Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 39
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
EMBO J ; : e104120, 2020 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-32128853

RESUMO

Protein prenylation is essential for many cellular processes including signal transduction, cytoskeletal reorganization, and membrane trafficking. Here, we identify a novel type of protein prenyltransferase, which we named geranylgeranyltransferase type-III (GGTase-III). GGTase-III consists of prenyltransferase alpha subunit repeat containing 1 (PTAR1) and the ß subunit of RabGGTase. Using a biotinylated geranylgeranyl analogue, we identified the Golgi SNARE protein Ykt6 as a substrate of GGTase-III. GGTase-III transfers a geranylgeranyl group to mono-farnesylated Ykt6, generating doubly prenylated Ykt6. The crystal structure of GGTase-III in complex with Ykt6 provides structural basis for Ykt6 double prenylation. In GGTase-III-deficient cells, Ykt6 remained in a singly prenylated form, and the Golgi SNARE complex assembly was severely impaired. Consequently, the Golgi apparatus was structurally disorganized, and intra-Golgi protein trafficking was delayed. Our findings reveal a fourth type of protein prenyltransferase that generates geranylgeranyl-farnesyl Ykt6. Double prenylation of Ykt6 is essential for the structural and functional organization of the Golgi apparatus.

2.
Proc Natl Acad Sci U S A ; 116(27): 13368-13373, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31217287

RESUMO

TMEM16K, a membrane protein carrying 10 transmembrane regions, has phospholipid scramblase activity. TMEM16K is localized to intracellular membranes, but whether it actually scrambles phospholipids inside cells has not been demonstrated, due to technical difficulties in studying intracellular lipid distributions. Here, we developed a freeze-fracture electron microscopy method that enabled us to determine the phosphatidylserine (PtdSer) distribution in the individual leaflets of cellular membranes. Using this method, we found that the endoplasmic reticulum (ER) of mammalian cells harbored abundant PtdSer in its cytoplasmic leaflet and much less in the luminal leaflet, whereas the outer and inner nuclear membranes (NMs) had equivalent amounts of PtdSer in both leaflets. The ER and NMs of budding yeast also harbored PtdSer in their cytoplasmic leaflet, but asymmetrical distribution in the ER was not observed. Treating mouse embryonic fibroblasts with the Ca2+ ionophore A23187 compromised the cytoplasmic leaflet-dominant PtdSer asymmetry in the ER and increased PtdSer in the NMs, especially in the nucleoplasmic leaflet of the inner NM. This Ca2+-induced PtdSer redistribution was not observed in TMEM16K-null fibroblasts, but was recovered in these cells by reexpressing TMEM16K. These results indicate that, similar to the plasma membrane, PtdSer in the ER of mammalian cells is predominantly localized to the cytoplasmic leaflet, and that TMEM16K directly or indirectly mediates Ca2+-dependent phospholipid scrambling in the ER.

3.
Nat Commun ; 10(1): 1230, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30862813

RESUMO

The original version of this Article contained errors in the Abstract and Introduction, whereby CCTα was incorrectly defined as an abbreviation of CDP-choline diacylglycerol phosphotransferase α, instead of CTP:phosphocholine cytidylyltransferase α. This has now been corrected in both the PDF and HTML versions of the Article.

4.
Oncogene ; 38(26): 5142-5157, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30894682

RESUMO

The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a transcriptional target of the lineage-survival oncogene NKX2-1/TTF-1 in lung adenocarcinomas. In addition to its kinase-dependent role, ROR1 functions as a scaffold protein to facilitate interaction between caveolin-1 (CAV1) and CAVIN1, and consequently maintains caveolae formation, which in turn sustains pro-survival signaling toward AKT from multiple receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR), MET (proto-oncogene, receptor tyrosine kinase), and IGF-IR (insulin-like growth factor receptor 1). Therefore, ROR1 is an attractive target for overcoming EGFR-TKI resistance due to various mechanisms such as EGFR T790M double mutation and bypass signaling from other RTKs. Here, we report that ROR1 possesses a novel scaffold function indispensable for efficient caveolae-dependent endocytosis. CAVIN3 was found to bind with ROR1 at a site distinct from sites for CAV1 and CAVIN1, a novel function required for proper CAVIN3 subcellular localization and caveolae-dependent endocytosis, but not caveolae formation itself. Furthermore, evidence of a mechanistic link between ROR1-CAVIN3 interaction and consequential caveolae trafficking, which was found to utilize a binding site distinct from those for ROR1 interactions with CAV1 and CAVIN1, with RTK-mediated pro-survival signaling towards AKT in early endosomes in lung adenocarcinoma cells was also obtained. The present findings warrant future study to enable development of novel therapeutic strategies for inhibiting the multifaceted scaffold functions of ROR1 in order to reduce the intolerable death toll from this devastating cancer.


