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1.
J Cell Sci ; 135(5)2022 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-34000034

RESUMO

Membrane phase separation to form micron-scale domains of lipids and proteins occurs in artificial membranes; however, a similar large-scale phase separation has not been reported in the plasma membrane of the living cells. We show here that a stable micron-scale protein-depleted region is generated in the plasma membrane of yeast mutants lacking phosphatidylserine at high temperatures. We named this region the 'void zone'. Transmembrane proteins and certain peripheral membrane proteins and phospholipids are excluded from the void zone. The void zone is rich in ergosterol, and requires ergosterol and sphingolipids for its formation. Such properties are also found in the cholesterol-enriched domains of phase-separated artificial membranes, but the void zone is a novel membrane domain that requires energy and various cellular functions for its formation. The formation of the void zone indicates that the plasma membrane in living cells has the potential to undergo phase separation with certain lipid compositions. We also found that void zones were frequently in contact with vacuoles, in which a membrane domain was also formed at the contact site.

2.
Mol Biol Cell ; : mbcE20110699, 2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-34038161

RESUMO

Sterols are important lipid components of the plasma membrane (PM) in eukaryotic cells, but it is unknown how the PM retains sterols at a high concentration. Phospholipids are asymmetrically distributed in the PM, and phospholipid flippases play an important role in generating this phospholipid asymmetry. Here, we provide evidence that phospholipid flippases are essential for retaining ergosterol in the PM of yeast. A mutant in three flippases, Dnf1-Lem3, Dnf2-Lem3, and Dnf3-Crf1, and a membrane protein, Sfk1, showed a severe growth defect. We recently identified Sfk1 as a PM protein involved in phospholipid asymmetry. The PM of this mutant showed high permeability and low density. Staining with the sterol probe filipin and the expression of a sterol biosensor revealed that ergosterol was not retained in the PM. Instead, ergosterol accumulated in an esterified form in lipid droplets. We propose that ergosterol is retained in the PM by the asymmetrical distribution of phospholipids and the action of Sfk1. Once phospholipid asymmetry is severely disrupted, sterols might be exposed on the cytoplasmic leaflet of the PM and actively transported to the endoplasmic reticulum by sterol transfer proteins.

3.
PLoS One ; 15(7): e0236520, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32730286

RESUMO

In eukaryotic cells, phospholipid flippases translocate phospholipids from the exoplasmic to the cytoplasmic leaflet of the lipid bilayer. Budding yeast contains five flippases, of which Cdc50p-Drs2p and Neo1p are primarily involved in membrane trafficking in endosomes and Golgi membranes. The ANY1/CFS1 gene was identified as a suppressor of growth defects in the neo1Δ and cdc50Δ mutants. Cfs1p is a membrane protein of the PQ-loop family and is localized to endosomal/Golgi membranes, but its relationship to phospholipid asymmetry remains unknown. The neo1Δ cfs1Δ mutant appears to function normally in membrane trafficking but may function abnormally in the regulation of phospholipid asymmetry. To identify a gene that is functionally relevant to NEO1 and CFS1, we isolated a mutation that is synthetically lethal with neo1Δ cfs1Δ and identified ERD1. Erd1p is a Golgi membrane protein that is involved in the transport of phosphate (Pi) from the Golgi lumen to the cytoplasm. The Neo1p-depleted cfs1Δ erd1Δ mutant accumulated plasma membrane proteins in the Golgi, perhaps due to a lack of phosphatidylinositol 4-phosphate. The Neo1p-depleted cfs1Δ erd1Δ mutant also exhibited abnormal structure of the endoplasmic reticulum (ER) and induced an unfolded protein response, likely due to defects in the retrieval pathway from the cis-Golgi region to the ER. Genetic analyses suggest that accumulation of Pi in the Golgi lumen is responsible for defects in Golgi functions in the Neo1p-depleted cfs1Δ erd1Δ mutant. Thus, the luminal ionic environment is functionally relevant to phospholipid asymmetry. Our results suggest that flippase-mediated phospholipid redistribution and luminal Pi concentration coordinately regulate Golgi membrane functions.


