RESUMEN
Lysosomal cholesterol egress requires two proteins, NPC1 and NPC2, whose defects are responsible for Niemann-Pick disease type C (NPC). Here, we present systematic structural characterizations that reveal the molecular basis for low-pH-dependent cholesterol delivery from NPC2 to the transmembrane (TM) domain of NPC1. At pH 8.0, similar structures of NPC1 were obtained in nanodiscs and in detergent at resolutions of 3.6 Å and 3.0 Å, respectively. A tunnel connecting the N-terminal domain (NTD) and the transmembrane sterol-sensing domain (SSD) was unveiled. At pH 5.5, the NTD exhibits two conformations, suggesting the motion for cholesterol delivery to the tunnel. A putative cholesterol molecule is found at the membrane boundary of the tunnel, and TM2 moves toward formation of a surface pocket on the SSD. Finally, the structure of the NPC1-NPC2 complex at 4.0 Å resolution was obtained at pH 5.5, elucidating the molecular basis for cholesterol handoff from NPC2 to NPC1(NTD).
Asunto(s)
Colesterol/metabolismo , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lisosomas/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Modelos Moleculares , Nanopartículas/química , Nanopartículas/ultraestructura , Proteína Niemann-Pick C1 , Dominios Proteicos , Homología Estructural de Proteína , Relación Estructura-ActividadRESUMEN
Cholesterol is dynamically transported among organelles, which is essential for multiple cellular functions. However, the mechanism underlying intracellular cholesterol transport has remained largely unknown. We established an amphotericin B-based assay enabling a genome-wide shRNA screen for delayed LDL-cholesterol transport and identified 341 hits with particular enrichment of peroxisome genes, suggesting a previously unappreciated pathway for cholesterol transport. We show dynamic membrane contacts between peroxisome and lysosome, which are mediated by lysosomal Synaptotagmin VII binding to the lipid PI(4,5)P2 on peroxisomal membrane. LDL-cholesterol enhances such contacts, and cholesterol is transported from lysosome to peroxisome. Disruption of critical peroxisome genes leads to cholesterol accumulation in lysosome. Together, these findings reveal an unexpected role of peroxisome in intracellular cholesterol transport. We further demonstrate massive cholesterol accumulation in human patient cells and mouse model of peroxisomal disorders, suggesting a contribution of abnormal cholesterol accumulation to these diseases.
Asunto(s)
Colesterol/metabolismo , Lisosomas/metabolismo , Peroxisomas/metabolismo , ARN Interferente Pequeño/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Adrenoleucodistrofia/metabolismo , Anfotericina B/farmacología , Animales , Transporte Biológico , Estudio de Asociación del Genoma Completo , Humanos , Ratones , Trastorno Peroxisomal/metabolismo , Trastorno Peroxisomal/patología , Fosfatidilinositol 4,5-Difosfato/metabolismo , Sinaptotagminas/metabolismo , Pez CebraRESUMEN
Cholesterol is highly enriched at the plasma membrane (PM), and lipid transfer proteins may deliver cholesterol to the PM in a nonvesicular manner. Here, through a mini-screen, we identified the oxysterol binding protein (OSBP)-related protein 2 (ORP2) as a novel mediator of selective cholesterol delivery to the PM. Interestingly, ORP2-mediated enrichment of PM cholesterol was coupled with the removal of phosphatidylinositol 4, 5-bisphosphate (PI(4,5)P2) from the PM. ORP2 overexpression or deficiency impacted the levels of PM cholesterol and PI(4,5)P2, and ORP2 efficiently transferred both cholesterol and PI(4,5)P2in vitro. We determined the structure of ORP2 in complex with PI(4,5)P2 at 2.7 Å resolution. ORP2 formed a stable tetramer in the presence of PI(4,5)P2, and tetramerization was required for ORP2 to transfer PI(4,5)P2. Our results identify a novel pathway for cholesterol delivery to the PM and establish ORP2 as a key regulator of both cholesterol and PI(4,5)P2 of the PM.
