of HDL or serum, we visualized 13C and 15N distribution by nanoscale secondary ion mass spectrometry (NanoSIMS). We observed substantial 13C enrichment in SMCs that were adjacent to [13C]cholesterol-loaded macrophages-including in cytosolic lipid droplets of SMCs. In follow-up studies, we depleted "accessible cholesterol" from the plasma membrane of [13C]cholesterol-loaded macrophages with MßCD before plating the macrophages onto the SMCs. After an overnight incubation, we again observed substantial 13C enrichment in the SMCs adjacent to macrophages. Thus, macrophages transfer cholesterol to adjacent cells in the absence of serum or HDL. We suspect that macrophages within tissues transfer cholesterol to adjacent cells, thereby contributing to the ability to unload surplus cholesterol.


Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Colesterol/metabolismo , Macrófagos/metabolismo , Transportador 1 de Cassete de Ligação de ATP/fisiologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Aterosclerose/metabolismo , Aterosclerose/fisiopatologia , Transporte Biológico , Células Espumosas/metabolismo , Metabolismo dos Lipídeos , Lipoproteínas HDL/metabolismo , Macrófagos/fisiologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Soro/metabolismo , beta-Ciclodextrinas/metabolismo
5.
Proc Natl Acad Sci U S A ; 117(27): 15827-15836, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571911

RESUMO

Bromine and peroxidasin (an extracellular peroxidase) are essential for generating sulfilimine cross-links between a methionine and a hydroxylysine within collagen IV, a basement membrane protein. The sulfilimine cross-links increase the structural integrity of basement membranes. The formation of sulfilimine cross-links depends on the ability of peroxidasin to use bromide and hydrogen peroxide substrates to produce hypobromous acid (HOBr). Once a sulfilimine cross-link is created, bromide is released into the extracellular space and becomes available for reutilization. Whether the HOBr generated by peroxidasin is used very selectively for creating sulfilimine cross-links or whether it also causes oxidative damage to bystander molecules (e.g., generating bromotyrosine residues in basement membrane proteins) is unclear. To examine this issue, we used nanoscale secondary ion mass spectrometry (NanoSIMS) imaging to define the distribution of bromine in mammalian tissues. We observed striking enrichment of bromine (79Br, 81Br) in basement membranes of normal human and mouse kidneys. In peroxidasin knockout mice, bromine enrichment of basement membranes of kidneys was reduced by ∼85%. Proteomic studies revealed bromination of tyrosine-1485 in the NC1 domain of α2 collagen IV from kidneys of wild-type mice; the same tyrosine was brominated in collagen IV from human kidney. Bromination of tyrosine-1485 was reduced by >90% in kidneys of peroxidasin knockout mice. Thus, in addition to promoting sulfilimine cross-links in collagen IV, peroxidasin can also brominate a bystander tyrosine. Also, the fact that bromine enrichment is largely confined to basement membranes implies that peroxidasin activity is largely restricted to basement membranes in mammalian tissues.


Assuntos
Membrana Basal/metabolismo , Bromo/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Peroxidase/metabolismo , Animais , Biópsia , Bromatos/metabolismo , Brometos , Células Cultivadas , Colágeno Tipo IV/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Iminas/metabolismo , Rim/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteômica , Peroxidasina
6.
Proc Natl Acad Sci U S A ; 115(36): E8499-E8508, 2018 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-30127022

RESUMO

Macrophages are generally assumed to unload surplus cholesterol through direct interactions between ABC transporters on the plasma membrane and HDLs, but they have also been reported to release cholesterol-containing particles. How macrophage-derived particles are formed and released has not been clear. To understand the genesis of macrophage-derived particles, we imaged mouse macrophages by EM and nanoscale secondary ion mass spectrometry (nanoSIMS). By scanning EM, we found that large numbers of 20- to 120-nm particles are released from the fingerlike projections (filopodia) of macrophages. These particles attach to the substrate, forming a "lawn" of particles surrounding macrophages. By nanoSIMS imaging we showed that these particles are enriched in the mobile and metabolically active accessible pool of cholesterol (detectable by ALO-D4, a modified version of a cholesterol-binding cytolysin). The cholesterol content of macrophage-derived particles was increased by loading the cells with cholesterol or by adding LXR and RXR agonists to the cell-culture medium. Incubating macrophages with HDL reduced the cholesterol content of macrophage-derived particles. We propose that release of accessible cholesterol-rich particles from the macrophage plasma membrane could assist in disposing of surplus cholesterol and increase the efficiency of cholesterol movement to HDL.


Assuntos
Micropartículas Derivadas de Células/metabolismo , Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Macrófagos/metabolismo , Animais , Micropartículas Derivadas de Células/ultraestrutura , Lipoproteínas HDL/ultraestrutura , Macrófagos/ultraestrutura , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Células RAW 264.7 , Espectrometria de Massa de Íon Secundário
7.
J Lipid Res ; 61(10): 1347-1359, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32690595

RESUMO

For three decades, the LPL-specific monoclonal antibody 5D2 has been used to investigate LPL structure/function and intravascular lipolysis. 5D2 has been used to measure LPL levels, block the triglyceride hydrolase activity of LPL, and prevent the propensity of concentrated LPL preparations to form homodimers. Two early studies on the location of the 5D2 epitope reached conflicting conclusions, but the more convincing report suggested that 5D2 binds to a tryptophan (Trp)-rich loop in the carboxyl terminus of LPL. The same loop had been implicated in lipoprotein binding. Using surface plasmon resonance, we showed that 5D2 binds with high affinity to a synthetic LPL peptide containing the Trp-rich loop of human (but not mouse) LPL. We also showed, by both fluorescence and UV resonance Raman spectroscopy, that the Trp-rich loop binds lipids. Finally, we used X-ray crystallography to solve the structure of the Trp-rich peptide bound to a 5D2 Fab fragment. The Trp-rich peptide contains a short α-helix, with two Trps projecting into the antigen recognition site. A proline substitution in the α-helix, found in mouse LPL, is expected to interfere with several hydrogen bonds, explaining why 5D2 cannot bind to mouse LPL.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Lipase Lipoproteica/química , Lipase Lipoproteica/imunologia , Animais , Sítios de Ligação , Humanos , Camundongos , Triptofano
8.
Proc Natl Acad Sci U S A ; 114(8): 2000-2005, 2017 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-28167768

RESUMO

Cholesterol is a crucial lipid within the plasma membrane of mammalian cells. Recent biochemical studies showed that one pool of cholesterol in the plasma membrane is "accessible" to binding by a modified version of the cytolysin perfringolysin O (PFO*), whereas another pool is sequestered by sphingomyelin and cannot be bound by PFO* unless the sphingomyelin is destroyed with sphingomyelinase (SMase). Thus far, it has been unclear whether PFO* and related cholesterol-binding proteins bind uniformly to the plasma membrane or bind preferentially to specific domains or morphologic features on the plasma membrane. Here, we used nanoscale secondary ion mass spectrometry (NanoSIMS) imaging, in combination with 15N-labeled cholesterol-binding proteins (PFO* and ALO-D4, a modified anthrolysin O), to generate high-resolution images of cholesterol distribution in the plasma membrane of Chinese hamster ovary (CHO) cells. The NanoSIMS images revealed preferential binding of PFO* and ALO-D4 to microvilli on the plasma membrane; lower amounts of binding were detectable in regions of the plasma membrane lacking microvilli. The binding of ALO-D4 to the plasma membrane was virtually eliminated when cholesterol stores were depleted with methyl-ß-cyclodextrin. When cells were treated with SMase, the binding of ALO-D4 to cells increased, largely due to increased binding to microvilli. Remarkably, lysenin (a sphingomyelin-binding protein) also bound preferentially to microvilli. Thus, high-resolution images of lipid-binding proteins on CHO cells can be acquired with NanoSIMS imaging. These images demonstrate that accessible cholesterol, as judged by PFO* or ALO-D4 binding, is not evenly distributed over the entire plasma membrane but instead is highly enriched on microvilli.


Assuntos
Toxinas Bacterianas/química , Membrana Celular/metabolismo , Colesterol/metabolismo , Proteínas Hemolisinas/química , Microvilosidades/metabolismo , Imagem Molecular/métodos , Nanotubos/química , Animais , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Células CHO , Técnicas de Cultura de Células/métodos , Membrana Celular/ultraestrutura , Cricetulus , Proteínas Hemolisinas/metabolismo , Marcação por Isótopo , Glicoproteínas de Membrana/metabolismo , Microscopia Confocal , Isótopos de Nitrogênio/química , Ligação Proteica , Espectrometria de Massa de Íon Secundário , Esfingomielina Fosfodiesterase/metabolismo , Esfingomielinas/metabolismo , beta-Ciclodextrinas/farmacologia
9.
Biochem Biophys Res Commun ; 504(4): 899-902, 2018 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-30224066

RESUMO

Heterogeneity in the metabolic properties of adipocytes in white adipose tissue has been well documented. We sought to investigate metabolic heterogeneity in adipocytes of brown adipose tissue (BAT), focusing on heterogeneity in nutrient uptake. To explore the possibility of metabolic heterogeneity in brown adipocytes, we used nanoscale secondary ion mass spectrometry (NanoSIMS) to quantify uptake of lipids in adipocytes interscapular BAT and perivascular adipose tissue (PVAT) after an intravenous injection of triglyceride-rich lipoproteins (TRLs) containing [2H]triglycerides (2H-TRLs). The uptake of deuterated lipids into brown adipocytes was quantified by NanoSIMS. We also examined 13C enrichment in brown adipocytes after administering [13C]glucose or 13C-labeled mixed fatty acids by gastric gavage. The uptake of 2H-TRLs-derived lipids into brown adipocytes was heterogeneous, with 2H enrichment in adjacent adipocytes varying by more than fourfold. We also observed substantial heterogeneity in 13C enrichment in adjacent brown adipocytes after administering [13C]glucose or [13C]fatty acids by gastric gavage. The uptake of nutrients by adjacent brown adipocytes within a single depot is variable, suggesting that there is heterogeneity in the metabolic properties of brown adipocytes.


Assuntos
Adipócitos Marrons/metabolismo , Nutrientes/farmacocinética , Espectrometria de Massa de Íon Secundário/métodos , Animais , Isótopos de Carbono/análise , Ácidos Graxos/farmacocinética , Glucose/farmacocinética , Lipídeos/farmacocinética , Lipoproteínas/administração & dosagem , Lipoproteínas/farmacocinética , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Receptores de Lipoproteínas/genética
10.
J Lipid Res ; 58(1): 208-215, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27875259

RESUMO

GPIHBP1, an endothelial cell protein, binds LPL in the interstitial spaces and shuttles it to its site of action inside blood vessels. For years, studies of human GPIHBP1 have been hampered by an absence of useful antibodies. We reasoned that monoclonal antibodies (mAbs) against human GPIHBP1 would be useful for 1) defining the functional relevance of GPIHBP1's Ly6 and acidic domains to the binding of LPL; 2) ascertaining whether human GPIHBP1 is expressed exclusively in capillary endothelial cells; and 3) testing whether GPIHBP1 is detectable in human plasma. Here, we report the development of a panel of human GPIHBP1-specific mAbs. Two mAbs against GPIHBP1's Ly6 domain, RE3 and RG3, abolished LPL binding, whereas an antibody against the acidic domain, RF4, did not. Also, mAbs RE3 and RG3 bound with reduced affinity to a mutant GPIHBP1 containing an Ly6 domain mutation (W109S) that abolishes LPL binding. Immunohistochemistry studies with the GPIHBP1 mAbs revealed that human GPIHBP1 is expressed only in capillary endothelial cells. Finally, we created an ELISA that detects GPIHBP1 in human plasma. That ELISA should make it possible for clinical lipidologists to determine whether plasma GPIHBP1 levels are a useful biomarker of metabolic or vascular disease.


Assuntos
Anticorpos Monoclonais/imunologia , Lipase Lipoproteica/imunologia , Receptores de Lipoproteínas/imunologia , Triglicerídeos/metabolismo , Animais , Sítios de Ligação/imunologia , Linhagem Celular , Drosophila , Células Endoteliais/enzimologia , Células Endoteliais/imunologia , Humanos , Lipase Lipoproteica/antagonistas & inibidores , Lipase Lipoproteica/isolamento & purificação , Camundongos , Receptores de Lipoproteínas/genética , Triglicerídeos/imunologia
11.
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
12.
Biochim Biophys Acta ; 1851(9): 1227-39, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26008578

RESUMO

Coq9 is a polypeptide subunit in a mitochondrial multi-subunit complex, termed the CoQ-synthome, required for biosynthesis of coenzyme Q (ubiquinone or Q). Deletion of COQ9 results in dissociation of the CoQ-synthome, but over-expression of Coq8 putative kinase stabilizes the CoQ-synthome in the coq9 null mutant and leads to the accumulation of two nitrogen-containing Q intermediates, imino-demethoxy-Q6 (IDMQ6) and 3-hexaprenyl-4-aminophenol (4-AP) when para-aminobenzoic acid (pABA) is provided as a ring precursor. To investigate whether Coq9 is responsible for deamination steps in Q biosynthesis, we utilized the yeast coq5-5 point mutant. The yeast coq5-5 point mutant is defective in the C-methyltransferase step of Q biosynthesis but retains normal steady-state levels of the Coq5 polypeptide. Here, we show that when high amounts of 13C6-pABA are provided, the coq5-5 mutant accumulates both 13C6-imino-demethyl-demethoxy-Q6 (13C6-IDDMQ6) and 13C6-demethyl-demethoxy-Q6 (13C6-DDMQ6). Deletion of COQ9 in the yeast coq5-5 mutant along with Coq8 over-expression and 13C6- pABA labeling leads to the absence of 13C6-DDMQ6, and the nitrogen-containing intermediates 13C6-4-AP and 13C6-IDDMQ6 persist. We describe a coq9 temperature-sensitive mutant and show that at the non-permissive temperature, steady-state polypeptide levels of Coq9-ts19 increased, while Coq4, Coq5, Coq6, and Coq7 decreased. The coq9-ts19 mutant had decreased Q6 content and increased levels of nitrogen-containing intermediates. These findings identify Coq9 as a multi-functional protein that is required for the function of Coq6 and Coq7 hydroxylases, for removal of the nitrogen substituent from pABA-derived Q intermediates, and is an essential component of the CoQ synthome.


Assuntos
Ácido 4-Aminobenzoico/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquinona/metabolismo , Desaminação , Metiltransferases/genética , Metiltransferases/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Modelos Moleculares , Mutação Puntual , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Temperatura , Ubiquinona/genética
14.
J Lipid Res ; 56(4): 909-19, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25681964

RESUMO

Coenzyme Q (Q or ubiquinone) is a redox-active polyisoprenylated benzoquinone lipid essential for electron and proton transport in the mitochondrial respiratory chain. The aromatic ring 4-hydroxybenzoic acid (4HB) is commonly depicted as the sole aromatic ring precursor in Q biosynthesis despite the recent finding that para-aminobenzoic acid (pABA) also serves as a ring precursor in Saccharomyces cerevisiae Q biosynthesis. In this study, we employed aromatic (13)C6-ring-labeled compounds including (13)C6-4HB, (13)C6-pABA, (13)C6-resveratrol, and (13)C6-coumarate to investigate the role of these small molecules as aromatic ring precursors in Q biosynthesis in Escherichia coli, S. cerevisiae, and human and mouse cells. In contrast to S. cerevisiae, neither E. coli nor the mammalian cells tested were able to form (13)C6-Q when cultured in the presence of (13)C6-pABA. However, E. coli cells treated with (13)C6-pABA generated (13)C6-ring-labeled forms of 3-octaprenyl-4-aminobenzoic acid, 2-octaprenyl-aniline, and 3-octaprenyl-2-aminophenol, suggesting UbiA, UbiD, UbiX, and UbiI are capable of using pABA or pABA-derived intermediates as substrates. E. coli, S. cerevisiae, and human and mouse cells cultured in the presence of (13)C6-resveratrol or (13)C6-coumarate were able to synthesize (13)C6-Q. Future evaluation of the physiological and pharmacological responses to dietary polyphenols should consider their metabolism to Q.


Assuntos
Ácidos Cumáricos/metabolismo , Estilbenos/metabolismo , Ubiquinona/biossíntese , Ubiquinona/química , Animais , Linhagem Celular Tumoral , Escherichia coli/metabolismo , Humanos , Camundongos , Propionatos , Resveratrol , Saccharomyces cerevisiae/metabolismo
15.
Biochim Biophys Acta ; 1841(4): 630-44, 2014 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-24406904

RESUMO

Coenzyme Q biosynthesis in yeast requires a multi-subunit Coq polypeptide complex. Deletion of any one of the COQ genes leads to respiratory deficiency and decreased levels of the Coq4, Coq6, Coq7, and Coq9 polypeptides, suggesting that their association in a high molecular mass complex is required for stability. Over-expression of the putative Coq8 kinase in certain coq null mutants restores steady-state levels of the sensitive Coq polypeptides and promotes the synthesis of late-stage Q-intermediates. Here we show that over-expression of Coq8 in yeast coq null mutants profoundly affects the association of several of the Coq polypeptides in high molecular mass complexes, as assayed by separation of digitonin extracts of mitochondria by two-dimensional blue-native/SDS PAGE. The Coq4 polypeptide persists at high molecular mass with over-expression of Coq8 in coq3, coq5, coq6, coq7, coq9, and coq10 mutants, indicating that Coq4 is a central organizer of the Coq complex. Supplementation with exogenous Q6 increased the steady-state levels of Coq4, Coq7, and Coq9, and several other mitochondrial polypeptides in select coq null mutants, and also promoted the formation of late-stage Q-intermediates. Q supplementation may stabilize this complex by interacting with one or more of the Coq polypeptides. The stabilizing effects of exogenously added Q6 or over-expression of Coq8 depend on Coq1 and Coq2 production of a polyisoprenyl intermediate. Based on the observed interdependence of the Coq polypeptides, the effect of exogenous Q6, and the requirement for an endogenously produced polyisoprenyl intermediate, we propose a new model for the Q-biosynthetic complex, termed the CoQ-synthome.


Assuntos
Proteínas Mitocondriais/genética , Respiração/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquinona/biossíntese , Suplementos Nutricionais , Regulação Fúngica da Expressão Gênica , Metiltransferases/química , Metiltransferases/genética , Metiltransferases/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Complexos Multiproteicos , Mutação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquinona/química , Ubiquinona/genética , Ubiquinona/metabolismo
16.
BMC Biol ; 12: 70, 2014 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-25159688

RESUMO

BACKGROUND: Steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domains were first identified from mammalian proteins that bind lipid/sterol ligands via a hydrophobic pocket. In plants, predicted START domains are predominantly found in homeodomain leucine zipper (HD-Zip) transcription factors that are master regulators of cell-type differentiation in development. Here we utilized studies of Arabidopsis in parallel with heterologous expression of START domains in yeast to investigate the hypothesis that START domains are versatile ligand-binding motifs that can modulate transcription factor activity. RESULTS: Our results show that deletion of the START domain from Arabidopsis Glabra2 (GL2), a representative HD-Zip transcription factor involved in differentiation of the epidermis, results in a complete loss-of-function phenotype, although the protein is correctly localized to the nucleus. Despite low sequence similarly, the mammalian START domain from StAR can functionally replace the HD-Zip-derived START domain. Embedding the START domain within a synthetic transcription factor in yeast, we found that several mammalian START domains from StAR, MLN64 and PCTP stimulated transcription factor activity, as did START domains from two Arabidopsis HD-Zip transcription factors. Mutation of ligand-binding residues within StAR START reduced this activity, consistent with the yeast assay monitoring ligand-binding. The D182L missense mutation in StAR START was shown to affect GL2 transcription factor activity in maintenance of the leaf trichome cell fate. Analysis of in vivo protein-metabolite interactions by mass spectrometry provided direct evidence for analogous lipid-binding activity in mammalian and plant START domains in the yeast system. Structural modeling predicted similar sized ligand-binding cavities of a subset of plant START domains in comparison to mammalian counterparts. CONCLUSIONS: The START domain is required for transcription factor activity in HD-Zip proteins from plants, although it is not strictly necessary for the protein's nuclear localization. START domains from both mammals and plants are modular in that they can bind lipid ligands to regulate transcription factor function in a yeast system. The data provide evidence for an evolutionarily conserved mechanism by which lipid metabolites can orchestrate transcription. We propose a model in which the START domain is used by both plants and mammals to regulate transcription factor activity.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Homeodomínio/genética , Fosfoproteínas/genética , Fatores de Transcrição/metabolismo , Animais , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Ligantes , Espectrometria de Massas , Camundongos , Organismos Geneticamente Modificados/genética , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética
17.
Mol Cell Biol ; 40(19)2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32719109

RESUMO

Recent studies have demonstrated the existence of a discrete pool of cholesterol in the plasma membranes (PM) of mammalian cells-referred to as the accessible cholesterol pool-that can be detected by the binding of modified versions of bacterial cytolysins (e.g., anthrolysin O). When the amount of accessible cholesterol in the PM exceeds a threshold level, the excess cholesterol moves to the endoplasmic reticulum (ER), where it regulates the SREBP2 pathway and undergoes esterification. We reported previously that the Aster/Gramd1 family of sterol transporters mediates nonvesicular movement of cholesterol from the PM to the ER in multiple mammalian cell types. Here, we investigated the PM pool of accessible cholesterol in cholesterol-loaded fibroblasts with a knockdown of Aster-A and in mouse macrophages from Aster-B and Aster-A/B-deficient mice. Nanoscale secondary ion mass spectrometry (NanoSIMS) analyses revealed expansion of the accessible cholesterol pool in cells lacking Aster expression. The increased accessible cholesterol pool in the PM was accompanied by reduced cholesterol movement to the ER, evidenced by increased expression of SREBP2-regulated genes. Cosedimentation experiments with liposomes revealed that the Aster-B GRAM domain binds to membranes in a cholesterol concentration-dependent manner and that the binding is facilitated by the presence of phosphatidylserine. These studies revealed that the Aster-mediated nonvesicular cholesterol transport pathway controls levels of accessible cholesterol in the PM, as well as the activity of the SREBP pathway.


Assuntos
Membrana Celular/metabolismo , Colesterol/metabolismo , Células 3T3-L1 , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Retículo Endoplasmático/metabolismo , Fibroblastos/metabolismo , Lipossomos/metabolismo , Macrófagos Peritoneais/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espectrometria de Massa de Íon Secundário/métodos , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo
18.
Cell Metab ; 30(1): 51-65, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31269429

RESUMO

Lipoprotein lipase (LPL), identified in the 1950s, has been studied intensively by biochemists, physiologists, and clinical investigators. These efforts uncovered a central role for LPL in plasma triglyceride metabolism and identified LPL mutations as a cause of hypertriglyceridemia. By the 1990s, with an outline for plasma triglyceride metabolism established, interest in triglyceride metabolism waned. In recent years, however, interest in plasma triglyceride metabolism has awakened, in part because of the discovery of new molecules governing triglyceride metabolism. One such protein-and the focus of this review-is GPIHBP1, a protein of capillary endothelial cells. GPIHBP1 is LPL's essential partner: it binds LPL and transports it to the capillary lumen; it is essential for lipoprotein margination along capillaries, allowing lipolysis to proceed; and it preserves LPL's structure and activity. Recently, GPIHBP1 was the key to solving the structure of LPL. These developments have transformed the models for intravascular triglyceride metabolism.


Assuntos
Células Endoteliais/metabolismo , Hiperlipoproteinemia Tipo I/metabolismo , Lipase Lipoproteica/metabolismo , Receptores de Lipoproteínas/metabolismo , Animais , Humanos , Hiperlipoproteinemia Tipo I/genética , Hipertrigliceridemia/genética , Hipertrigliceridemia/metabolismo , Lipase Lipoproteica/genética
19.
Elife ; 82019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31486771

RESUMO

Cultured mouse peritoneal macrophages release large numbers of ~30-nm cholesterol-rich particles. Here, we show that those particles represent fragments of the plasma membrane that are pulled away and left behind during the projection and retraction of filopodia and lamellipodia. Consistent with this finding, the particles are enriched in proteins found in focal adhesions, which attach macrophages to the substrate. The release of particles is abolished by blocking cell movement (either by depolymerizing actin with latrunculin A or by inhibiting myosin II with blebbistatin). Confocal microscopy and NanoSIMS imaging studies revealed that the plasma membrane-derived particles are enriched in 'accessible cholesterol' (a mobile pool of cholesterol detectable with the modified cytolysin ALO-D4) but not in sphingolipid-sequestered cholesterol [a pool detectable with ostreolysin A (OlyA)]. The discovery that macrophages release cholesterol-rich particles during cellular locomotion is likely relevant to cholesterol efflux and could contribute to extracellular cholesterol deposition in atherosclerotic plaques.


Assuntos
Membrana Celular/metabolismo , Movimento Celular , Micropartículas Derivadas de Células/química , Micropartículas Derivadas de Células/metabolismo , Colesterol/análise , Macrófagos Peritoneais/metabolismo , Pseudópodes/metabolismo , Animais , Células Cultivadas , Camundongos , Proteínas/análise
20.
Elife ; 82019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31169500

RESUMO

GPIHBP1, a GPI-anchored protein of capillary endothelial cells, binds lipoprotein lipase (LPL) within the subendothelial spaces and shuttles it to the capillary lumen. GPIHBP1-bound LPL is essential for the margination of triglyceride-rich lipoproteins (TRLs) along capillaries, allowing the lipolytic processing of TRLs to proceed. In peripheral tissues, the intravascular processing of TRLs by the GPIHBP1-LPL complex is crucial for the generation of lipid nutrients for adjacent parenchymal cells. GPIHBP1 is absent from the capillaries of the brain, which uses glucose for fuel; however, GPIHBP1 is expressed in the capillaries of mouse and human gliomas. Importantly, the GPIHBP1 in glioma capillaries captures locally produced LPL. We use NanoSIMS imaging to show that TRLs marginate along glioma capillaries and that there is uptake of TRL-derived lipid nutrients by surrounding glioma cells. Thus, GPIHBP1 expression in gliomas facilitates TRL processing and provides a source of lipid nutrients for glioma cells.


Assuntos
Glioma/metabolismo , Lipoproteínas/metabolismo , Receptores de Lipoproteínas/metabolismo , Animais , Encéfalo/irrigação sanguínea , Encéfalo/patologia , Capilares/metabolismo , Isótopos de Carbono/metabolismo , Células Endoteliais/metabolismo , Ácidos Graxos/metabolismo , Glioma/irrigação sanguínea , Glioma/patologia , Glioma/ultraestrutura , Glucose/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Humanos , Lipase Lipoproteica/metabolismo , Camundongos Endogâmicos C57BL , Triglicerídeos/metabolismo
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