Assuntos
Complexo de Golgi/metabolismo , Fosfatos/metabolismo , Fosfolipídeos/metabolismo , Saccharomyces cerevisiae/genética , Adenosina Trifosfatases/genética , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Mutação , Proteínas de Transferência de Fosfolipídeos/genética , Receptores Citoplasmáticos e Nucleares/genética , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Resposta a Proteínas não Dobradas
5.
Intern Med ; 59(12): 1515-1517, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32188808

RESUMO

Vasovagal reactions are the most common type of adverse reaction after blood donation; however, there are no reports of ischemic colitis as an adverse reaction after blood donation. A previously healthy 55-year-old woman suffered loss of consciousness at the end of her first plasma donation. She was diagnosed with a vasovagal reaction and received hydration. However, she developed persistent left flank pain and watery diarrhea, followed by bloody diarrhea. Abdominal computed tomography confirmed ischemic colitis. She was asked to fast and was eventually discharged 7 days later. We should consider the possibility of ischemic colitis if patients develop persistent abdominal pain after transient hypotension, such as that observed during a vasovagal reaction.


Assuntos
Doadores de Sangue , Colite Isquêmica/complicações , Síncope Vasovagal/complicações , Testes Diagnósticos de Rotina , Feminino , Humanos , Pessoa de Meia-Idade , Tomografia Computadorizada por Raios X
6.
FASEB J ; 34(5): 6185-6197, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32162745

RESUMO

During adhesion, cells develop filopodia to facilitate the attachment to the extracellular matrix. The small guanosine triphosphate (GTP)-binding protein, Cdc42, plays a central role in the formation of filopodia. It has been reported that Cdc42 activity is regulated by cholesterol (Chol). We examined Chol distribution in filopodia using Chol-binding domain 4 (D4) fragment of bacterial toxin, perfringolysin O that senses high membrane concentration of Chol. Our results indicate that fluorescent D4 was enriched at the tip of the outer leaflet of filopodia in the initiation phase of cell adhesion. This enrichment was accompanied by a defect of D4 labeling in the inner leaflet. Steady phase adhered cell experiment indicated that both Cdc42 and ATP-binding cassette transporter, ABCA1, were involved in the binding of D4 to the cell surface. Depletion of Chol activated Cdc42. Our results suggest that asymmetric distribution of Chol at the tip of filopodia induces activation of Cdc42, and thus, facilitates filopodia formation.

7.
Sci Rep ; 10(1): 3161, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-32081926

RESUMO

This study aimed to explore novel microRNAs in plasma for predicting chemoresistance in adjuvant chemotherapy for patients with gastric cancer (GC). We used the Toray 3D-Gene microRNA array-based approach to compare preoperative plasma microRNA levels between GC patients with and without recurrences after curative gastrectomy. All patients underwent adjuvant chemotherapy with S-1, an oral fluoropyrimidine. Of 2566 candidates, six candidate microRNAs (miR-1229-3p, 1249-5p, 762, 711, 1268a and 1260b), which were highly expressed in the preoperative plasma of patients with subsequent recurrences, were selected. In a large-scale validation analysis by quantitative RT-PCR, we focused on high plasma levels of miR-1229-3p, which was an independent poor prognostic factor for recurrence free survival (P = 0.009, HR = 3.71). Overexpression of miR-1229-3p in GC cells induced significant chemoresistance to 5-fluorouracil (5-FU), up-regulation of thymidylate synthase (TS) and dihydroprimidine dehydrogenase (DPD) and down-regulation of SLC22A7 both in vitro and in vivo. Intraperitoneal injection of miR-1229-3p in mice induced significant chemoresistance to 5-FU, accompanied by high levels of miR-1229-3p in plasma and tumor tissue. These findings suggest that plasma miR-1229-3p might be a clinically useful biomarker for predicting chemoresistance to S-1 and selecting other or combined intensive chemotherapy regimens in GC patients.

8.
Gan To Kagaku Ryoho ; 45(4): 706-708, 2018 Apr.
Artigo em Japonês | MEDLINE | ID: mdl-29650842

RESUMO

Anastomotic recurrence in 6 colorectal cancer cases during the postoperative surveillance between 2008 and 2015 was evaluated retrospectively. Five cases had undergone DST anastomosis for sigmoidectomy and proctectomy. They had a pathological tendency to have T3 tumor and deeper, positive lymph node metastases and positive lymphatic and vascular invasion. There were 2 cases with the anastomotic recurrence diagnosis 6 to 8 months after the primary tumor resection while 4 resected cases had recurrent tumor depth of T3, though 3 cases were diagnosed 1 year after the primary tumor resection. Anastomotic recurrence should be considered a few months after primary tumor resection.