Assuntos
Adenocarcinoma de Pulmão/patologia , Cavéolas/fisiologia , Endocitose , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/patologia , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Animais , Células COS , Cavéolas/metabolismo , Sobrevivência Celular/genética , Células Cultivadas , Endocitose/genética , Células HEK293 , Células HeLa , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ligação Proteica/fisiologia , Células Sf9 , Transdução de Sinais/genética , Spodoptera
5.
Nat Commun ; 10(1): 473, 2019 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-30692541

RESUMO

The origin and physiological significance of lipid droplets (LDs) in the nucleus is not clear. Here we show that nuclear LDs in hepatocytes are derived from apolipoprotein B (ApoB)-free lumenal LDs, a precursor to very low-density lipoproprotein (VLDL) generated in the ER lumen by microsomal triglyceride transfer protein. ApoB-free lumenal LDs accumulate under ER stress, grow within the lumen of the type I nucleoplasmic reticulum, and turn into nucleoplasmic LDs by disintegration of the surrounding inner nuclear membrane. Oleic acid with or without tunicamycin significantly increases the formation of nucleoplasmic LDs, to which CDP-choline diacylglycerol phosphotransferase α (CCTα) is recruited, resulting in activation of phosphatidylcholine (PC) synthesis. Perilipin-3 competes with CCTα in binding to nucleoplasmic LDs, and thus, knockdown and overexpression of perilipin-3 increases and decreases PC synthesis, respectively. The results indicate that nucleoplasmic LDs in hepatocytes constitute a feedback mechanism to regulate PC synthesis in accordance with ER stress.


Assuntos
Núcleo Celular/metabolismo , Gotículas Lipídicas/metabolismo , Lipoproteínas/metabolismo , Fosfatidilcolinas/biossíntese , Precursores de Proteínas/metabolismo , Células A549 , Animais , Linhagem Celular Tumoral , Colina-Fosfato Citidililtransferase/metabolismo , Células HEK293 , Células HeLa , Hepatócitos/metabolismo , Humanos , Ácido Oleico/metabolismo , Perilipina-3/metabolismo , Ratos
6.
EMBO J ; 37(21)2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30237312

RESUMO

PGAM5, a mitochondrial protein phosphatase that is genetically and biochemically linked to PINK1, facilitates mitochondrial division by dephosphorylating the mitochondrial fission factor Drp1. At the onset of mitophagy, PGAM5 is cleaved by PARL, a rhomboid protease that degrades PINK1 in healthy cells, and the cleaved form facilitates the engulfment of damaged mitochondria by autophagosomes by dephosphorylating the mitophagy receptor FUNDC1. Here, we show that the function and localization of PGAM5 are regulated by syntaxin 17 (Stx17), a mitochondria-associated membrane/mitochondria protein implicated in mitochondrial dynamics in fed cells and autophagy in starved cells. In healthy cells, loss of Stx17 causes PGAM5 aggregation within mitochondria and thereby failure of the dephosphorylation of Drp1, leading to mitochondrial elongation. In Parkin-mediated mitophagy, Stx17 is prerequisite for PGAM5 to interact with FUNDC1. Our results reveal that the Stx17-PGAM5 axis plays pivotal roles in mitochondrial division and PINK1/Parkin-mediated mitophagy.


