論文 - 大﨑　雄樹

All-trans-retinol generated by rhodopsin photobleaching induces rapid recruitment of TIP47 to lipid droplets in the retinal pigment epithelium

Eiko Tsuiki, Akikazu Fujita, Yuki Ohsaki, Jinglei Cheng, Toshiaki Irie, Kiwamu Yoshikawa, Haruki Senoo, Kazuaki Mishima, Takashi Kitaoka, Toyoshi Fujimoto

INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE ( ASSOC RESEARCH VISION OPHTHALMOLOGY INC )  48 ( 6 ) 2858 - 2867  2007年06月  [査読有り]  [国際誌]

　概要を見る

Purpose. To investigate the effect of light stimulation on lipid droplets (LDs) and LD proteins in the retinal pigment epithelium (RPE). Methods. Dark-adapted mouse eyes were exposed to intense flashes of light, and ARPE-19 cells were treated with all-trans-retinol. The two specimens were labeled with BODIPY493/503 for LDs and with antibodies for three LD proteins: adipocyte differentiation-related protein (ADRP), TIP47, and Rab18. The labeling intensity in fluorescence microscopy was quantified by image analysis. Localization of mutated TIP47 was also examined. Immunoelectron microscopy was performed for ADRP in mouse RPE. Expression of TIP47 in ARPE-19 cells was knocked down by RNA interference (RNAi), and its effect on retinyl ester storage was measured by HPLC. Results. Both flashes of light on mouse eyes and all-trans-retinol on ARPE-19 cells caused rapid translocation of TIP47 from the cytosol to LDs, whereas ADRP distributed constitutively in LDs. The density of LDs did not show visible changes by any treatment. The localization of TIP47 to LDs was abolished when either the amino-terminal or the carboxyl-terminal half of the molecule was deleted, but was enhanced by a short deletion in the carboxyl terminus. Manipulation of TIP47 expression by RNAi or cDNA transfection did not affect the retinyl ester amounts in ARPE-19 cells significantly. Conclusions. All-trans-retinol generated by photobleaching in the retina induces rapid translocation of TIP47 to LDs in the RPE.

DOI PubMed

Endosomal accumulation of Toll-like receptor 4 causes constitutive secretion of cytokines and activation of signal transducers and activators of transcription in Niemann-Pick disease type C (NPC) fibroblasts: A potential basis for glial cell activation in the NPC brain

Michitaka Suzuki, Yuko Sugimoto, Yuki Ohsaki, Makoto Ueno, Shinsuke Kato, Yukisato Kitamura, Hiroshi Hosokawa, Joanna P. Davies, Yiannis A. Ioannou, Marie T. Vanier, Kousaku Ohno, Haruaki Ninomiya

JOURNAL OF NEUROSCIENCE ( SOC NEUROSCIENCE )  27 ( 8 ) 1879 - 1891  2007年02月  [査読有り]  [国際誌]

　概要を見る

Niemann-Pick disease type C (NPC) is an inherited lipid storage disorder caused by mutations in NPC1 or NPC2 genes. Loss of function of either protein results in the endosomal accumulation of cholesterol and other lipids, progressive neurodegeneration, and robust glial cell activation. Here, we report that cultured human NPC fibroblasts secrete interferon-beta, interleukin- 6 (IL-6), and IL-8, and contain increased levels of signal transducers and activators of transcription (STATs). These cells also contained increased levels of Toll-like receptor 4 (TLR4) that accumulated in cholesterol-enriched endosomes/lysosomes, and small interfering RNA knockdown of this receptor reduced cytokine secretion. In the NPC1(-/-) mouse brain, glial cells expressed TLR4 and IL-6, whereas both glial and neuronal cells expressed STATs. Genetic deletion of TLR4 in NPC1(-/-) mice reduced IL-6 secretion by cultured fibroblasts but failed to alter STAT levels or glial cell activation in the brain. In contrast, genetic deletion of IL-6 normalized STAT levels and suppressed glial cell activation. These findings indicate that constitutive cytokine secretion leads to activation of STATs in NPC fibroblasts and that this secretion is partly caused by an endosomal accumulation of TLR4. These results also suggest that similar signaling events may underlie glial cell activation in the NPC1(-/-) mouse brain.

DOI PubMed

Cholesterol depletion induces autophagy.