Assuntos
Neoplasias Colorretais/diagnóstico , Idoso , Anastomose Cirúrgica , Neoplasias Colorretais/cirurgia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Estudos Retrospectivos
9.
Mol Biol Cell ; 29(10): 1203-1218, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29540528

RESUMO

Phospholipid flippase (type 4 P-type ATPase) plays a major role in the generation of phospholipid asymmetry in eukaryotic cell membranes. Loss of Lem3p-Dnf1/2p flippases leads to the exposure of phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the cell surface in yeast, resulting in sensitivity to PS- or PE-binding peptides. We isolated Sfk1p, a conserved membrane protein in the TMEM150/FRAG1/DRAM family, as a multicopy suppressor of this sensitivity. Overexpression of SFK1 decreased PS/PE exposure in lem3Δ mutant cells. Consistent with this, lem3Δ sfk1Δ double mutant cells exposed more PS/PE than the lem3Δ mutant. Sfk1p was previously implicated in the regulation of the phosphatidylinositol-4 kinase Stt4p, but the effect of Sfk1p on PS/PE exposure in lem3Δ was independent of Stt4p. Surprisingly, Sfk1p did not facilitate phospholipid flipping but instead repressed it, even under ATP-depleted conditions. We propose that Sfk1p negatively regulates transbilayer movement of phospholipids irrespective of directions. In addition, we showed that the permeability of the plasma membrane was dramatically elevated in the lem3Δ sfk1Δ double mutant in comparison with the corresponding single mutants. Interestingly, total ergosterol was decreased in the lem3Δ sfk1Δ mutant. Our results suggest that phospholipid asymmetry is required for the maintenance of low plasma membrane permeability.


Assuntos
Permeabilidade da Membrana Celular , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Fosfolipídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Depsipeptídeos/farmacologia , Ergosterol/farmacologia , Bicamadas Lipídicas/metabolismo , Fluidez de Membrana/efeitos dos fármacos , Proteínas de Membrana/química , Modelos Biológicos , Mutação/genética , Fosfatidiletanolaminas/metabolismo , Fosfatidilinositóis/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Saccharomyces cerevisiae/química , Estresse Fisiológico/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
10.
Gan To Kagaku Ryoho ; 45(2): 288-290, 2018 Feb.
Artigo em Japonês | MEDLINE | ID: mdl-29483423

RESUMO

Re-laparotomy with resection of the mesh after abdominal incisional hernia repair may cause recurrence of the hernia and infection of the mesh. In the present study, we performed laparoscopic distal gastrectomy(LDG)for early gastric cancer without the resection of the mesh in such a case. A 82-year-old man who had undergone abdominal vascular replacement, cholecystectomy, abdominal incisional hernia repair with the mesh, sigmoidectomy had local recurrence of gastric cancer after endoscopic submucosal resection. We diagnosed as cStage IA and performed LDG without resection of the mesh. He had no recurrence of hernia nor infection of the mesh. Minimizing damage to the abdominal wall by laparoscopic surgery can prevent them.


Assuntos
Abdome/cirurgia , Hérnia Ventral/cirurgia , Laparoscopia , Neoplasias Gástricas/cirurgia , Idoso de 80 Anos ou mais , Gastrectomia , Hérnia Ventral/complicações , Humanos , Laparoscopia/instrumentação , Laparoscopia/métodos , Laparotomia , Masculino , Neoplasias Gástricas/complicações , Telas Cirúrgicas
11.
FASEB J ; 31(4): 1301-1322, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27492925