Asunto(s)
Membrana Celular/metabolismo , Colesterol/metabolismo , Hepatocitos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Receptores de Esteroides/metabolismo , Transporte Biológico , Línea Celular Tumoral , Células HEK293 , Humanos , Modelos Moleculares , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Receptores de Esteroides/química , Receptores de Esteroides/genética , Relación Estructura-ActividadRESUMEN
As members of the membrane-bound O-acyltransferase (MBOAT) enzyme family, acyl-coenzyme A:cholesterol acyltransferases (ACATs) catalyse the transfer of an acyl group from acyl-coenzyme A to cholesterol to generate cholesteryl ester, the primary form in which cholesterol is stored in cells and transported in plasma1. ACATs have gained attention as potential drug targets for the treatment of diseases such as atherosclerosis, Alzheimer's disease and cancer2-7. Here we present the cryo-electron microscopy structure of human ACAT1 as a dimer of dimers. Each protomer consists of nine transmembrane segments, which enclose a cytosolic tunnel and a transmembrane tunnel that converge at the predicted catalytic site. Evidence from structure-guided mutational analyses suggests that acyl-coenzyme A enters the active site through the cytosolic tunnel, whereas cholesterol may enter from the side through the transmembrane tunnel. This structural and biochemical characterization helps to rationalize the preference of ACAT1 for unsaturated acyl chains, and provides insight into the catalytic mechanism of enzymes within the MBOAT family8.
Asunto(s)
Biocatálisis , Microscopía por Crioelectrón , Esterol O-Aciltransferasa/química , Esterol O-Aciltransferasa/metabolismo , Dominio Catalítico , Humanos , Modelos Moleculares , Multimerización de Proteína , Esterol O-Aciltransferasa/ultraestructura , Especificidad por SustratoRESUMEN
Cholesterol is essential for both normal cell viability and cancer cell proliferation. Aberrant activity of squalene monooxygenase (SM, also known as squalene epoxidase), the rate-limiting enzyme of the committed cholesterol synthesis pathway, is accordingly implicated in a growing list of cancers. We previously reported that hypoxia triggers the truncation of SM to a constitutively active form, thus preserving sterol synthesis during oxygen shortfalls. Here, we show SM truncation is upregulated and correlates with the magnitude of hypoxia in endometrial cancer tissues, supporting the in vivo relevance of our earlier work. To further investigate the pathophysiological consequences of SM truncation, we examined its lipid droplet-localized pool using complementary immunofluorescence and cell fractionation approaches and found that it exclusively comprises the truncated enzyme. This partitioning is facilitated by the loss of an endoplasmic reticulum-embedded region at the SM N terminus, whereas the catalytic domain containing membrane-associated C-terminal helices is spared. Moreover, we determined multiple amphipathic helices contribute to the lipid droplet localization of truncated SM. Taken together, our results expand on the striking differences between the two forms of SM and suggest upregulated truncation may contribute to SM-related oncogenesis.