Jinglei Cheng, Yuki Ohsaki, Kumi Tauchi-Sato, Akikazu Fujita, Toyoshi Fujimoto

Biochemical and biophysical research communications   351 ( 1 ) 246 - 52  2006年12月  [国際誌]

　概要を見る

Autophagy is a mechanism to digest cells' own components, and its importance in many physiological and pathological processes is being recognized. But the molecular mechanism that regulates autophagy is not understood in detail. In the present study, we found that cholesterol depletion induces macroautophagy. The cellular cholesterol in human fibroblasts was depleted either acutely using 5mM methyl-beta-cyclodextrin or 10-20microg/ml nystatin for 1h, or metabolically by 20microM mevastatin and 200microM mevalonolactone along with 10% lipoprotein-deficient serum for 2-3 days. By any of these protocols, marked increase of LC3-II was detected by immunoblotting and by immunofluorescence microscopy, and the increase was more extensive than that caused by amino acid starvation, i.e., incubation in Hanks' solution for several hours. The induction of autophagic vacuoles by cholesterol depletion was also observed in other cell types, and the LC3-positive membranes were often seen as long tubules, >50microm in length. The increase of LC3-II by methyl-beta-cyclodextrin was suppressed by phosphatidylinositol 3-kinase inhibitors and was accompanied by dephosphorylation of mammalian target of rapamycin. By electron microscopy, autophagic vacuoles induced by cholesterol depletion were indistinguishable from those seen after amino acid starvation. These results demonstrate that a decrease in cholesterol activates autophagy by a phosphatidylinositol 3-kinase-dependent mechanism.

PubMed

Cheng J, Ohsaki Y, Tauchi-Sato K, Fujita A, Fujimoto T.

Cheng J, Ohsaki Y, Tauchi-Sato K, Fujita A, Fujimoto T

Biochem. Biophys. Res. Commun.   351 ( 1 ) 246-252  2006年12月  [査読有り]

　概要を見る

Autophagy is a mechanism to digest cells' own components, and its importance in many physiological and pathological processes is being recognized. But the molecular mechanism that regulates autophagy is not understood in detail. In the present study, we found that cholesterol depletion induces macroautophagy. The cellular cholesterol in human fibroblasts was depleted either acutely using 5mM methyl-beta-cyclodextrin or 10-20microg/ml nystatin for 1h, or metabolically by 20microM mevastatin and 200microM mevalonolactone along with 10% lipoprotein-deficient serum for 2-3 days. By any of these protocols, marked increase of LC3-II was detected by immunoblotting and by immunofluorescence microscopy, and the increase was more extensive than that caused by amino acid starvation, i.e., incubation in Hanks' solution for several hours. The induction of autophagic vacuoles by cholesterol depletion was also observed in other cell types, and the LC3-positive membranes were often seen as long tubules, >50microm in length. The increase of LC3-II by methyl-beta-cyclodextrin was suppressed by phosphatidylinositol 3-kinase inhibitors and was accompanied by dephosphorylation of mammalian target of ra

Recruitment of TIP47 to lipid droplets is controlled by the putative hydrophobic cleft

Yuki Ohsaki, Takashi Maeda, Marl Maeda, Kumi Tauchi-Sato, Toyoshi Fujimoto

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ( ACADEMIC PRESS INC ELSEVIER SCIENCE )  347 ( 1 ) 279 - 287  2006年08月  [査読有り]  [国際誌]

担当区分：   筆頭著者

　概要を見る

Adipose differentiation-related protein (ADRP) and TIP47 show sequence similarity, particularly in their N-terminal PAT-1 domain. Under standard culture conditions, ADRP existed in most lipid droplets (LDs), whereas TIP47 was observed only in some LDs and recruited to LDs on treatment with fatty acids. By analyzing deletion mutants, we found that the C-terminal half of TIP47, or more specifically the putative hydrophobic cleft [S.J. Hickenbottom, A.R. Kimmel, C. Londos, J.H. Hurley, Structure of a lipid droplet protein the PAT family member TIP47, Structure (Camb) 12 (2004) 1199-1207.], was involved in LD targeting and responsiveness to fatty acids. The result contrasted with that observed for ADRP and implied a distinct LD-targeting mechanism for TIP47. Consistent with this, overexpression of Rab18 decreased ADRP, but not TIP47, from LDs, and TIP47 did not displace pre-existing ADRP from LDs. But ADRP may be a factor to control the TIP47 behavior, because TIP47 in LDs increased upon down-regulation of ADRP. The results suggested that the putative hydrophobic cleft is critical for the unique characteristics of TIP47. (c) 2006 Elsevier Inc. All rights reserved.