RESUMO

We identified a novel, nontoxic mushroom protein that specifically binds to a complex of sphingomyelin (SM), a major sphingolipid in mammalian cells, and cholesterol (Chol). The purified protein, termed nakanori, labeled cell surface domains in an SM- and Chol-dependent manner and decorated specific lipid domains that colocalized with inner leaflet small GTPase H-Ras, but not K-Ras. The use of nakanori as a lipid-domain-specific probe revealed altered distribution and dynamics of SM/Chol on the cell surface of Niemann-Pick type C fibroblasts, possibly explaining some of the disease phenotype. In addition, that nakanori treatment of epithelial cells after influenza virus infection potently inhibited virus release demonstrates the therapeutic value of targeting specific lipid domains for anti-viral treatment.-Makino, A., Abe, M., Ishitsuka, R., Murate, M., Kishimoto, T., Sakai, S., Hullin-Matsuda, F., Shimada, Y., Inaba, T., Miyatake, H., Tanaka, H., Kurahashi, A., Pack, C.-G., Kasai, R. S., Kubo, S., Schieber, N. L., Dohmae, N., Tochio, N., Hagiwara, K., Sasaki, Y., Aida, Y., Fujimori, F., Kigawa, T., Nishibori, K., Parton, R. G., Kusumi, A., Sako, Y., Anderluh, G., Yamashita, M., Kobayashi, T., Greimel, P., Kobayashi, T. A novel sphingomyelin/cholesterol domain-specific probe reveals the dynamics of the membrane domains during virus release and in Niemann-Pick type C.


Assuntos
Colesterol/metabolismo , Proteínas Fúngicas/farmacologia , Grifola/química , Microdomínios da Membrana/efeitos dos fármacos , Doença de Niemann-Pick Tipo C/metabolismo , Esfingomielinas/metabolismo , Sítios de Ligação , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Células HeLa , Humanos , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/virologia , Ligação Proteica , Liberação de Vírus
12.
Gan To Kagaku Ryoho ; 44(12): 1320-1322, 2017 Nov.
Artigo em Japonês | MEDLINE | ID: mdl-29394620

RESUMO

Retroperitoneal liposarcoma is a relatively rare tumor. The only established therapy is surgical resection and the tumor often recurs. This paper deals with a case of a retroperitoneal liposarcoma in which frequent surgical resections for recurrent tumors have provided relatively long-term survival for the patient. The patient was a 70-year-old woman who had undergone surgical resection for a right retroperitoneal tumor. The pathological diagnosis was dedifferentiated liposarcoma. Thereafter she experienced frequent recurrences which required 3 surgical resections. By means of positive margin for the last surgery, chemotherapy with eribulin was administered. There has been no recurrence 13 months after the last surgery.


Assuntos
Lipossarcoma/cirurgia , Neoplasias Retroperitoneais/cirurgia , Idoso , Feminino , Humanos , Lipossarcoma/secundário , Prognóstico , Neoplasias Retroperitoneais/secundário , Fatores de Tempo
13.
Gan To Kagaku Ryoho ; 44(12): 1874-1876, 2017 Nov.
Artigo em Japonês | MEDLINE | ID: mdl-29394805

RESUMO

A 67-year-old male was referred to our hospital for further investigation of fecal occult blood. We diagnosed him with rectal cancer with osseous metastasis. Chemo-and radiation therapy were administered following resection of the rectal cancer. There were no other lesions except for the osseous metastasis remaining after these interventions. The osseous lesion was then resected. There have no signs of recurrence for 1 year and 9 months since the last operation. We report a case of successful resection of osseous metastasis from rectal cancer.


Assuntos
Adenocarcinoma/terapia , Neoplasias Ósseas/terapia , Neoplasias Retais/terapia , Adenocarcinoma/secundário , Adenocarcinoma/cirurgia , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Ósseas/secundário , Neoplasias Ósseas/cirurgia , Quimiorradioterapia , Humanos , Masculino , Neoplasias Retais/patologia , Neoplasias Retais/cirurgia
14.
Clin Case Rep ; 4(9): 896-8, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27648270

RESUMO

Cervical lymphangioma can cause airway obstruction secondary to enlargement following infection. Physicians should be aware that the airway obstruction can progress rapidly when patients with cervical lymphangioma have respiratory symptoms. Sclerotherapy for lymphangioma can cause both transient swelling and airway obstruction; thus, prophylactic and elective tracheostomy should be considered.