Assuntos
Dinâmica Mitocondrial , Proteínas Mitocondriais/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Qa-SNARE/metabolismo , Transdução de Sinais , Autofagossomos/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Metaloproteases/genética , Metaloproteases/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Mitocondriais/genética , Fosfoproteínas Fosfatases/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteólise , Proteínas Qa-SNARE/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
7.
Acta Histochem Cytochem ; 50(5): 141-147, 2017 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-29276316

RESUMO

Phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2] is a phosphoinositide that plays important roles in signal transduction, endocytosis, and cell migration among others. The intracellular distribution of PtdIns(3,4)P2 has mainly been studied by observing the distribution of GFP-tagged PtdIns(3,4)P2-binding protein domains in live cells and by labeling with anti-PtdIns(3,4)P2 antibody in fixed cell samples, but these methods only offer low spatial resolution results and may have pitfalls. In the present study, we developed an electron microscopic method to observe the PtdIns(3,4)P2 distribution using the SDS-treated freeze-fracture replica labeling method. The recombinant GST-tagged pleckstrin homology (PH) domain of TAPP1 was used as the binding probe, and its binding to PtdIns(3,4)P2 in the freeze-fracture replica was confirmed by using liposomes containing different phosphoinositides and by the lack of labeling by a mutant probe, in which one amino acid in the PH domain was substituted. The method was applied to NIH3T3 cell samples and showed that the increase of PtdIns(3,4)P2 in cells treated with hydrogen peroxide occurs in the cytoplasmic leaflet of the plasma membrane, except in the caveolar membrane. The present method can define the distribution of PtdIns(3,4)P2 at a high spatial resolution and will facilitate our understanding of the physiological function of this less studied phosphoinositide.

8.
Elife ; 62017 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-28590904

RESUMO

Niemann-Pick type C is a storage disease caused by dysfunction of NPC proteins, which transport cholesterol from the lumen of lysosomes to the limiting membrane of that compartment. Using freeze fracture electron microscopy, we show here that the yeast NPC orthologs, Ncr1p and Npc2p, are essential for formation and expansion of raft-like domains in the vacuolar (lysosome) membrane, both in stationary phase and in acute nitrogen starvation. Moreover, the expanded raft-like domains engulf lipid droplets by a microautophagic mechanism. We also found that the multivesicular body pathway plays a crucial role in microautophagy in acute nitrogen starvation by delivering sterol to the vacuole. These data show that NPC proteins promote microautophagy in stationary phase and under nitrogen starvation conditions, likely by increasing sterol in the limiting membrane of the vacuole.


Assuntos
Autofagia , Proteínas de Transporte/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Leveduras/fisiologia , Colesterol/metabolismo , Microscopia Crioeletrônica , Vacúolos/ultraestrutura , Leveduras/ultraestrutura
9.
J Cell Biol ; 212(1): 29-38, 2016 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-26728854

RESUMO

Lipid droplets (LDs) in the nucleus of hepatocyte-derived cell lines were found to be associated with premyelocytic leukemia (PML) nuclear bodies (NBs) and type I nucleoplasmic reticulum (NR) or the extension of the inner nuclear membrane. Knockdown of PML isoform II (PML-II) caused a significant decrease in both nuclear LDs and type I NR, whereas overexpression of PML-II increased both. Notably, these effects were evident only in limited types of cells, in which a moderate number of nuclear LDs exist intrinsically, and PML-II was targeted not only at PML NBs, but also at the nuclear envelope, excluding lamins and SUN proteins. Knockdown of SUN proteins induced a significant increase in the type I NR and nuclear LDs, but these effects were cancelled by simultaneous knockdown of PML-II. Nuclear LDs harbored diacylglycerol O-acyltransferase 2 and CTP:phosphocholine cytidylyltransferase α and incorporated newly synthesized lipid esters. These results corroborated that PML-II plays a critical role in generating nuclear LDs in specific cell types.