DOI PubMed

Cholesterol depletion facilitates ubiquitylation of NPC1 and its association with SKD1/Vps4

Y Ohsaki, Y Sugimoto, M Suzuki, H Hosokawa, T Yoshimori, JP Davies, YA Ioannou, MT Vanier, K Ohno, H Ninomiya

JOURNAL OF CELL SCIENCE ( COMPANY OF BIOLOGISTS LTD )  119 ( 13 ) 2643 - 2653  2006年07月  [査読有り]  [国際誌]

担当区分：   筆頭著者

　概要を見る

Niemann-Pick disease type C (NPC) is an inherited lipid storage disorder caused by mutations in NPC1 or NPC2. NPC1 is a polytopic glycoprotein that contains a sterol-sensing domain, whereas NPC2 is a soluble protein that contains an MD-2-like lipid-recognition domain. In the current study, we addressed the hypothesis that ubiquitylation of NPC1 might be regulated by cholesterol. We found that depletion of cellular cholesterol facilitated ubiquitylation of NPC1 expressed in COS cells. A loss-of-function mutant, NPC1( P691S), which contains an amino acid substitution in the sterol-sensing domain, failed to respond to cholesterol depletion. Another mutant, NPC1(delta LLNF), which lacks the endosomal-targeting motif, also failed to respond. SKD1(E235Q), a dominant-negative mutant of SKD1/Vps4 that inhibits disassembly of the endosomal sorting complex required for transport (ESCRT), caused an accumulation of ubiquitylated NPC1. SKD1( E235Q) associated with NPC1 on the endosomal membrane, whereas wild-type SKD1 associated with NPC1 only when cells were depleted of cholesterol. Similarly, in control human skin fibroblasts, cholesterol depletion facilitated ubiquitylation of endogenous NPC1. In patient cells that lack NPC2 function, NPC1 was ubiquitylated regardless of cellular cholesterol levels, suggesting that NPC2 is required to prevent NPC1 ubiquitylation under cholesterol-rich conditions. These results suggest that ubiquitylation of NPC1 and its association with the ESCRT complex are controlled by endosomal cholesterol levels utilizing a mechanism that involves NPC2.

DOI PubMed

Cytoplasmic lipid droplets are sites of convergence of proteasomal and autophagic degradation of apolipoprotein B

Yuki Ohsaki, Jinglei Cheng, Akikazu Fujita, Toshinobu Tokumoto, Toyoshi Fujimoto

MOLECULAR BIOLOGY OF THE CELL ( AMER SOC CELL BIOLOGY )  17 ( 6 ) 2674 - 2683  2006年06月  [査読有り]  [国際誌]

担当区分：   筆頭著者  , 責任著者

　概要を見る

Lipid esters stored in cytoplasmic lipid droplets (CLDs) of hepatocytes are used to synthesize very low-density lipoproteins (VLDLs), into which apolipoprotein B (ApoB) is integrated cotranslationally. In the present study, by using Huh7 cells, derived from human hepatoma and competent for VLDL secretion, we found that ApoB is highly concentrated around CLDs to make "ApoB-crescents." ApoB-crescents were seen in < 10% of Huh7 cells under normal conditions, but the ratio increased to nearly 50% after 12 h of proteasomal inhibition by N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal. Electron microscopy showed ApoB to be localized to a cluster of electron-lucent particles 50-100 nm in diameter adhering to CLDs. ApoB, proteasome subunits, and ubiquitinated proteins were detected in the CLD fraction, and this ApoB was ubiquitinated. Interestingly, proteasome inhibition also caused increases in autophagic vacuoles and ApoB in lysosomes. ApoB-crescents began to decrease after 12-24 h of proteasomal. inhibition, but the decrease was blocked by an autophagy inhibitor, 3-methyladenine. Inhibition of autophagy alone caused an increase in ApoB-crescents. These observations indicate that both proteasomal and autophagy/lysosomal degradation of ApoB occur around CLDs and that the CLD surface functions as a unique platform for convergence of the two pathways.

DOI PubMed

Cytoplasmic lipid droplets - Rediscovery of an old structure as a unique platform

Toyoshi Fujimoto, Yuki Ohsaki

INTEGRATED MOLECULAR MEDICINE FOR NEURONAL AND NEOPLASTIC DISORDERS ( BLACKWELL PUBLISHING )  1086   104 - 115  2006年  [査読有り]  [国際誌]

　概要を見る

Cytoplasmic lipid droplets (LDs) exist in virtually any kind of cell. They have a core of lipid esters covered by the surface phospholipid monolayer. A number of proteins related to various cell functions are present in LDs and/or LD-rich subcellular fractions, suggesting that LDs are an independent organelle that is engaged in various cellular activities. Furthermore, a recent study suggested that LDs are a platform where the proteasomal and autophagic pathways converge. The molecular composition and architecture of LDs are discussed here, with special reference to the technical difficulties encountered when analyzing this unique organelle.

DOI PubMed

Proteasomal and autophagic pathways converge on lipid droplets

Toyoshi Fujimoto, Yuki Ohsaki

Autophagy ( Taylor and Francis Inc. )  2 ( 4 ) 299 - 301  2006年  [査読有り]  [招待有り]

　概要を見る

Apolipoprotein B (apoB) is the primary protein of very low-density lipoproteins (VLDL). We found that apoB accumulated on the surface of cytoplasmic lipid droplets (LDs) of hepatocytes when the proteasomal or autophagic processes were suppressed. ApoB associated with LDs was poly-ubiquitinated and surrounded by autophagic vacuoles. Moreover, proteasomal subunits were concentrated around LDs. Our data suggest that apoB that is destined to be degraded remains adhered to LDs until it is broken down by the proteasomal and autophagic pathways. We speculate that the LD surface serves as a platform to prevent hydrophobic apoB from forming aggregates, and that LDs may play a similar role for other aggregation-prone hydrophobic proteins. ©2006 Landes Bioscience.

DOI PubMed

Fixation and permeabilization protocol is critical for the immunolabeling of lipid droplet proteins

Y Ohsaki, T Maeda, T Fujimoto

HISTOCHEMISTRY AND CELL BIOLOGY ( SPRINGER )  124 ( 5 ) 445 - 452  2005年11月  [査読有り]  [国際誌]

担当区分：   筆頭著者

　概要を見る

The number of proteins known to be associated with lipid droplets (LDs) is increasing. However, the reported distribution of a given protein in the LDs was, in some cases, found not reproduced by other groups. We report here that the choice of the fixation and permeabilization method is important in order to observe LD proteins using immunofluorescence microscopy. Formaldehyde fixation followed by treatment with Triton X-100, one of the most frequently used protocols for the immunolabeling of cultured cells, was not appropriate to label adipocyte differentiation-related protein (ADRP), TIP47, or Rab18 in LDs. Formaldehyde fixation followed by treatment with digitonin or saponin, allowed the visualization of all these proteins in LDs. When cells were fixed with glutaraldehyde, permeabilization by Triton X-100 could also be used for ADRP. These observations suggest that LD proteins are likely to be solubilized by some detergents, and strong cross-linkage to the surrounding protein matrix or mild permeabilization is necessary for their retention on the LD surface.

DOI PubMed

カベオラ，ラフトと血管

藤本豊士, 藤田豊, 大崎雄樹

血管医学   5   455-461  2004年10月  [招待有り]

Accumulation of cholera toxin and GM1 ganglioside in the early endosome of niemann-pick C1-deficient cells

Yuko Sugimoto, Haruaki Ninomiya, Yuki Ohsaki, Katsumi Higaki, Joanna P. Davies, Yiannis A. Ioannou, Kousaku Ohno

Proceedings of the National Academy of Sciences of the United States of America   98 ( 22 ) 12391 - 12396  2001年10月

　概要を見る

We investigated intracellular trafficking of GM1 ganglioside in Niemann-Pick C1 (NPC1)-deficient Chinese hamster ovary cells [NPC1(-) cells] by using cholera toxin (CT) as a probe. Both the holotoxin and the B subunit (CTB) accumulated in GM1-enriched intracellular vesicles of NPC1(-) cells. CTB-labeled vesicles contained the early endosome marker Rab5 but not lysosome-associated membrane protein 2 and were not labeled with either Texas red-transferrin or Lysotracker, indicating that they represent early endosomes. Similarly, CT accumulated in intracellular vesicles of human NPC fibroblasts that contained both Rab5 and early endosomal antigen 1. CTB accumulation in NPC1(-) cells was abolished by expression of wild-type NPC1 but not by mutant proteins with a mutation either in the NPC domain or the sterol-sensing domain. A part of these mutant NPC1 proteins expressed in NPC1(-) cells was localized on CTB-labeled vesicles. U18666A treatment of "knock in" cells [NPC1(-) cells that stably expressed wild-type NPC1] caused CTB accumulation similar to that in NPC1(-) cells, and a part of wild-type NPC1was localized on CTB-labeled vesicles in drug-treated cells. Finally, CT tracer experiments in NPC1(-) cells revealed retarded excretion of internalized toxin into the culture medium and an increase in the intracellular release of A subunits. In accordance with the latter result, CT was more effective in stimulating cAMP formation in NPC1(-) than in wild-type cells. These results suggest that transport of CT/GM1 complexes from the early endosome to the plasma membrane depends on the function of NPC1, whereas transport to the Golgi apparatus/ endoplasmic reticulum does not.

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