15.
Proc Natl Acad Sci U S A ; 113(28): 7834-9, 2016 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-27342861

RESUMO

Lipid membrane curvature plays important roles in various physiological phenomena. Curvature-regulated dynamic membrane remodeling is achieved by the interaction between lipids and proteins. So far, several membrane sensing/sculpting proteins, such as Bin/amphiphysin/Rvs (BAR) proteins, are reported, but there remains the possibility of the existence of unidentified membrane-deforming proteins that have not been uncovered by sequence homology. To identify new lipid membrane deformation proteins, we applied liposome-based microscopic screening, using unbiased-darkfield microscopy. Using this method, we identified phospholipase Cß1 (PLCß1) as a new candidate. PLCß1 is well characterized as an enzyme catalyzing the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2). In addition to lipase activity, our results indicate that PLCß1 possessed the ability of membrane tubulation. Lipase domains and inositol phospholipids binding the pleckstrin homology (PH) domain of PLCß1 were not involved, but the C-terminal sequence was responsible for this tubulation activity. Computational modeling revealed that the C terminus displays the structural homology to the BAR domains, which is well known as a membrane sensing/sculpting domain. Overexpression of PLCß1 caused plasma membrane tubulation, whereas knockdown of the protein reduced the number of caveolae and induced the evagination of caveolin-rich membrane domains. Taken together, our results suggest a new function of PLCß1: plasma membrane remodeling, and in particular, caveolae formation.


Assuntos
Cavéolas/fisiologia , Fosfolipase C beta/metabolismo , Animais , Lipossomos , Camundongos , Camundongos Endogâmicos C57BL , Células Swiss 3T3
16.
Diabetes ; 65(6): 1648-59, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26953164

RESUMO

VAMP7 is a SNARE protein that mediates specific membrane fusions in intracellular trafficking and was recently reported to regulate autophagosome formation. However, its function in pancreatic ß-cells is largely unknown. To elucidate the physiological role of VAMP7 in ß-cells, we generated pancreatic ß-cell-specific VAMP7 knockout (Vamp7(flox/Y);Cre) mice. VAMP7 deletion impaired glucose-stimulated ATP production and insulin secretion, though VAMP7 was not localized to insulin granules. VAMP7-deficient ß-cells showed defective autophagosome formation and reduced mitochondrial function. p62/SQSTM1, a marker protein for defective autophagy, was selectively accumulated on mitochondria in VAMP7-deficient ß-cells. These findings suggest that accumulation of dysfunctional mitochondria that are degraded by autophagy caused impairment of glucose-stimulated ATP production and insulin secretion in Vamp7(flox/Y);Cre ß-cells. Feeding a high-fat diet to Vamp7(flox/Y);Cre mice exacerbated mitochondrial dysfunction, further decreased ATP production and insulin secretion, and consequently induced glucose intolerance. Moreover, we found upregulated VAMP7 expression in wild-type mice fed a high-fat diet and in db/db mice, a model for diabetes. Thus our data indicate that VAMP7 regulates autophagy to maintain mitochondrial quality and insulin secretion in response to pathological stress in ß-cells.


Assuntos
Autofagia/fisiologia , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Mitocôndrias/fisiologia , Proteínas R-SNARE/fisiologia , Trifosfato de Adenosina/biossíntese , Animais , Dieta Hiperlipídica/efeitos adversos , Glucose/metabolismo , Intolerância à Glucose/metabolismo , Homeostase , Secreção de Insulina , Masculino , Camundongos , Camundongos Knockout , Proteínas R-SNARE/deficiência
17.
Biochim Biophys Acta ; 1861(8 Pt B): 812-829, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26993577

RESUMO

Although sphingomyelin and cholesterol are major lipids of mammalian cells, the detailed distribution of these lipids in cellular membranes remains still obscure. However, the recent development of protein probes that specifically bind sphingomyelin and/or cholesterol provides new information about the landscape of the lipid domains that are enriched with sphingomyelin or cholesterol or both. Here, we critically summarize the tools to study distribution and dynamics of sphingomyelin and cholesterol. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.


Assuntos
Colesterol/metabolismo , Lipídeos de Membrana/análise , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Técnicas de Sonda Molecular , Esfingomielinas/metabolismo , Animais , Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Humanos , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Sondas Moleculares/análise , Sondas Moleculares/química , Peso Molecular
18.
J Anesth ; 30(1): 20-5, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26545801

RESUMO

PURPOSE: Palatoplasty carries a high risk of airway obstruction as a postoperative complication. Since 2007, the protocol in our hospital has been to leave an endotracheal tube in place after surgery while the patient is moved to the pediatric intensive care unit. Extubation is then performed after achievement of hemostasis and recovery of consciousness. We compared the cases over the 5-year periods before and after the introduction of this revised postsurgical management plan to investigate its effect on postoperative complications. METHODS: This was a retrospective cohort study involving a single pediatric hospital. The subjects were 199 children aged 1-3 years, who underwent palatoplasty between January 2002 and July 2012. Changes in the incidence rates of postoperative complications were statistically examined. RESULTS: There were significantly more postoperative complications among the patients who were extubated in the operating room than among those extubated in the intensive care unit (operating room group, 22/94 cases; intensive care unit group, 10/105 cases; P < 0.01). Serious complications, such as hypoxemia and airway obstruction, also occurred more frequently in the operating room group. CONCLUSION: Extubation in an intensive care unit was possibly associated with a reduction in postoperative complications.


Assuntos
Extubação/efeitos adversos , Fissura Palatina/cirurgia , Intubação Intratraqueal/efeitos adversos , Complicações Pós-Operatórias/etiologia , Obstrução das Vias Respiratórias/complicações , Pré-Escolar , Estudos de Coortes , Feminino , Humanos , Lactente , Unidades de Terapia Intensiva Pediátrica , Masculino , Salas Cirúrgicas , Estudos Retrospectivos , Risco
19.
J Biol Chem ; 290(39): 23464-77, 2015 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-26198636

RESUMO

Cellular cholesterol homeostasis involves sterol sensing at the endoplasmic reticulum (ER) and sterol export from the plasma membrane (PM). Sterol sensing at the ER requires efficient sterol delivery from the PM; however, the macromolecules that facilitate retrograde sterol transport at the PM have not been identified. ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol and phospholipid export to apolipoprotein A-I for the assembly of high density lipoprotein (HDL). Mutations in ABCA1 cause Tangier disease, a familial HDL deficiency. Several lines of clinical and experimental evidence suggest a second function of ABCA1 in cellular cholesterol homeostasis in addition to mediating cholesterol efflux. Here, we report the unexpected finding that ABCA1 also plays a key role in facilitating retrograde sterol transport from the PM to the ER for sterol sensing. Deficiency in ABCA1 delays sterol esterification at the ER and activates the SREBP-2 cleavage pathway. The intrinsic ATPase activity in ABCA1 is required to facilitate retrograde sterol transport. ABCA1 deficiency causes alternation of PM composition and hampers a clathrin-independent endocytic activity that is required for ER sterol sensing. Our finding identifies ABCA1 as a key macromolecule facilitating bidirectional sterol movement at the PM and shows that ABCA1 controls retrograde sterol transport by modulating a certain clathrin-independent endocytic process.


Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Retículo Endoplasmático/metabolismo , Esteróis/metabolismo , Animais , Transporte Biológico , Células Cultivadas , Metabolismo dos Lipídeos , Camundongos , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo
20.
FASEB J ; 29(9): 3920-34, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26060215

RESUMO

Ceramide phosphoethanolamine (CPE), a sphingomyelin analog, is a major sphingolipid in invertebrates and parasites, whereas only trace amounts are present in mammalian cells. In this study, mushroom-derived proteins of the aegerolysin family­pleurotolysin A2 (PlyA2; K(D) = 12 nM), ostreolysin (Oly; K(D) = 1.3 nM), and erylysin A (EryA; K(D) = 1.3 nM)­strongly associated with CPE/cholesterol (Chol)-containing membranes, whereas their low affinity to sphingomyelin/Chol precluded establishment of the binding kinetics. Binding specificity was determined by multilamellar liposome binding assays, supported bilayer assays, and solid-phase studies against a series of neutral and negatively charged lipid classes mixed 1:1 with Chol or phosphatidylcholine. No cross-reactivity was detected with phosphatidylethanolamine. Only PlyA2 also associated with CPE, independent of Chol content (K(D) = 41 µM), rendering it a suitable tool for visualizing CPE in lipid-blotting experiments and biologic samples from sterol auxotrophic organisms. Visualization of CPE enrichment in the CNS of Drosophila larvae (by PlyA2) and in the bloodstream form of the parasite Trypanosoma brucei (by EryA) by fluorescence imaging demonstrated the versatility of aegerolysin family proteins as efficient tools for detecting and visualizing CPE.


Assuntos
Proteínas Fúngicas/química , Proteínas Hemolisinas/química , Esfingomielinas/química , Esfingomielinas/metabolismo , Animais , Drosophila melanogaster , Larva/química , Larva/metabolismo
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