Assuntos
Núcleo Celular/metabolismo , Gotículas Lipídicas/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Endogâmicos , Proteína da Leucemia Promielocítica , Isoformas de Proteínas/metabolismo
10.
Nat Commun ; 7: 10060, 2016 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-26725982

RESUMO

The receptor tyrosine kinase-like orphan receptor 1 (ROR1) sustains prosurvival signalling directly downstream of the lineage-survival oncogene NKX2-1/TTF-1 in lung adenocarcinoma. Here we report an unanticipated function of this receptor tyrosine kinase (RTK) as a scaffold of cavin-1 and caveolin-1 (CAV1), two essential structural components of caveolae. This kinase-independent function of ROR1 facilitates the interactions of cavin-1 and CAV1 at the plasma membrane, thereby preventing the lysosomal degradation of CAV1. Caveolae structures and prosurvival signalling towards AKT through multiple RTKs are consequently sustained. These findings provide mechanistic insight into how ROR1 inhibition can overcome EGFR-tyrosine kinase inhibitor (TKI) resistance due to bypass signalling via diverse RTKs such as MET and IGF-IR, which is currently a major clinical obstacle. Considering its onco-embryonic expression, inhibition of the scaffold function of ROR1 in patients with lung adenocarcinoma is an attractive approach for improved treatment of this devastating cancer.


Assuntos
Caveolina 1/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Adenocarcinoma/terapia , Antineoplásicos/farmacologia , Caveolina 1/genética , Linhagem Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Neoplasias Pulmonares/terapia , Fosforilação , Análise Serial de Proteínas , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Transdução de Sinais
11.
Traffic ; 17(2): 154-67, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26563567

RESUMO

Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2 ) has critical functions in endosomes and lysosomes. We developed a method to define nanoscale distribution of PtdIns(3,5)P2 using freeze-fracture electron microscopy. GST-ATG18-4×FLAG was used to label PtdIns(3,5)P2 and its binding to phosphatidylinositol 3-phosphate (PtdIns(3)P) was blocked by an excess of the p40(phox) PX domain. In yeast exposed to hyperosmotic stress, PtdIns(3,5)P2 was concentrated in intramembrane particle (IMP)-deficient domains in the vacuolar membrane, which made close contact with adjacent membranes. The IMP-deficient domain was also enriched with PtdIns(3)P, but was deficient in Vph1p, a liquid-disordered domain marker. In yeast lacking either PtdIns(3,5)P2 or its effector, Atg18p, the IMP-deficient, PtdIns(3)P-rich membranes were folded tightly to make abnormal tubular structures, thus showing where the vacuolar fragmentation process is arrested when PtdIns(3,5)P2 metabolism is defective. In HeLa cells, PtdIns(3,5)P2 was significantly enriched in the vesicular domain of RAB5- and RAB7-positive endosome/lysosomes of the tubulo-vesicular morphology. This biased distribution of PtdIns(3,5)P2 was also observed using fluorescence microscopy, which further showed enrichment of a retromer component, VPS35, in the tubular domain. This is the first report to show segregation of PtdIns(3,5)P2 -rich and -deficient domains in endosome/lysosomes, which should be important for endosome/lysosome functionality.


Assuntos
Membrana Celular/metabolismo , Endossomos/metabolismo , Lisossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Animais , Células COS , Linhagem Celular , Linhagem Celular Tumoral , Células HeLa , Humanos , Estrutura Terciária de Proteína , Vacúolos/metabolismo , Leveduras/metabolismo
12.
Mol Biol Cell ; 26(12): 2333-42, 2015 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-25904333

RESUMO

Adipocyte triglyceride lipase (ATGL) is the major enzyme involved in the hydrolysis of triglycerides. The Arf1-coat protein complex I (COPI) machinery is known to be engaged in the recruitment of ATGL to lipid droplets (LDs), but the regulatory mechanism has not been clarified. In the present study, we found that ELMOD2, a putative noncanonical Arf-GTPase activating protein (GAP) localizing in LDs, plays an important role in controlling ATGL transport to LDs. We showed that knockdown of ELMOD2 by RNA interference induced an increase in the amount of ATGL existing in LDs and decreased the total cellular triglycerides. These effects of ELMOD2 knockdown were canceled by transfection of small interfering RNA-resistant cDNA of wild-type ELMOD2 but not by that of mutated ELMOD2 lacking the Arf-GAP activity. ELMOD2 was distributed in the endoplasmic reticulum and mitochondria as well as in LDs, but palmitoylation was required only for distribution to LDs. An ELMOD2 mutant deficient in palmitoylation failed to reconstitute the ATGL transport after the ELMOD2 knockdown, indicating that distribution in LDs is indispensable to the functionality of ELMOD2. These results indicate that ELMOD2 regulates ATGL transport and cellular lipid metabolism by modulating the Arf1-COPI activity in LDs.