Asunto(s)
Colesterol , Neoplasias Endometriales , Gotas Lipídicas , Escualeno-Monooxigenasa , Femenino , Humanos , Línea Celular Tumoral , Colesterol/metabolismo , Colesterol/biosíntesis , Neoplasias Endometriales/metabolismo , Neoplasias Endometriales/patología , Neoplasias Endometriales/genética , Retículo Endoplásmico/metabolismo , Regulación Neoplásica de la Expresión Génica , Gotas Lipídicas/metabolismo , Escualeno-Monooxigenasa/metabolismo , Escualeno-Monooxigenasa/genética , Regulación hacia ArribaRESUMEN
Beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), also known as ß-secretase, is an aspartic protease. The sorting of this enzyme into Rab11-positive recycling endosomes regulates the BACE1-mediated cleavage of its substrates, however, the mechanisms underlying this targeting remain poorly understood. The neural cell adhesion molecule 2 (NCAM2) is a substrate of BACE1. We show that BACE1 cleaves NCAM2 in cultured hippocampal neurons and NCAM2-transfected CHO cells. The C-terminal fragment of NCAM2 that comprises the intracellular domain and a small portion of NCAM2's extracellular domain, associates with BACE1. This association is not affected in cells with inhibited endocytosis, indicating that the interaction of NCAM2 and BACE1 precedes the targeting of BACE1 from the cell surface to endosomes. In neurons and CHO cells, this fragment and BACE1 co-localize in Rab11-positive endosomes. Overexpression of full-length NCAM2 or a recombinant NCAM2 fragment containing the transmembrane and intracellular domains but lacking the extracellular domain leads to an increase in BACE1 levels in these organelles. In NCAM2-deficient neurons, the levels of BACE1 are increased at the cell surface and reduced in intracellular organelles. These effects are correlated with increased levels of the soluble extracellular domain of BACE1 in the brains of NCAM2-deficient mice, suggesting increased shedding of BACE1 from the cell surface. Of note, shedding of the extracellular domain of Sez6, a protein cleaved exclusively by BACE1, is reduced in NCAM2-deficient animals. These results indicate that the BACE1-generated fragment of NCAM2 regulates BACE1 activity by promoting the targeting of BACE1 to Rab11-positive endosomes.
Asunto(s)
Enfermedad de Alzheimer , Secretasas de la Proteína Precursora del Amiloide , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/metabolismo , Cricetinae , Cricetulus , Endosomas/metabolismo , Ratones , Proteínas del Tejido Nervioso/metabolismo , Moléculas de Adhesión de Célula Nerviosa/genética , Moléculas de Adhesión de Célula Nerviosa/metabolismoRESUMEN
Oxysterol-binding protein (OSBP) and its related proteins (ORPs) constitute a large, evolutionarily conserved family of lipid-binding proteins that are associated with a wide range of cellular activities. The core function of OSBP/ORPs appears to be moving lipids between cellular membranes in a non-vesicular manner. Recent studies have unveiled a novel, counter-transport mechanism of cellular lipid transfer mediated by OSBP/ORPs at the membrane contact sites that involves phosphatidylinositol 4-phosphate. Importantly, the OSBP/ORPs family has also been implicated in cell signalling pathways and cancer development. Here, we summarize recent progress in understanding the role of OSBP/ORPs in cancer development, and discuss how the lipid transfer function of OSBP/ORPs may underpin their role in tumorigenesis.
Asunto(s)
Metabolismo de los Lípidos/fisiología , Neoplasias/metabolismo , Receptores de Esteroides/fisiología , Transducción de Señal/fisiología , Animales , Transporte Biológico , Membrana Celular/metabolismo , Oxiesteroles/metabolismo , Fosfatos de Fosfatidilinositol/metabolismoRESUMEN
Cholesterol plays essential structural and signaling roles in mammalian cells, but too much cholesterol can cause cytotoxicity. Acyl-CoA:cholesterol acyltransferases 1 and 2 (ACAT1/2) convert cholesterol into its storage form, cholesteryl esters, regulating a key step in cellular cholesterol homeostasis. Adipose tissue can store >50% of whole-body cholesterol. Interestingly, however, almost no ACAT activity is present in adipose tissue, and most adipose cholesterol is stored in its free form. We therefore hypothesized that increased cholesterol esterification may have detrimental effects on adipose tissue function. Here, using several approaches, including protein overexpression, quantitative RT-PCR, immunofluorescence, and various biochemical assays, we found that ACAT1 expression is significantly increased in the adipose tissue of the ob/ob mice. We further demonstrated that ACAT1/2 overexpression partially inhibited the differentiation of 3T3-L1 preadipocytes. In mature adipocytes, increased ACAT activity reduced the size of lipid droplets (LDs) and inhibited lipolysis and insulin signaling. Paradoxically, the amount of free cholesterol increased on the surface of LDs in ACAT1/2-overexpressing adipocytes, accompanied by increased LD localization of caveolin-1. Moreover, cholesterol depletion in adipocytes by treating the cells with cholesterol-deficient media or ß-cyclodextrins induced changes in cholesterol distribution that were similar to those caused by ACAT1/2 overexpression. Our results suggest that ACAT1/2 overexpression increases the level of free cholesterol on the LD surface, thereby impeding adipocyte function. These findings provide detailed insights into the role of free cholesterol in LD and adipocyte function and suggest that ACAT inhibitors have potential utility for managing disorders associated with extreme obesity.
