RESUMO
SREBP transcription factors are central regulators of lipid metabolism. Their proteolytic activation requires ER to the Golgi translocation and subsequent cleavage by site-1-protease (S1P). Produced as a proprotein, S1P undergoes autocatalytic cleavage from its precursor S1PA to mature S1PC form. Here, we report that SPRING (previously C12ORF29) and S1P interact through their ectodomains, and that this facilitates the autocatalytic cleavage of S1PA into its mature S1PC form. Reciprocally, we identified a S1P recognition-motif in SPRING and demonstrate that S1P-mediated cleavage leads to secretion of the SPRING ectodomain in cells, and in liver-specific Spring knockout (LKO) mice transduced with AAV-mSpring. By reconstituting SPRING variants into SPRINGKO cells we show that the SPRING ectodomain supports proteolytic maturation of S1P and SREBP signaling, but that S1P-mediated SPRING cleavage is not essential for these processes. Absence of SPRING modestly diminishes proteolytic maturation of S1PAâC and trafficking of S1PC to the Golgi. However, despite reaching the Golgi in SPRINGKO cells, S1PC fails to rescue SREBP signaling. Remarkably, whereas SREBP signaling was severely attenuated in SPRINGKO cells and LKO mice, that of ATF6, another S1P substrate, was unaffected in these models. Collectively, our study positions SPRING as a dedicated licensing factor for SREBP-specific activation by S1P.
Assuntos
Pró-Proteína Convertases , Serina Endopeptidases , Animais , Humanos , Camundongos , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Células HEK293 , Fígado/metabolismo , Camundongos Knockout , Pró-Proteína Convertases/metabolismo , Pró-Proteína Convertases/genética , Proteólise , Serina Endopeptidases/metabolismo , Serina Endopeptidases/genética , Transdução de Sinais , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Proteínas de Ligação a Elemento Regulador de Esterol/genéticaRESUMO
The sterol regulatory element binding proteins (SREBPs) are transcription factors that govern cholesterol and fatty acid metabolism. We recently identified SPRING as a post-transcriptional regulator of SREBP activation. Constitutive or inducible global ablation of Spring in mice is not tolerated, and we therefore develop liver-specific Spring knockout mice (LKO). Transcriptomics and proteomics analysis reveal attenuated SREBP signaling in livers and hepatocytes of LKO mice. Total plasma cholesterol is reduced in male and female LKO mice in both the low-density lipoprotein and high-density lipoprotein fractions, while triglycerides are unaffected. Loss of Spring decreases hepatic cholesterol and triglyceride content due to diminished biosynthesis, which coincides with reduced very-low-density lipoprotein secretion. Accordingly, LKO mice are protected from fructose diet-induced hepatosteatosis. In humans, we find common genetic SPRING variants that associate with circulating high-density lipoprotein cholesterol and ApoA1 levels. This study positions SPRING as a core component of hepatic SREBP signaling and systemic lipid metabolism in mice and humans.
Assuntos
Metabolismo dos Lipídeos , Fígado , Humanos , Feminino , Masculino , Animais , Camundongos , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Metabolismo dos Lipídeos/genética , Hepatócitos , Lipoproteínas HDLRESUMO
Lysoplasmalogens are a class of vinyl ether bioactive lipids that have a central role in plasmalogen metabolism and membrane fluidity. The liver X receptor (LXR) transcription factors are important determinants of cellular lipid homeostasis owing to their ability to regulate cholesterol and fatty acid metabolism. However, their role in governing the composition of lipid species such as lysoplasmalogens in cellular membranes is less well studied. Here, we mapped the lipidome of bone marrow-derived macrophages (BMDMs) following LXR activation. We found a marked reduction in the levels of lysoplasmalogen species in the absence of changes in the levels of plasmalogens themselves. Transcriptional profiling of LXR-activated macrophages identified the gene encoding transmembrane protein 86a (TMEM86a), an integral endoplasmic reticulum protein, as a previously uncharacterized sterol-regulated gene. We demonstrate that TMEM86a is a direct transcriptional target of LXR in macrophages and microglia and that it is highly expressed in TREM2+/lipid-associated macrophages in human atherosclerotic plaques, where its expression positively correlates with other LXR-regulated genes. We further show that both murine and human TMEM86a display active lysoplasmalogenase activity that can be abrogated by inactivating mutations in the predicted catalytic site. Consequently, we demonstrate that overexpression of Tmem86a in BMDM markedly reduces lysoplasmalogen abundance and membrane fluidity, while reciprocally, silencing of Tmem86a increases basal lysoplasmalogen levels and abrogates the LXR-dependent reduction of this lipid species. Collectively, our findings implicate TMEM86a as a sterol-regulated lysoplasmalogenase in macrophages that contributes to sterol-dependent membrane remodeling.
Assuntos
Macrófagos , Esteróis , Animais , Humanos , Camundongos , Receptores X do Fígado/metabolismo , Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Receptores Imunológicos , Esteróis/metabolismo , Fatores de Transcrição/metabolismoRESUMO
OBJECTIVE: Four-and-a-Half-LIM-domain-protein 2 (FHL2) modulates multiple signal transduction pathways but has not been implicated in obesity or energy metabolism. In humans, methylation and expression of the FHL2 gene increases with age, and high FHL2 expression is associated with increased body weight in humans and mice. This led us to hypothesize that FHL2 is a determinant of diet-induced obesity. METHODS: FHL2-deficient (FHL2-/-) and wild type male mice were fed a high-fat diet. Metabolic phenotyping of these mice, as well as transcriptional analysis of key metabolic tissues was performed. Correlation of the expression of FHL2 and relevant genes was assessed in datasets from white adipose tissue of individuals with and without obesity. RESULTS: FHL2 Deficiency protects mice from high-fat diet-induced weight gain, whereas glucose handling is normal. We observed enhanced energy expenditure, which may be explained by a combination of changes in multiple tissues; mild activation of brown adipose tissue with increased fatty acid uptake, increased cardiac glucose uptake and browning of white adipose tissue. Corroborating our findings in mice, expression of FHL2 in human white adipose tissue positively correlates with obesity and negatively with expression of browning-associated genes. CONCLUSION: Our results position FHL2 as a novel regulator of obesity and energy expenditure in mice and human. Given that FHL2 expression increases during aging, we now show that low FHL2 expression associates with a healthy metabolic state.
Assuntos
Proteínas com Homeodomínio LIM/genética , Proteínas Musculares/genética , Obesidade/genética , Fatores de Transcrição/genética , Adipogenia/genética , Tecido Adiposo Branco/metabolismo , Adulto , Idoso , Animais , Biomarcadores/metabolismo , Dieta Hiperlipídica , Feminino , Predisposição Genética para Doença , Humanos , Proteínas com Homeodomínio LIM/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Proteínas Musculares/metabolismo , Obesidade/diagnóstico , Obesidade/metabolismo , Fatores de Transcrição/metabolismo , Aumento de Peso/genéticaRESUMO
The lysosomal storage disease Niemann-Pick type C (NPC) is caused by impaired cholesterol efflux from lysosomes, which is accompanied by secondary lysosomal accumulation of sphingomyelin and glucosylceramide (GlcCer). Similar to Gaucher disease (GD), patients deficient in glucocerebrosidase (GCase) degrading GlcCer, NPC patients show an elevated glucosylsphingosine and glucosylated cholesterol. In livers of mice lacking the lysosomal cholesterol efflux transporter NPC1, we investigated the expression of established biomarkers of lipid-laden macrophages of GD patients, their GCase status, and content on the cytosol facing glucosylceramidase GBA2 and lysosomal integral membrane protein type B (LIMP2), a transporter of newly formed GCase to lysosomes. Livers of 80-week-old Npc1-/- mice showed a partially reduced GCase protein and enzymatic activity. In contrast, GBA2 levels tended to be reciprocally increased with the GCase deficiency. In Npc1-/- liver, increased expression of lysosomal enzymes (cathepsin D, acid ceramidase) was observed as well as increased markers of lipid-stressed macrophages (GPNMB and galectin-3). Immunohistochemistry showed that the latter markers are expressed by lipid laden Kupffer cells. Earlier reported increase of LIMP2 in Npc1-/- liver was confirmed. Unexpectedly, immunohistochemistry showed that LIMP2 is particularly overexpressed in the hepatocytes of the Npc1-/- liver. LIMP2 in these hepatocytes seems not to only localize to (endo)lysosomes. The recent recognition that LIMP2 harbors a cholesterol channel prompts the speculation that LIMP2 in Npc1-/- hepatocytes might mediate export of cholesterol into the bile and thus protects the hepatocytes.
Assuntos
Glucosilceramidase/metabolismo , Fígado/metabolismo , Proteínas de Membrana Lisossomal/metabolismo , Doença de Niemann-Pick Tipo C/metabolismo , Receptores Depuradores/metabolismo , Animais , Transporte Biológico/fisiologia , Catepsina D/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Doença de Gaucher/metabolismo , Glucosilceramidas/metabolismo , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Lisossomos/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Células RAW 264.7 , Esfingomielinas/metabolismoRESUMO
Deficiency of glucocerebrosidase (GBA), a lysosomal ß-glucosidase, causes Gaucher disease. The enzyme hydrolyzes ß-glucosidic substrates and transglucosylates cholesterol to cholesterol-ß-glucoside. Here we show that recombinant human GBA also cleaves ß-xylosides and transxylosylates cholesterol. The xylosyl-cholesterol formed acts as an acceptor for the subsequent formation of di-xylosyl-cholesterol. Common mutant forms of GBA from patients with Gaucher disease with reduced ß-glucosidase activity were similarly impaired in ß-xylosidase, transglucosidase, and transxylosidase activities, except for a slightly reduced xylosidase/glucosidase activity ratio of N370S GBA and a slightly reduced transglucosylation/glucosidase activity ratio of D409H GBA. XylChol was found to be reduced in spleen from patients with Gaucher disease. The origin of newly identified XylChol in mouse and human tissues was investigated. Cultured human cells exposed to exogenous ß-xylosides generated XylChol in a manner dependent on active lysosomal GBA but not the cytosol-facing ß-glucosidase GBA2. We later sought an endogenous ß-xyloside acting as donor in transxylosylation reactions, identifying xylosylated ceramide (XylCer) in cells and tissues that serve as donor in the formation of XylChol. UDP-glucosylceramide synthase (GCS) was unable to synthesize XylChol but could catalyze the formation of XylCer. Thus, food-derived ß-D-xyloside and XylCer are potential donors for the GBA-mediated formation of XylChol in cells. The enzyme GCS produces XylCer at a low rate. Our findings point to further catalytic versatility of GBA and prompt a systematic exploration of the distribution and role of xylosylated lipids.
Assuntos
GlucosilceramidaseRESUMO
BACKGROUND AND AIMS: Cholesterol metabolism is tightly regulated by transcriptional and post-transcriptional mechanisms. Accordingly, dysregulation of cholesterol metabolism is a major risk factor for the development of coronary artery disease and associated complications. In recent years, it has become apparent that next to the liver, the intestine plays a key role in systemic cholesterol metabolism by governing cholesterol absorption, secretion, and incorporation into lipoprotein particles. We have previously demonstrated that the Liver X receptor (LXR)-regulated E3 ubiquitin ligase inducible degrader of LDLR (IDOL) is a regulator of cholesterol uptake owing to its ability to promote the ubiquitylation of the low-density lipoprotein receptor (LDLR). However, whether the LXR-IDOL-LDLR axis regulates the LDLR in the intestine and whether this influences intestinal cholesterol homeostasis is not known. METHODS: In this study, we evaluated the role of the LXR-IDOL-LDLR axis in enterocyte cell models and in primary enterocytes isolated from Idol(-/-) and wild type mice. Furthermore, we studied the regulation of intestinal LDLR in Idol(-/-) and in wild type mice treated with the LXR agonist GW3965. Finally, we assessed ezetimibe-induced trans-intestinal cholesterol efflux in Idol(-/-) mice. RESULTS: We show that in a wide range of intestinal cell lines LXR activation decreases LDLR protein abundance, cell surface occupancy, and LDL uptake in an IDOL-dependent manner. Similarly, we find that pharmacological dosing of C57BL6/N mice with the LXR agonist GW3965 increases Idol expression across the intestine with a concomitant reduction in Ldlr protein. Conversely, primary enterocytes isolated from Idol(-/-) mice have elevated Ldlr. To test whether these changes contribute to trans-intestinal cholesterol efflux, we measured fecal cholesterol in mice following ezetimibe dosing, but found no differences between Idol(-/-) and control mice in this setting. CONCLUSIONS: In conclusion, our study establishes that the LXR-IDOL-LDLR axis is active in the intestine and is part of the molecular circuitry that maintains cholesterol homeostasis in enterocytes.
Assuntos
Receptores Nucleares Órfãos , Receptores de LDL , Animais , Intestinos , Receptores X do Fígado , Camundongos , Receptores Nucleares Órfãos/genética , Receptores de LDL/genética , Receptores de LDL/metabolismo , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
The sterol-regulatory element binding proteins (SREBP) are central transcriptional regulators of lipid metabolism. Using haploid genetic screens we identify the SREBP Regulating Gene (SPRING/C12ORF49) as a determinant of the SREBP pathway. SPRING is a glycosylated Golgi-resident membrane protein and its ablation in Hap1 cells, Hepa1-6 hepatoma cells, and primary murine hepatocytes reduces SREBP signaling. In mice, Spring deletion is embryonic lethal yet silencing of hepatic Spring expression also attenuates the SREBP response. Mechanistically, attenuated SREBP signaling in SPRINGKO cells results from reduced SREBP cleavage-activating protein (SCAP) and its mislocalization to the Golgi irrespective of the cellular sterol status. Consistent with limited functional SCAP in SPRINGKO cells, reintroducing SCAP restores SREBP-dependent signaling and function. Moreover, in line with the role of SREBP in tumor growth, a wide range of tumor cell lines display dependency on SPRING expression. In conclusion, we identify SPRING as a previously unrecognized modulator of SREBP signaling.
Assuntos
Colesterol/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Animais , Linhagem Celular , Desenvolvimento Embrionário/genética , Retículo Endoplasmático/metabolismo , Expressão Gênica , Complexo de Golgi/metabolismo , Haploidia , Hepatócitos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metabolismo dos Lipídeos/genética , Fígado/metabolismo , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Ligação a Elemento Regulador de Esterol/genéticaRESUMO
Obesity is taking on worldwide epidemic proportions, yet effective pharmacological agents with long-term efficacy remain unavailable. Previously, we designed the iminosugar N-adamantine-methyloxypentyl-deoxynojirimycin (AMP-DNM), which potently improves glucose homeostasis by lowering excessive glycosphingolipids. Here we show that AMP-DNM promotes satiety and activates brown adipose tissue (BAT) in obese rodents. Moreover, we demonstrate that the mechanism mediating these favorable actions depends on oral, but not central, administration of AMP-DNM, which ultimately stimulates systemic glucagon-like peptide 1 (GLP1) secretion. We evidence an essential role of brain GLP1 receptors (GLP1r), as AMP-DNM fails to promote satiety and activate BAT in mice lacking the brain GLP1r as well as in mice treated intracerebroventricularly with GLP1r antagonist exendin-9. In conclusion, AMP-DNM markedly ameliorates metabolic abnormalities in obese rodents by restoring satiety and activating BAT through central GLP1r, while improving glucose homeostasis by mechanisms independent of central GLP1r.
Assuntos
1-Desoxinojirimicina/análogos & derivados , Adamantano/análogos & derivados , Tecido Adiposo Marrom/efeitos dos fármacos , Peptídeo 1 Semelhante ao Glucagon/fisiologia , Saciação/efeitos dos fármacos , 1-Desoxinojirimicina/farmacologia , Adamantano/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/fisiologia , Glucose/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacosRESUMO
The endocannabinoid system (ECS) is considered to be an endogenous protective system in various neurodegenerative diseases. Niemann-Pick type C (NPC) is a neurodegenerative disease in which the role of the ECS has not been studied yet. Most of the endocannabinoid enzymes are serine hydrolases, which can be studied using activity-based protein profiling (ABPP). Here, we report the serine hydrolase activity in brain proteomes of a NPC mouse model as measured by ABPP. Two ABPP methods are used: a gel-based method and a chemical proteomics method. The activities of the following endocannabinoid enzymes were quantified: diacylglycerol lipase (DAGL) α, α/ß-hydrolase domain-containing protein 4, α/ß-hydrolase domain-containing protein 6, α/ß-hydrolase domain-containing protein 12, fatty acid amide hydrolase, and monoacylglycerol lipase. Using the gel-based method, two bands were observed for DAGL α. Only the upper band corresponding to this enzyme was significantly decreased in the NPC mouse model. Chemical proteomics showed that three lysosomal serine hydrolase activities (retinoid-inducible serine carboxypeptidase, cathepsin A, and palmitoyl-protein thioesterase 1) were increased in Niemann-Pick C1 protein knockout mouse brain compared to wild-type brain, whereas no difference in endocannabinoid hydrolase activity was observed. We conclude that these targets might be interesting therapeutic targets for future validation studies.
RESUMO
Objective- The E3 ubiquitin ligase IDOL (inducible degrader of the LDLR [LDL (low-density lipoprotein) receptor]) is a post-transcriptional regulator of LDLR abundance. Model systems and human genetics support a role for IDOL in regulating circulating LDL levels. Whether IDOL plays a broader metabolic role and affects development of metabolic syndrome-associated comorbidities is unknown. Approach and Results- We studied WT (wild type) and Idol(-/-) (Idol-KO) mice in 2 models: physiological aging and diet-induced obesity. In both models, deletion of Idol protected mice from metabolic dysfunction. On a Western-type diet, Idol loss resulted in decreased circulating levels of cholesterol, triglycerides, glucose, and insulin. This was accompanied by protection from weight gain in short- and long-term dietary challenges, which could be attributed to reduced hepatosteatosis and fat mass in Idol-KO mice. Although feeding and intestinal fat uptake were unchanged in Idol-KO mice, their brown adipose tissue was protected from lipid accumulation and had elevated expression of UCP1 (uncoupling protein 1) and TH (tyrosine hydroxylase). Indirect calorimetry indicated a marked increase in locomotion and suggested a trend toward increased cumulative energy expenditure and fat oxidation. An increase in in vivo clearance of reconstituted lipoprotein particles in Idol-KO mice may sustain this energetic demand. In the BXD mouse genetic reference population, hepatic Idol expression correlates with multiple metabolic parameters, thus providing support for findings in the Idol-KO mice. Conclusions- Our study uncovers an unrecognized role for Idol in regulation of whole body metabolism in physiological aging and on a Western-type diet. These findings support Idol inhibition as a therapeutic strategy to target multiple metabolic syndrome-associated comorbidities.
Assuntos
Dieta Hiperlipídica , Metabolismo Energético , Fígado/enzimologia , Síndrome Metabólica/prevenção & controle , Obesidade/prevenção & controle , Ubiquitina-Proteína Ligases/deficiência , Adipogenia , Tecido Adiposo Marrom/enzimologia , Adiposidade , Fatores Etários , Envelhecimento , Animais , Biomarcadores/sangue , Glicemia/metabolismo , Colesterol/sangue , Modelos Animais de Doenças , Feminino , Insulina/sangue , Locomoção , Masculino , Síndrome Metabólica/sangue , Síndrome Metabólica/enzimologia , Síndrome Metabólica/genética , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Obesidade/sangue , Obesidade/enzimologia , Obesidade/genética , Triglicerídeos/sangue , Tirosina 3-Mono-Oxigenase/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteína Desacopladora 1/metabolismoRESUMO
During obesity, adipose tissue macrophages (ATM) are increased in concert with local inflammation and insulin resistance. Since the levels of sphingolipid (SLs) in adipose tissue (AT) are altered during obesity we investigated the potential impact of SLs on ATMs. For this, we first analyzed expression of SL metabolizing genes in ATMs isolated from obese mice. A marked induction of sphingosine kinase 1 (Sphk1) expression was observed in obese ATM when compared to lean ATM. This induction was observed in both MGL-ve (M1) and MGL1+ve (M2) macrophages from obese WAT. Next, RAW264.7 cells were exposed to excessive palmitate, resulting in a similar induction of Sphk1. This Sphk1 induction was also observed when cells were treated with chloroquine, a lysosomotropic amine impacting lysosome function. Simultaneous incubation of RAW cells with palmitate and the Sphk1 inhibitor SK1-I promoted cell death, suggesting a protective role of Sphk1 during lipotoxic conditions. Interestingly, a reduction of endoplasmic reticulum (ER) stress related genes was detected in obese ATM and was found to be associated with elevated Sphk1 expression. Altogether, our data suggest that lipid overload in ATM induces Sphk1, which promotes cell viability.
Assuntos
Tecido Adiposo/citologia , Sobrevivência Celular/fisiologia , Macrófagos/metabolismo , Obesidade/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Animais , Antígeno CD11b/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cloroquina/farmacologia , Análise por Conglomerados , Dieta Hiperlipídica/efeitos adversos , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Ácido Palmítico/farmacologia , Células RAW 264.7 , Esfingolipídeos/metabolismoRESUMO
Glucocerebrosidase (GBA) is a lysosomal ß-glucosidase that degrades glucosylceramide. Its deficiency results in Gaucher disease (GD). We examined the effects of active site occupancy of GBA on its structural stability. For this, we made use of cyclophellitol-derived activity-based probes (ABPs) that bind irreversibly to the catalytic nucleophile (E340), and for comparison, we used the potent reversible inhibitor isofagomine. We demonstrate that cyclophellitol ABPs improve the stability of GBA in vitro, as revealed by thermodynamic measurements (Tm increase by 21 °C), and introduce resistance to tryptic digestion. The stabilizing effect of cell-permeable cyclophellitol ABPs is also observed in intact cultured cells containing wild-type GBA, N370S GBA (labile in lysosomes), and L444P GBA (exhibits impaired ER folding): all show marked increases in lysosomal forms of GBA molecules upon exposure to ABPs. The same stabilization effect is observed for endogenous GBA in the liver of wild-type mice injected with cyclophellitol ABPs. Stabilization effects similar to those observed with ABPs were also noted at high concentrations of the reversible inhibitor isofagomine. In conclusion, we provide evidence that the increase in cellular levels of GBA by ABPs and by the reversible inhibitor is in part caused by their ability to stabilize GBA folding, which increases the resistance of GBA against breakdown by lysosomal proteases. These effects are more pronounced in the case of the amphiphilic ABPs, presumably due to their high lipophilic potential, which may promote further structural compactness of GBA through hydrophobic interactions. Our study provides further rationale for the design of chaperones for GBA to ameliorate Gaucher disease.
Assuntos
Domínio Catalítico/fisiologia , Estabilidade Enzimática/fisiologia , Glucosilceramidase/química , Glucosilceramidase/metabolismo , Imino Piranoses/metabolismo , Animais , Sítios de Ligação , Estabilidade Enzimática/efeitos dos fármacos , Imino Piranoses/química , Imino Piranoses/farmacologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Camundongos , Estrutura Molecular , TemperaturaRESUMO
Deficiency of glucocerebrosidase (GBA) causes Gaucher disease (GD). In the common non-neuronopathic GD type I variant, glucosylceramide accumulates primarily in the lysosomes of visceral macrophages. Supplementing storage cells with lacking enzyme is accomplished via chronic intravenous administration of recombinant GBA containing mannose-terminated N-linked glycans, mediating the selective uptake by macrophages expressing mannose-binding lectin(s). Two recombinant GBA preparations with distinct N-linked glycans are registered in Europe for treatment of type I GD: imiglucerase (Genzyme), contains predominantly Man(3) glycans, and velaglucerase (Shire PLC) Man(9) glycans. Activity-based probes (ABPs) enable fluorescent labeling of recombinant GBA preparations through their covalent attachment to the catalytic nucleophile E340 of GBA. We comparatively studied binding and uptake of ABP-labeled imiglucerase and velaglucerase in isolated dendritic cells, cultured human macrophages and living mice, through simultaneous detection of different GBAs by paired measurements. Uptake of ABP-labeled rGBAs by dendritic cells was comparable, as well as the bio-distribution following equimolar intravenous administration to mice. ABP-labeled rGBAs were recovered largely in liver, white-blood cells, bone marrow and spleen. Lungs, brain and skin, affected tissues in severe GD types II and III, were only poorly supplemented. Small, but significant differences were noted in binding and uptake of rGBAs in cultured human macrophages, in the absence and presence of mannan. Mannan-competed binding and uptake were largest for velaglucerase, when determined with single enzymes or as equimolar mixtures of both enzymes. Vice versa, imiglucerase showed more prominent binding and uptake not competed by mannan. Uptake of recombinant GBAs by cultured macrophages seems to involve multiple receptors, including several mannose-binding lectins. Differences among cells from different donors (n = 12) were noted, but the same trends were always observed. Our study suggests that further insight in targeting and efficacy of enzyme therapy of individual Gaucher patients could be obtained by the use of recombinant GBA, trace-labeled with an ABP, preferably equipped with an infrared fluorophore or other reporter tag suitable for in vivo imaging.
Assuntos
Células Dendríticas/enzimologia , Corantes Fluorescentes/química , Glucosilceramidase/metabolismo , Macrófagos/enzimologia , Monócitos/enzimologia , Animais , Benzofuranos/química , Células Cultivadas , Imunofluorescência , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Polissacarídeos/metabolismoRESUMO
Cellular cholesterol metabolism is subject to tight regulation to maintain adequate levels of this central lipid molecule. Herein, the sterol-responsive Liver X Receptors (LXRs) play an important role owing to their ability to reduce cellular cholesterol load. In this context, identifying the full set of LXR-regulated genes will contribute to our understanding of their role in cholesterol metabolism. Using global transcriptional analysis we report here the identification of RNF145 as an LXR-regulated target gene. We demonstrate that RNF145 is regulated by LXRs in both human and mouse primary cells and cell lines, and in vivo in mice. Regulation of RNF145 by LXR depends on a functional LXR-element in its proximal promotor. Consistent with LXR-dependent regulation of Rnf145 we show that regulation is lost in macrophages and fibroblasts from Lxrαß(-/-) mice, and also in vivo in livers of Lxrα(-/-) mice treated with the LXR synthetic ligand T0901317. RNF145 is closely related to RNF139/TRC8, an E3 ligase implicated in control of SREBP processing. However, silencing of RNF145 in HepG2 or HeLa cells does not impair SREBP1/2 processing and sterol-responsive gene expression in these cells. Similar to TRC8, we demonstrate that RNF145 is localized to the ER and that it possesses intrinsic E3 ubiquitin ligase activity. In summary, we report the identification of RNF145 as an ER-resident E3 ubiquitin ligase that is transcriptionally controlled by LXR.
Assuntos
Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica , Receptores X do Fígado/genética , Proteínas de Membrana/genética , Transcrição Gênica , Animais , Linhagem Celular , Colesterol/metabolismo , Humanos , Hidrocarbonetos Fluorados/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Receptores X do Fígado/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas , Sulfonamidas/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Galactosylceramidase (GALC) is the lysosomal ß-galactosidase responsible for the hydrolysis of galactosylceramide. Inherited deficiency in GALC causes Krabbe disease, a devastating neurological disorder characterized by accumulation of galactosylceramide and its deacylated counterpart, the toxic sphingoid base galactosylsphingosine (psychosine). We report the design and application of a fluorescently tagged activity-based probe (ABP) for the sensitive and specific labeling of active GALC molecules from various species. The probe consists of a ß-galactopyranose-configured cyclophellitol-epoxide core, conferring specificity for GALC, equipped with a BODIPY fluorophore at C6 that allows visualization of active enzyme in cells and tissues. Detection of residual GALC in patient fibroblasts holds great promise for laboratory diagnosis of Krabbe disease. We further describe a procedure for in situ imaging of active GALC in murine brain by intra-cerebroventricular infusion of the ABP. In conclusion, this GALC-specific ABP should find broad applications in diagnosis, drug development, and evaluation of therapy for Krabbe disease.
Assuntos
Galactosilceramidase/genética , Galactosilceramidase/metabolismo , Leucodistrofia de Células Globoides/enzimologia , Sondas Moleculares , Deficiências Nutricionais/enzimologia , Deficiências Nutricionais/genética , Galactosilceramidase/antagonistas & inibidores , Leucodistrofia de Células Globoides/diagnóstico , Leucodistrofia de Células Globoides/genética , Doenças por Armazenamento dos Lisossomos/enzimologia , Doenças por Armazenamento dos Lisossomos/genética , Estrutura Molecular , MutaçãoRESUMO
Gaucher disease is caused by inherited deficiency of lysosomal glucocerebrosidase. Proteome analysis of laser-dissected splenic Gaucher cells revealed increased amounts of glycoprotein nonmetastatic melanoma protein B (gpNMB). Plasma gpNMB was also elevated, correlating with chitotriosidase and CCL18, which are established markers for human Gaucher cells. In Gaucher mice, gpNMB is also produced by Gaucher cells. Correction of glucocerebrosidase deficiency in mice by gene transfer or pharmacological substrate reduction reverses gpNMB abnormalities. In conclusion, gpNMB acts as a marker for glucosylceramide-laden macrophages in man and mouse and gpNMB should be considered as candidate biomarker for Gaucher disease in treatment monitoring.
RESUMO
Aberrant mitochondrial fission plays a pivotal role in the pathogenesis of skeletal muscle insulin resistance. However, fusion-fission dynamics are physiologically regulated by inherent tissue-specific and nutrient-sensitive processes that may have distinct or even opposing effects with respect to insulin sensitivity. Based on a combination of mouse population genetics and functional in vitro assays, we describe here a regulatory circuit in which peroxisome proliferator-activated receptor γ (PPARγ), the adipocyte master regulator and receptor for the thiazolidinedione class of antidiabetic drugs, controls mitochondrial network fragmentation through transcriptional induction of Bnip3. Short hairpin RNA-mediated knockdown of Bnip3 in cultured adipocytes shifts the balance toward mitochondrial elongation, leading to compromised respiratory capacity, heightened fatty acid ß-oxidation-associated mitochondrial reactive oxygen species generation, insulin resistance, and reduced triacylglycerol storage. Notably, the selective fission/Drp1 inhibitor Mdivi-1 mimics the effects of Bnip3 knockdown on adipose mitochondrial bioenergetics and glucose disposal. We further show that Bnip3 is reciprocally regulated in white and brown fat depots of diet-induced obesity and leptin-deficient ob/ob mouse models. Finally, Bnip3(-/-) mice trade reduced adiposity for increased liver steatosis and develop aggravated systemic insulin resistance in response to high-fat feeding. Together, our data outline Bnip3 as a key effector of PPARγ-mediated adipose mitochondrial network fragmentation, improving insulin sensitivity and limiting oxidative stress.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , PPAR gama/metabolismo , Células 3T3-L1 , Adipócitos/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Feminino , Glucose/metabolismo , Immunoblotting , Imuno-Histoquímica , Insulina/metabolismo , Resistência à Insulina/genética , Resistência à Insulina/fisiologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/genética , Dinâmica Mitocondrial/fisiologia , Proteínas Mitocondriais/genética , Obesidade/genética , Obesidade/metabolismo , PPAR gama/genética , Ensaio de Radioimunoprecipitação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We developed a mass spectrometric procedure to quantify sphingosine-1-phosphate (S1P) in biological materials. The use of newly synthesized (13)C5 C18-S1P and commercial C17-S1P as internal standards rendered very similar results with respect to linearity, limit of detection and limit of quantitation. Caution is warranted with determination of plasma S1P levels. Earlier it was reported that S1P is elevated in plasma of Fabry disease patients. We investigated this with the improved quantification. No clear conclusion could be drawn for patient plasma samples given the lack of uniformity of blood collection and plasma preparation. To still obtain insight, plasma and tissues were identically collected from α-galactosidase A deficient Fabry mice and matched control animals. No significant difference was observed in plasma S1P levels. A significant 2.3 fold increase was observed in kidney of Fabry mice, but not in liver and heart. Comparative analysis of S1P in cultured fibroblasts from normal subjects and classically affected Fabry disease males revealed no significant difference. In conclusion, accurate quantification of S1P in biological materials is feasible by mass spectrometry using the internal standards (13)C5 C18-S1P or C17-S1P. Significant local increases of S1P in the kidney might occur in Fabry disease as suggested by the mouse model.
Assuntos
Doença de Fabry/sangue , Lisofosfolipídeos/sangue , Esfingosina/análogos & derivados , Espectrometria de Massas em Tandem/normas , Animais , Isótopos de Carbono , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Doença de Fabry/patologia , Feminino , Fibroblastos/patologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Padrões de Referência , Esfingosina/sangueRESUMO
The membrane lipid glucosylceramide (GlcCer) is continuously formed and degraded. Cells express two GlcCer-degrading ß-glucosidases, glucocerebrosidase (GBA) and GBA2, located in and outside the lysosome, respectively. Here we demonstrate that through transglucosylation both GBA and GBA2 are able to catalyze in vitro the transfer of glucosyl-moieties from GlcCer to cholesterol, and vice versa. Furthermore, the natural occurrence of 1-O-cholesteryl-ß-D-glucopyranoside (GlcChol) in mouse tissues and human plasma is demonstrated using LC-MS/MS and (13)C6-labeled GlcChol as internal standard. In cells, the inhibition of GBA increases GlcChol, whereas inhibition of GBA2 decreases glucosylated sterol. Similarly, in GBA2-deficient mice, GlcChol is reduced. Depletion of GlcCer by inhibition of GlcCer synthase decreases GlcChol in cells and likewise in plasma of inhibitor-treated Gaucher disease patients. In tissues of mice with Niemann-Pick type C disease, a condition characterized by intralysosomal accumulation of cholesterol, marked elevations in GlcChol occur as well. When lysosomal accumulation of cholesterol is induced in cultured cells, GlcChol is formed via lysosomal GBA. This illustrates that reversible transglucosylation reactions are highly dependent on local availability of suitable acceptors. In conclusion, mammalian tissues contain GlcChol formed by transglucosylation through ß-glucosidases using GlcCer as donor. Our findings reveal a novel metabolic function for GlcCer.