Assuntos
Adipócitos/metabolismo , Proteínas do Citoesqueleto/metabolismo , Lipase/metabolismo , Gotículas Lipídicas/metabolismo , Lipoilação , Fator 1 de Ribosilação do ADP/metabolismo , Adipócitos/enzimologia , Complexo I de Proteína do Envoltório/metabolismo , Regulação da Expressão Gênica , Humanos , Lipase/genética , Triglicerídeos/metabolismo
13.
Dev Cell ; 32(3): 304-17, 2015 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-25619926

RESUMO

Recent evidence suggests that endoplasmic reticulum (ER) tubules mark the sites where the GTPase Drp1 promotes mitochondrial fission via a largely unknown mechanism. Here, we show that the SNARE protein syntaxin 17 (Syn17) is present on raft-like structures of ER-mitochondria contact sites and promotes mitochondrial fission by determining Drp1 localization and activity. The hairpin-like C-terminal hydrophobic domain, including Lys-254, but not the SNARE domain, is important for this regulation. Syn17 also regulates ER Ca(2+) homeostasis and interferes with Rab32-mediated regulation of mitochondrial dynamics. Starvation disrupts the Syn17-Drp1 interaction, thus favoring mitochondrial elongation during autophagy. Because we also demonstrate that Syn17 is an ancient SNARE, our findings suggest that Syn17 is one of the original key regulators for ER-mitochondria contact sites present in the last eukaryotic common ancestor. As such, Syn17 acts as a switch that responds to nutrient conditions and integrates functions for the ER and autophagosomes with mitochondrial dynamics.


Assuntos
Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/fisiologia , Proteínas Qa-SNARE/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Células HeLa , Humanos , Proteínas Mitocondriais/metabolismo , Fagossomos/metabolismo
14.
Artigo em Inglês | MEDLINE | ID: mdl-25451556

RESUMO

To address preventive effects of n-3 PUFAs/LC n-3 PUFAs on CRTs, a randomized controlled trial was conducted. One-hundred four experimental group participants were advised to increase intake of n-3 PUFAs, including fish/shell fish, fish oil supplements and perilla oils, and to decrease consumption of n-6 PUFAs and fats/oils as a whole for 24 months. One-hundred one control group participants were only cautioned to reduce consumption of fats/oils as a whole. Random allocation was satisfactorily attained, and participants sufficiently complied with our regimen. Intakes, plasma concentrations, and compositions of the RBC and sigmoid colon membranes of n-3 PUFAs, LC n-3 PUFAs, EPA and DHA increased, and the ratios of n-6 PUFAs/n-3 PUFAs and AA/LC n-3 PUFAs decreased without any adverse response. Twenty-four months after the intervention, the multivariate-adjusted hazard ratio (95% confidence intervals) was estimated to be 0.805 (0.536-1.209) with a signal towards the reduced CRT incidence.


Assuntos
Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/prevenção & controle , Gorduras Insaturadas na Dieta/administração & dosagem , Ácidos Graxos Ômega-3/administração & dosagem , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Neoplasias Colorretais/sangue , Neoplasias Colorretais/cirurgia , Gorduras Insaturadas na Dieta/sangue , Seguimentos , Humanos , Adesão à Medicação , Resultado do Tratamento
15.
ACS Chem Biol ; 9(10): 2217-22, 2014 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-25122546