Asunto(s)
Acetil-CoA C-Acetiltransferasa/metabolismo , Adipocitos/metabolismo , Colesterol/metabolismo , Gotas Lipídicas/metabolismo , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Lipid droplets (LDs) are evolutionarily conserved organelles that play critical roles in mammalian lipid storage and metabolism. However, the molecular mechanisms governing the biogenesis and growth of LDs remain poorly understood. Phosphatidic acid (PA) is a precursor of phospholipids and triacylglycerols and substrate of CDP-diacylglycerol (CDP-DAG) synthase 1 (CDS1) and CDS2, which catalyze the formation of CDP-DAG. Here, using siRNA-based gene knockdowns and CRISPR/Cas9-mediated gene knockouts, along with immunological, molecular, and fluorescence microscopy approaches, we examined the role of CDS1 and CDS2 in LD biogenesis and growth. Knockdown of either CDS1 or CDS2 expression resulted in the formation of giant or supersized LDs in cultured mammalian cells. Interestingly, down-regulation of cell death-inducing DFF45-like effector C (CIDEC), encoding a prominent regulator of LD growth in adipocytes, restored LD size in CDS1- but not in CDS2-deficient cells. On the other hand, reducing expression of two enzymes responsible for triacylglycerol synthesis, diacylglycerol O-acyltransferase 2 (DGAT2) and glycerol-3-phosphate acyltransferase 4 (GPAT4), rescued the LD phenotype in CDS2-deficient, but not CDS1-deficient, cells. Moreover, CDS2 deficiency, but not CDS1 deficiency, promoted the LD association of DGAT2 and GPAT4 and impaired initial LD maturation. Finally, although both CDS1 and CDS2 appeared to regulate PA levels on the LD surface, CDS2 had a stronger effect. We conclude that CDS1 and CDS2 regulate LD dynamics through distinct mechanisms.
Asunto(s)
Diacilglicerol Colinafosfotransferasa/metabolismo , Gotas Lipídicas/metabolismo , Línea Celular , Diacilglicerol Colinafosfotransferasa/deficiencia , Diacilglicerol Colinafosfotransferasa/genética , Diacilglicerol O-Acetiltransferasa/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Ácidos Fosfatidicos/metabolismoRESUMEN
Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a large family of proteins that mainly function in lipid transport and sensing. ORP5 is an endoplasmic reticulum (ER)-anchored protein implicated in lipid transfer at the contact sites between the ER and other membranes. Recent studies indicate that ORP5 is also involved in cancer cell invasion and tumor progression. However, the molecular mechanism underlying ORP5's involvement in cancer is unclear. Here, we report that ORP5 promotes cell proliferation and motility of HeLa cells, an effect that depends on its functional OSBP-related domain (ORD). We also found that ORP5 depletion or substitutions of key residues located within ORP5-ORD and responsible for interactions with lipids interfered with cell proliferation, migration, and invasion. ORP5 interacted with the protein mechanistic target of rapamycin (mTOR), and this interaction also required ORP5-ORD. Of note, whereas ORP5 overexpression induced mTOR complex 1 (mTORC1) activity, ORP5 down-regulation had the opposite effect. Finally, ORP5-depleted cells exhibited impaired mTOR localization to lysosomes, which may have accounted for the blunted mTORC1 activation. Together, our results suggest that ORP5 expression is positively correlated with mTORC1 signaling and that ORP5 stimulates cell proliferation, at least in part, by activating mTORC1.
Asunto(s)
Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/agonistas , Neoplasias/metabolismo , Receptores de Esteroides/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Regulación hacia Arriba , Sustitución de Aminoácidos , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Activación Enzimática , Eliminación de Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Lisosomas/enzimología , Lisosomas/patología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Invasividad Neoplásica , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/enzimología , Neoplasias/patología , Mutación Puntual , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Interferencia de ARN , Receptores de Esteroides/antagonistas & inhibidores , Receptores de Esteroides/química , Receptores de Esteroides/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Adipose tissue can store over 50% of whole-body cholesterol; however, the physiological role of adipose tissue in cholesterol metabolism and atherogenesis has not been directly assessed. Here, we examined lipoprotein metabolism and atherogenesis in a unique mouse model of severe lipodystrophy: the Seipin(-/-) mice, and also in mice deficient in both low-density lipoprotein receptor (Ldlr) and Seipin: the Ldlr(-/-)Seipin(-/-) mice. Plasma cholesterol was moderately increased in the Seipin(-/-) mice when fed an atherogenic diet. Strikingly, plasma cholesterol reached ~6000 mg/dl in the Seipin(-/-)Ldlr(-/-) mice on an atherogenic diet, as compared to ~1000 mg/dl in the Ldlr(-/-) mice on the same diet. The Seipin(-/-)Ldlr(-/-) mice also developed spontaneous atherosclerosis on chow diet and severe atherosclerosis on an atherogenic diet. Rosiglitazone treatment significantly reduced the hypercholesterolemia of the Seipin(-/-)Ldlr(-/-) mice, and also alleviated the severity of atherosclerosis. Our results provide direct evidence, for the first time, that the adipose tissue plays a critical role in the clearance of plasma cholesterol. Our results also reveal a previously unappreciated strong link between adipose tissue and LDLR in plasma cholesterol metabolism.
Asunto(s)
Tejido Adiposo/metabolismo , Adiposidad , Aterosclerosis/metabolismo , Hiperlipidemias/metabolismo , Receptores de LDL/deficiencia , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/patología , Tejido Adiposo/fisiopatología , Animales , Aterosclerosis/genética , Aterosclerosis/patología , Aterosclerosis/fisiopatología , Aterosclerosis/prevención & control , Colesterol/sangre , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Subunidades gamma de la Proteína de Unión al GTP , Predisposición Genética a la Enfermedad , Proteínas de Unión al GTP Heterotriméricas/deficiencia , Proteínas de Unión al GTP Heterotriméricas/genética , Hiperlipidemias/genética , Hiperlipidemias/patología , Hiperlipidemias/fisiopatología , Hiperlipidemias/prevención & control , Hipolipemiantes/farmacología , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Receptores de LDL/genética , Rosiglitazona , Índice de Severidad de la Enfermedad , Tiazolidinedionas/farmacología , Factores de TiempoRESUMEN
The expansion of lipid droplets (LDs) and the differentiation of preadipocytes are two important aspects of mammalian lipid storage. In this study, we examined the role of CDP-diacylglycerol (DAG) synthases (CDSs), encoded by CDS1 and CDS2 genes in mammals, in lipid storage. CDS enzymes catalyze the formation of CDP-DAG from phosphatidic acid (PA). Knocking down either CDS1 or CDS2 resulted in the formation of giant or supersized LDs in cultured cells. Moreover, depleting CDS1 almost completely blocked the differentiation of 3T3-L1 preadipocytes, whereas depleting CDS2 had a moderate inhibitory effect on adipocyte differentiation. The levels of many PA species were significantly increased upon knocking down CDS1 In contrast, only a small number of PA species were increased upon depleting CDS2 Importantly, the amount of PA in the endoplasmic reticulum was dramatically increased upon knocking down CDS1 or CDS2 Our results suggest that the changes in PA level and localization may underlie the formation of giant LDs as well as the block in adipogenesis in CDS-deficient cells. We have therefore identified CDS1 and CDS2 as important novel regulators of lipid storage, and these results highlight the crucial role of phospholipids in mammalian lipid storage.
Asunto(s)
Adipocitos/enzimología , Diacilglicerol Colinafosfotransferasa/fisiología , Gotas Lipídicas/enzimología , Células 3T3-L1 , Animales , Diferenciación Celular , Expresión Génica , Células HeLa , Humanos , Metabolismo de los Lípidos , Ratones , Fosfatidato Fosfatasa/genética , Fosfatidato Fosfatasa/metabolismo , Fosfolípidos/metabolismo , Transporte de Proteínas , Triglicéridos/metabolismoRESUMEN
Null mutations of the Niemann-Pick type C1 (NPC1) gene cause NPC disease, a lysosomal storage disorder characterized by cholesterol accumulation in late endosomes (LE) and lysosomes (Ly). Nascent or mutated NPC1 is degraded through the ubiquitin-proteasome pathway, but how NPC1 degradation is regulated remains currently unknown. In the present study, we demonstrated a link between NPC1 degradation and the Akt (protein kinase B)/mTOR [mammalian (or mechanistic) target of rapamycin] signalling pathway in cervical cancer cell lines. We provided evidence that activated Akt/mTOR pathway increased NPC1 degradation by â¼50% in C33A cells when compared with SiHa or HeLa cells. NPC1 degradation in C33A cells was reversed when Akt/mTOR activation was blocked by specific inhibitors or when mTORC1 (mTOR complex 1) was disrupted by regulatory associated protein of mTOR (Raptor) knockdown. Importantly, inhibition of the Akt/mTOR pathway led to decreased NPC1 ubiquitination in C33A cells, pointing to a role of Akt/mTOR in the proteasomal degradation of NPC1. Moreover, we found that NPC1 depletion in several cancer cell lines inhibited cell proliferation and migration. Our results uncover Akt as a key regulator of NPC1 degradation and link NPC1 to cancer cell proliferation and migration.
Asunto(s)
Proteínas Portadoras/metabolismo , Colesterol/metabolismo , Glicoproteínas de Membrana/metabolismo , Neoplasias/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Portadoras/genética , Movimiento Celular/genética , Proliferación Celular/genética , Colesterol/genética , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Diana Mecanicista del Complejo 1 de la Rapamicina , Glicoproteínas de Membrana/genética , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Neoplasias/genética , Neoplasias/patología , Proteína Niemann-Pick C1 , Complejo de la Endopetidasa Proteasomal/genética , Proteínas Proto-Oncogénicas c-akt/genética , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The exit of low-density lipoprotein derived cholesterol (LDL-C) from late endosomes (LE)/lysosomes (Ly) is mediated by Niemann-Pick C1 (NPC1), a multipass integral membrane protein on the limiting membranes of LE/Ly, and by NPC2, a cholesterol-binding protein in the lumen of LE/Ly. NPC2 delivers cholesterol to the N-terminal domain of NPC1, which is believed to insert cholesterol into the limiting membrane for subsequent transport to other subcellular organelles. Few cytoplasmic factors have been identified to govern cholesterol efflux from LE/Ly, and much less is known about the underlying molecular mechanisms. Here we establish VPS4, an AAA ATPase that has a well-established role in disassembling the ESCRT (endosomal sorting complex required for transport)-III polymer, as an important regulator of endosomal cholesterol transport. Knocking down VPS4 in HeLa cells resulted in prominent accumulation of LDL-C in LE/Ly, and disrupted cholesterol homeostatic responses at the endoplasmic reticulum. The level and localization of NPC1 and NPC2 appeared to be normal in VPS4 knockdown cells. Importantly, depleting any of the ESCRT-III components did not exert a significant effect on endosomal cholesterol transport. Our results thus identify an important cytoplasmic regulator of endosomal cholesterol trafficking and represent the first functional separation of VPS4 from ESCRT-III.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , LDL-Colesterol/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Endosomas/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas/genética , Proteínas Portadoras/metabolismo , Retículo Endoplásmico/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Glicoproteínas/metabolismo , Células HeLa , Homeostasis , Humanos , Péptidos y Proteínas de Señalización Intracelular , Lisosomas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteína Niemann-Pick C1 , Transporte de Proteínas , ARN Interferente Pequeño , Proteínas de Transporte VesicularRESUMEN
The integrity of the plasma membrane is critical to cell function and survival. Cells have developed multiple mechanisms to repair damaged plasma membranes. A key process during plasma membrane repair is to limit the size of the damage, which is facilitated by the presence of tetraspanin-enriched rings surrounding damage sites. Here, we identify phosphatidylserine-enriched rings surrounding damaged sites of the plasma membrane, resembling tetraspanin-enriched rings. Importantly, the formation of both the phosphatidylserine- and tetraspanin-enriched rings requires phosphatidylserine and its transfer proteins ORP5 and ORP9. Interestingly, ORP9, but not ORP5, is recruited to the damage sites, suggesting cells acquire phosphatidylserine from multiple sources upon plasma membrane damage. We further demonstrate that ORP9 contributes to efficient plasma membrane repair. Our results thus unveil a role for phosphatidylserine and its transfer proteins in facilitating the formation of tetraspanin-enriched macrodomains and plasma membrane repair.
Asunto(s)
Membrana Celular , Fosfatidilserinas , Tetraspaninas , Humanos , Células HeLa , Proteínas de la Membrana/metabolismo , Receptores de Esteroides/metabolismoRESUMEN
The delivery of low-density lipoprotein-derived cholesterol (LDL-C) from endosomal compartments to the plasma membrane and the endoplasmic reticulum (ER) is an important yet poorly understood cellular process. Niemann-Pick C1 (NPC1), a multi-pass integral membrane protein on the limiting membranes of late endosomes (LE)/lysosomes (Ly), is known to insert lumenal LDL-C to the limiting membrane of LE/Ly. Recent progress has identified novel cytoplasmic proteins that regulate the exit of LDL-C from LE/Ly, such as ORP5, a member of the oxysterol-binding protein-related protein (ORPs) family, and Hrs/VPS27, a well-established regulator of the endosomal sorting complex required for transport pathway. Whereas ORP5/ORPs may serve as cytosolic cholesterol carriers and deliver cholesterol in a non-vesicular manner, how Hrs/VPS27 regulate endosomal cholesterol sorting remains enigmatic. We discuss the functional relationship between NPC1, Hrs, and ORP5, and formulate possible schemes on how LDL-C may be moved from endosomal compartments to other cellular organelles.
Asunto(s)
Colesterol/metabolismo , Endosomas/metabolismo , Proteínas/metabolismo , Animales , Transporte Biológico , Compartimento Celular , HumanosRESUMEN
Ubiquitous yet unique, lipid droplets are intracellular organelles that are increasingly being recognized for their versatility beyond energy storage. Advances uncovering the intricacies of their biogenesis and the diversity of their physiological and pathological roles have yielded new insights into lipid droplet biology. Despite these insights, the mechanisms governing the biogenesis and functions of lipid droplets remain incompletely understood. Moreover, the causal relationship between the biogenesis and function of lipid droplets and human diseases is poorly resolved. Here, we provide an update on the current understanding of the biogenesis and functions of lipid droplets in health and disease, highlighting a key role for lipid droplet biogenesis in alleviating cellular stresses. We also discuss therapeutic strategies of targeting lipid droplet biogenesis, growth or degradation that could be applied in the future to common diseases, such as cancer, hepatic steatosis and viral infection.
Asunto(s)
Gotas Lipídicas , Metabolismo de los Lípidos , Humanos , Gotas Lipídicas/metabolismo , Metabolismo de los Lípidos/fisiología , LipogénesisRESUMEN
Oxysterol-binding protein (OSBP) mediates lipid exchange between organelles at membrane contact sites, thereby regulating lipid dynamics and homeostasis. How OSBP's lipid transfer function impacts health and disease remain to be elucidated. In this review, we first summarize the structural characteristics and lipid transport functions of OSBP, and then focus on recent progresses linking OSBP with fatty liver disease, diabetes, lysosome-related diseases, cancer and viral infections, with the aim of discovering novel therapeutic strategies for common human diseases.
Asunto(s)
Transporte Biológico , Metabolismo de los Lípidos , Oxiesteroles , Humanos , Metabolismo de los Lípidos/fisiología , Lípidos , Oxiesteroles/metabolismoRESUMEN
Cholesterol synthesis is both energy- and oxygen-intensive, yet relatively little is known of the regulatory effects of hypoxia on pathway enzymes. We previously showed that the rate-limiting and first oxygen-dependent enzyme of the committed cholesterol synthesis pathway, squalene monooxygenase (SM), can undergo partial proteasomal degradation that renders it constitutively active. Here, we show hypoxia is a physiological trigger for this truncation, which occurs through a two-part mechanism: (1) increased targeting of SM to the proteasome via stabilization of the E3 ubiquitin ligase MARCHF6 and (2) accumulation of the SM substrate, squalene, which impedes the complete degradation of SM and liberates its truncated form. This preserves SM activity and downstream pathway flux during hypoxia. These results uncover a feedforward mechanism that allows SM to accommodate fluctuating substrate levels and may contribute to its widely reported oncogenic properties.
Cells need cholesterol to work properly but too much cholesterol is harmful and can contribute to atherosclerosis (narrowing of blood vessels), cancer and other diseases. Cells therefore carefully control the activity of the enzymes that are involved in making cholesterol, including an enzyme known as squalene monooxygenase. When the level of cholesterol in a cell rises, a protein called MARCHF6 adds molecules of ubiquitin to squalene monooxygenase. These molecules act as tags that direct the enzyme to be destroyed by a machine inside cells, known as the proteasome, thereby preventing further (unnecessary) production of cholesterol. Previous studies found that squalene monooxygenase is sometimes only partially broken down to make a shorter (truncated) form of the enzyme that is permanently active, even when the level of cholesterol in the cell is high. However, it was unclear what triggers this partial breakdown. The process of making cholesterol uses a lot of oxygen, yet many cancer cells thrive in tumours with low levels of oxygen. Here, Coates et al. used biochemical and cell biology approaches to study the effect of low oxygen levels on the activity of squalene monooxygenase in human cells. The experiments revealed that low oxygen levels trigger squalene monooxygenase to be partially degraded to make the truncated form of the enzyme. Firstly, MARCHF6 accumulates and adds ubiquitin to the enzyme to accelerate its delivery to the proteasome. Secondly, as the proteasome starts to degrade the enzyme, a build-up of squalene molecules impedes further breakdown of the enzyme. This mechanism preserves squalene monooxygenase activity when oxygen levels drop in cells, which may compensate for temporary oxygen shortfalls and allow cells to continue to make cholesterol. Squalene monooxygenase is overactive in individuals with a wide variety of diseases including fatty liver and prostate cancer. Drugs that block squalene monooxygenase activity have been shown to stop cancer cells from growing, but unfortunately these drugs are also toxic to mammals. These findings suggest that reducing the activity of squalene monooxygenase in more subtle ways, such as stopping it from being partially degraded, may be a more viable treatment strategy for cancer and other diseases associated with high levels of cholesterol.