RESUMO

Choline-containing phospholipids (Cho-PLs) are major components of all cellular membranes. We developed an electron microscopic technique to investigate the poorly understood problem of how Cho-PLs are distributed between membrane leaflets. Our method relies on generating freeze-fracture replicas of cells metabolically labeled with the choline analog, propargylcholine, followed by "click" reaction to conjugate biotin to propargylcholine head groups, and immunodetection of biotin with colloidal gold. Using this method in budding yeast, we found that, surprisingly, the Golgi and plasma membrane display a cytoplasmic leaflet-dominant asymmetry in Cho-PL distribution; in contrast, Cho-PLs are evenly distributed between the exoplasmic and cytoplasmic leaflets of other organelle membranes. In mammalian culture cells, the plasma membrane shows symmetrical Cho-PL distribution between leaflets, suggesting a fundamental difference between yeast and mammals. Our method should be expandable to other classes of lipids and will be useful for deciphering the mechanism responsible for generating lipid asymmetry in biological membranes.


Assuntos
Carcinoma Hepatocelular/metabolismo , Colina/metabolismo , Química Click/métodos , Técnica de Fratura por Congelamento/métodos , Neoplasias Hepáticas/metabolismo , Microscopia Eletrônica/métodos , Fosfolipídeos/metabolismo , Saccharomyces cerevisiae/metabolismo , Membrana Celular/metabolismo , Cromatografia em Camada Delgada , Citoplasma/metabolismo , Complexo de Golgi/metabolismo , Humanos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Células Tumorais Cultivadas
16.
Nat Commun ; 5: 3207, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24492518

RESUMO

Phosphatidylinositol 3-kinase is indispensable for autophagy but it is not well understood how its product, phosphatidylinositol 3-phosphate (PtdIns(3)P), participates in the biogenesis of autophagic membranes. Here, by using quick-freezing and freeze-fracture replica labelling, which enables determination of the nanoscale distributions of membrane lipids, we show that PtdIns(3)P in yeast autophagosomes is more abundant in the luminal leaflet (the leaflet facing the closed space between the outer and inner autophagosomal membranes) than in the cytoplasmic leaflet. This distribution is drastically different from that of the mammalian autophagosome in which PtdIns(3)P is confined to the cytoplasmic leaflet. In mutant yeast lacking two cytoplasmic phosphatases, ymr1Δ and sjl3Δ, PtdIns(3)P in the autophagosome is equally abundant in the two membrane leaflets, suggesting that the PtdIns(3)P asymmetry in wild-type yeast is generated by unilateral hydrolysis. The observed differences in PtdIns(3)P distribution suggest that autophagy in yeast and mammals may involve substantially different processes.


Assuntos
Autofagia , Fagossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Saccharomyces cerevisiae/fisiologia , Coloração e Rotulagem/métodos , Técnica de Fratura por Congelamento , Humanos , Membranas Intracelulares/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo
17.
Hepatology ; 59(4): 1591-9, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24214142

RESUMO

UNLABELLED: Autophagy can degrade aggregate-prone proteins, but excessive autophagy can have adverse effects. It would be beneficial if autophagy could be enhanced in a cell type-specific manner, but this has been difficult because the basic mechanism of autophagy is common. In the present study we found that inhibition of Niemann-Pick-type C1-like 1 (NPC1L1) by ezetimibe activates autophagy only in hepatocytes and small intestinal epithelia, but not in other cells. Ezetimibe induced accumulation of free cholesterol in the late endosome/lysosome and increased partitioning of a Ragulator component, LAMTOR1, in rafts. The latter change led to down-regulation of mammalian target of rapamycin (mTOR)C1 activity by decreasing mTOR recruitment to the late endosome/lysosome and activated autophagy. A primary effect of ezetimibe was found to be a decrease of free cholesterol in the plasma membrane, because all the results caused by ezetimibe were suppressed by supplementation of cholesterol as a methyl-ß-cyclodextrin complex. By enhancing autophagy in human primary hepatocytes with ezetimibe, insoluble mutant α1-antitrypsin Z was reduced significantly. CONCLUSION: Inhibition of NPC1L1 by ezetimibe activates autophagy in human hepatocytes by modulating cholesterol homeostasis. Ezetimibe may be used to ameliorate liver degeneration in α1-antitrypsin deficiency.


Assuntos
Autofagia/efeitos dos fármacos , Azetidinas/farmacologia , Hepatócitos/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Mutação/genética , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , Anticolesterolemiantes/farmacologia , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Colesterol/metabolismo , Ezetimiba , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Homeostase/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lisossomos/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo
18.
FEBS Lett ; 587(22): 3696-702, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-24100140

RESUMO

ApoB-crescent, an endoplasmic reticulum (ER)-lipid droplet amalgamation structure, is a useful marker to indicate aberrant lipidated apolipoprotein B accumulation in the hepatocyte ER. Blockade of the ER-to-Golgi transport by either vesicle transport inhibitors or dominant-negative Arf1 caused a significant increase in ApoB-crescents. However, a low concentration of Brefeldin A induced the same result without affecting protein secretion, suggesting ADP-ribosylation as an additional mechanism. ADP-ribosylation inhibitors not only suppressed the increase of ApoB-crescents, but also rapidly dissolved existing ApoB-crescents. These results implicate the involvement of ADP-ribosylation in the ApoB-crescent formation and maintenance process at the ER.


Assuntos
Adenosina Difosfato Ribose/metabolismo , Apolipoproteínas B/metabolismo , Retículo Endoplasmático/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , 3-Iodobenzilguanidina/farmacologia , Brefeldina A/farmacologia , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Células Hep G2 , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipoilação , Transporte Proteico/efeitos dos fármacos
19.
Methods Cell Biol ; 116: 227-51, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24099296

RESUMO

The lipid droplet (LD) is different from other cellular organelles in that most of its volume is made of lipid esters and its surface is lined by a phospholipid monolayer. This uniquely lipid-dominant structure poses a problem for electron microscopy (EM) because the aldehydes commonly used as a fixative do not react with most lipids. To circumvent this difficulty and utilize the high resolving power of EM, many methods have been developed. In this chapter, we discuss methods that have been used and/or are potentially useful to study LDs. The methods include conventional EM to observe the LD core, cryoelectron microscopy to observe the LD surface, freeze-substitution, immunoelectron microscopy (pre-embedding, post-embedding, and cryosectioning methods), and freeze-fracture. Each method has strong and weak points and therefore some caution is necessary in interpreting the obtained results. In combination with methods of other disciplines, the electron microscopic techniques should contribute significantly to solving the remaining questions on LDs.


Assuntos
Microscopia Crioeletrônica , Corpos de Inclusão/ultraestrutura , Triglicerídeos/química , Aldeídos/química , Animais , Linhagem Celular , Substituição ao Congelamento , Humanos , Corpos de Inclusão/química , Corpos de Inclusão/metabolismo , Compostos Organometálicos/química , Tetróxido de Ósmio/química
20.
PLoS One ; 7(8): e42379, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22879956

RESUMO

Lipid droplets (LDs) in non-adipocytes contain triglycerides (TG) and cholesterol esters (CE) in variable ratios. TG-rich LDs are generated when unsaturated fatty acids are administered, but the conditions that induce CE-rich LD formation are less well characterized. In the present study, we found that protein translation inhibitors such as cycloheximide (CHX) induced generation of CE-rich LDs and that TIP47 (perilipin 3) was recruited to the LDs, although the expression of this protein was reduced drastically. Electron microscopy revealed that LDs formed in CHX-treated cells possess a distinct electron-dense rim that is not found in TG-rich LDs, whose formation is induced by oleic acid. CHX treatment caused upregulation of mTORC1, but the CHX-induced increase in CE-rich LDs occurred even when rapamycin or Torin1 was given along with CHX. Moreover, the increase in CE was seen in both wild-type and autophagy-deficient Atg5-null mouse embryonic fibroblasts, indicating that mTORC1 activation and suppression of autophagy are not necessary to induce the observed phenomenon. The results showed that translation inhibitors cause a significant change in the lipid ester composition of LDs by a mechanism independent of mTORC1 signaling and autophagy.


Assuntos
Ésteres do Colesterol/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Animais , Autofagia/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Cicloeximida/farmacologia , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Complexos Multiproteicos , Perilipina-3 , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , Serina-Treonina Quinases TOR
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA