Details of a Researcher - OOSAKI Yuuki

Papers - OOSAKI Yuuki

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Lipid droplets: size matters

Michitaka Suzuki, Yuki Shinohara, Yuki Ohsaki, Toyoshi Fujimoto

JOURNAL OF ELECTRON MICROSCOPY ( OXFORD UNIV PRESS ) 60 ( 1 ) S101 - S116 2011.08 [Refereed] [Domestic journal]

The lipid droplet (LD), an organelle that exists ubiquitously in various organisms, from bacteria to mammals, has attracted much attention from both medical and cell biology fields. The LD in white adipocytes is often treated as the prototype LD, but is rather a special example, considering that its size, intracellular localization and molecular composition are vastly different from those of non-adipocyte LDs. These differences confer distinct properties on adipocyte and non-adipocyte LDs. In this article, we address the current understanding of LDs by discussing the differences between adipocyte and non-adipocyte LDs.

Role of ORPs in Sterol Transport from Plasma Membrane to ER and Lipid Droplets in Mammalian Cells

Maurice Jansen, Yuki Ohsaki, Laura Rita Rega, Robert Bittman, Vesa M. Olkkonen, Elina Ikonen

TRAFFIC ( WILEY-BLACKWELL PUBLISHING, INC ) 12 ( 2 ) 218 - 231 2011.02 [Refereed] [International journal]

In this study, we investigated the mechanisms of sterol transport from the plasma membrane (PM) to the endoplasmic reticulum (ER) and lipid droplets (LDs) in HeLa cells. By overexpressing all mammalian oxysterol-binding protein-related proteins (ORPs), we found that especially ORP1S and ORP2 enhanced PM-to-LD sterol transport. This reflected the stimulation of transport from the PM to the ER, rather than from the ER to LDs. Double knockdown of ORP1S and ORP2 inhibited sterol transport from the PM to the ER and LDs, suggesting a physiological role for these ORPs in the process. A two phenylalanines in an acidic tract (FFAT) motif in ORPs that mediates interaction with VAMP-associated proteins (VAPs) in the ER was not necessary for the enhancement of sterol transport by ORPs. However, VAP-A and VAP-B silencing slowed down PM-to-LD sterol transport. This was accompanied by enhanced degradation of ORP2 and decreased levels of several FFAT motif-containing ORPs, suggesting a role for VAPs in sterol transport by stabilization of ORPs.

Lysosomal accumulation of mTOR is enhanced by rapamycin

Yuki Ohsaki, Michitaka Suzuki, Yuki Shinohara, Toyoshi Fujimoto

HISTOCHEMISTRY AND CELL BIOLOGY ( SPRINGER ) 134 ( 6 ) 537 - 544 2010.12 [Refereed] [International journal]

Authorship： Lead author , Corresponding author

The mammalian target of rapamycin (mTOR) is a key regulator of cell growth that integrates signals from growth factors and nutrients. Recent studies have shown that an mTOR-containing complex, mTORC1, is targeted to lysosomes in the presence of amino acids and activated by Rheb GTPase resident in that compartment. In this study, we found that treatment with the mTOR inhibitors rapamycin and Torin1 significantly enhanced lysosomal accumulation of mTOR and Raptor. This phenomenon was not observed in the absence of amino acids but was restored upon addition of l-leucine or protein synthesis inhibitors. mTOR was not concentrated in autophagosomes that were induced by rapamycin. These results suggest that the lysosome harbors both active and inactive forms of mTOR in the presence of amino acids.

Lipoprotein lipase and hepatic triglyceride lipase reduce the infectivity of hepatitis C virus (HCV) through their catalytic activities on HCV-associated lipoproteins

Yuko Shimizu, Takayuki Hishiki, Kazuo Sugiyama, Kazuya Ogawa, Kenji Funami, Atsushi Kato, Yuki Ohsaki, Toyoshi Fujimoto, Hiroshi Takaku, Kunitada Shimotohno

VIROLOGY ( ACADEMIC PRESS INC ELSEVIER SCIENCE ) 407 ( 1 ) 152 - 159 2010.11 [Refereed] [International journal]

The effect of lipolysis by lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) on hepatitis C virus (HCV) infection was evaluated. First, medium from HuH7.5 cells bearing HCV genome replication was treated with LPL. LPL treatment led to reduced HCV infectivity, shifted HCV to higher densities, and lowered the amount of apolipoprotein E-associated HCV. The effect of endogenous HTGL secreted from HuH7.5 on HCV infectivity was next examined. Neutralization of HTGL by an anti-HTGL antibody resulted in suppression of LPL-induced reduction in infectivity of HCV-bearing medium, while knockdown of HTGL by siRNA led to increased HCV infectivity irrespective of LPL HCV in medium from HTGL knockdown cells was found in fractions with a lower density. These results indicate that changes in the nature of HCV-associated lipoproteins by LPL and/or HTGL affect HCV infectivity, suggesting that association of HCV with specific lipoproteins is important for HCV infectivity. (C) 2010 Elsevier Inc. All rights reserved.

Infectivity of Hepatitis C Virus Is Influenced by Association with Apolipoprotein E Isoforms

Takayuki Hishiki, Yuko Shimizu, Reiri Tobita, Kazuo Sugiyama, Kazuya Ogawa, Kenji Funami, Yuki Ohsaki, Toyoshi Fujimoto, Hiroshi Takaku, Takaji Wakita, Thomas F. Baumert, Yusuke Miyanari, Kunitada Shimotohno

JOURNAL OF VIROLOGY ( AMER SOC MICROBIOLOGY ) 84 ( 22 ) 12048 - 12057 2010.11 [Refereed] [International journal]

Hepatitis C virus (HCV) is a causative agent of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV in circulating blood associates with lipoproteins such as very low density lipoprotein (VLDL) and low-density lipoprotein (LDL). Although these associations suggest that lipoproteins are important for HCV infectivity, the roles of lipoproteins in HCV production and infectivity are not fully understood. To clarify the roles of lipoprotein in the HCV life cycle, we analyzed the effect of apolipoprotein E (ApoE), a component of lipoprotein, on virus production and infectivity. The production of infectious HCV was significantly reduced by the knockdown of ApoE. When an ApoE mutant that fails to be secreted into the culture medium was used, the amount of infectious HCV in the culture medium was dramatically reduced; the infectious HCV accumulated inside these cells, suggesting that infectious HCV must associate with ApoE prior to virus release. We performed rescue experiments in which ApoE isoforms were ectopically expressed in cells depleted of endogenous ApoE. The ectopic expression of the ApoE2 isoform, which has low affinity for the LDL receptor (LDLR), resulted in poor recovery of infectious HCV, whereas the expression of other isoforms, ApoE3 and ApoE4, rescued the production of infectious virus, raising it to an almost normal level. Furthermore, we found that the infectivity of HCV required both the LDLR and scavenger receptor class B, member I (SR-BI), ligands for ApoE. These findings indicate that ApoE is an essential apolipoprotein for HCV infectivity.

A pitfall in using BODIPY dyes to label lipid droplets for fluorescence microscopy

Yuki Ohsaki, Yuki Shinohara, Michitaka Suzuki, Toyoshi Fujimoto

HISTOCHEMISTRY AND CELL BIOLOGY ( SPRINGER ) 133 ( 4 ) 477 - 480 2010.04 [Refereed] [International journal]

Authorship： Lead author

The lipid droplet (LD) has become a focus of intense research. Fluorescence labeling is indispensable for the cell biological analysis of the LD, and a lipophilic fluorescence dye, BODIPY 493/503, which emits bright green fluorescence has been used extensively for LD labeling. The dye is convenient for double fluorescence labeling, but we noticed that it emits red fluorescence under certain conditions, which could lead to erroneous interpretations. We propose a protocol to preclude such a possibility.

Quantitative electron microscopy shows uniform incorporation of triglycerides into existing lipid droplets

Jinglei Cheng, Akikazu Fujita, Yuki Ohsaki, Michitaka Suzuki, Yuki Shinohara, Toyoshi Fujimoto

HISTOCHEMISTRY AND CELL BIOLOGY ( SPRINGER ) 132 ( 3 ) 281 - 291 2009.09 [Refereed] [International journal]

The lipid droplet (LD) is an organelle with a lipid ester core and a surface phospholipid monolayer. The mechanism of LD biogenesis is not well understood. The present study aimed to elucidate the LD growth process, for which we developed a new electron microscopic method that quantifies the proportion of existing and newly synthesized triglycerides in individual LDs. Our method takes advantage of the reactivity of unsaturated fatty acids and osmium tetroxide, which imparts LDs an electron density that reflects fatty acid composition. With this method, existing triglyceride-rich LDs in 3Y1 fibroblasts were observed to incorporate newly synthesized triglycerides at a highly uniform rate. This uniformity and its persistence even after microtubules were depolymerized suggest that triglycerides in fibroblasts are synthesized in the local vicinity of individual LDs and then incorporated. In contrast, LDs in 3T3-L1 adipocytes showed heterogeneity in the rate at which lipid esters were incorporated, indicating different mechanisms of LD growth in fibroblasts and adipocytes.

Biogenesis of cytoplasmic lipid droplets: From the lipid ester globule in the membrane to the visible structure

Yuki Ohsaki, Jinglei Cheng, Michitaka Suzuki, Yuki Shinohara, Akikazu Fujita, Toyoshi Fujimoto

BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR AND CELL BIOLOGY OF LIPIDS ( ELSEVIER SCIENCE BV ) 1791 ( 6 ) 399 - 407 2009.06 [Refereed] [International journal]

Authorship： Lead author

The cytoplasmic lipid droplet (CLD) and very low-density lipoprotein are generated from the lipid ester synthesized in the endoplasmic reticulum. The lipid ester deposited between the two membrane leaflets is supposed to bulge toward the cytoplasm to make a nascent CLD, but its size must be below the resolution limit of conventional techniques and the detectable CLD should only form after acquisition of additional lipid esters. The CLD is different from vesicular organelles in that the internal content is highly hydrophobic and the shape is invariably spherical. Due to its unique characteristics, quantitative discordance between the surface and the volume may occur in the growth and/or involution processes of the CLD. The possibility that these processes may give rise to the structural and functional diversities of the CLD is discussed. (C) 2008 Elsevier B.V. All rights reserved.

脂質を観察する方法

藤田秋一, 大崎雄樹, 藤本豊士

日本組織細胞化学会編組織細胞化学2009 35-43 2009

Lipid Droplet-Associated Proteins Protect Renal Tubular Cells from Fatty Acid-Induced Apoptosis

Yoshimichi Urahama, Yuki Ohsaki, Yutaka Fujita, Shoichi Maruyama, Yukio Yuzawa, Seiichi Matsuo, Toyoshi Fujimoto

AMERICAN JOURNAL OF PATHOLOGY ( ELSEVIER SCIENCE INC ) 173 ( 5 ) 1286 - 1294 2008.11 [Refereed] [International journal]

Proteinuria is a major cause of tubulointerstitial kidney damage, and free fatty acids bound to albumin are thought to play an important role in its pathogenesis. However, the mechanism whereby proteinuria causes tubulointerstitial damage to the kidney is unclear. Using primary human renal proximal tubular cells, we observed that albumin replete with fatty acids (rBSA) and defatted albumin (dBSA) complexed with linoleic acid (LA) induced significantly more apoptosis than did defatted albumin alone. Oxidative stress was partially involved in apoptotic induction by LA/dBSA but not by rBSA. Administration of fatty acid-bound BSA increased the number of lipid droplets (LDs) and the LD-associated proteins, adipocyte differentiation-related protein and TIP47. LDs are organelles that store esterified fatty acids, and the LD-associated proteins are presumed to facilitate LD formation. Knockdown of adipocyte differentiation-related protein or TIP47 by RNA interference enhanced induction of apoptosis by both rBSA and LA/dBSA. Apoptotic induction was observed similarly when either rBSA or LA/dBSA was applied to only the apical surfaces of polarized LLC-PK1 cells. The present results suggest that LDs and LD-associated proteins have protective effects against apoptosis induced by fatty acid-bound albumin by sequestering free fatty acids. Therapeutic manipulation of these LD-associated proteins could aid in the amelioration of nephritic diseases. (Anti J Pathol 2008, 173:1286-1294; DOI: 10.2353/ajpath.2008.080137)

Lipid droplets: a classic organelle with new outfits

Toyoshi Fujimoto, Yuki Ohsaki, Jinglei Cheng, Michitaka Suzuki, Yuki Shinohara

HISTOCHEMISTRY AND CELL BIOLOGY ( SPRINGER ) 130 ( 2 ) 263 - 279 2008.08 [Refereed] [International journal]

Lipid droplets are depots of neutral lipids that exist virtually in any kind of cell. Recent studies have revealed that the lipid droplet is not a mere lipid blob, but a major contributor not only to lipid homeostasis but also to diverse cellular functions. Because of the unique structure as well as the functional importance in relation to obesity, steatosis, and other prevailing diseases, the lipid droplet is now reborn as a brand new organelle, attracting interests from researchers of many disciplines.

*Lipid droplets are arrested in the ER membrane by tight binding of lipidated apolipoprotein B-100

Ohsaki, Y. Cheng, J. Suzuki, M. Fujita, A. Fujimoto, T

J Cell Sci 121 ( 14 ) 2415-22 - 22 2008.06 [Refereed] [International journal]

Authorship： Lead author

Apolipoprotein B-100 (ApoB) is a major component of very-low-density lipoproteins, and is deposited in a region around lipid droplets (LDs) called the ;ApoB-crescent'. The ApoB-crescent is thought to be related to ApoB degradation because it drastically increases when proteasome or autophagy is inhibited. In the present study, we found that ApoB-crescents were significantly reduced when ApoB lipidation was suppressed by either the inhibition or knockdown of the microsomal triglyceride-transfer protein. By contrast, ApoB-crescents increased under conditions that are presumed to cause lipidated ApoB abnormalities in secretory compartments. By electron microscopic analyses, we identified the ApoB-crescent as a thin cholesterol-rich ER cistern fused to an LD, and - topologically - this structure is equivalent to a lipid-ester globule between the two leaflets of the ER membrane. ApoB localized in the thin cisternal lumen, and its binding to LDs was resistant to alkaline treatment. Overexpression of ADRP or TIP47 suppressed the increase in the number of ApoB-crescents, whereas knockdown of these proteins had the opposite effect. From these results, we inferred that the ApoB-crescent is f

Platelet-derived growth factor signals play critical roles in differentiation of longitudinal smooth muscle cells in mouse embryonic gut.

Kurahashi, M, Niwa, Y, Cheng, J, Ohsaki, Y, Fujita, A, Goto, H, Fujimoto, Y, Torihashi, S

Neurogastroenterol Motil. 20 ( 5 ) 521-531 2008.01 [Refereed]

In the development of mouse gut, longitudinal smooth muscle cells (LMC) and interstitial cells of Cajal (ICC) originate from common precursor cells expressing c-Kit. Recently, some gastrointestinal stromal tumours, which develop from smooth muscle layers of the gut and have gain-of-function mutations of c-kit, have been reported to have gain-of-function mutations of platelet-derived growth factor (PDGF) receptor alpha gene. These data raise the possibility that PDGF signalling might be involved in the development of LMC. Therefore, we examined the expression pattern of the PDGF signal family of embryonic gut by immunohistochemistry and in situ hybridization, and investigated the role of PDGF signals in the development of smooth muscle layers in mouse gut using a new organ culture system. During embryonic development, the circular muscle layer expressed PDGF-A, enteric neurons expressed PDGF-B and common precursor cells of LMC and ICC expressed both PDGF receptor alpha and beta. The selective PDGF receptor inhibitor AG1295 suppressed the differentiation of LMC in gut explants. We conclude that PDGF signals play critical roles in the differentiation of LMC in mouse embryonic gut.

脂肪滴形成のメカニズム

藤本豊士, 大崎雄樹, 鈴木倫毅

膜 MEMBRANE 33 ( 1 ) 17-23 2008.01 [Invited]

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 2007.06 [Refereed] [International journal]

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.

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 2007.02 [Refereed] [International journal]

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.

Cholesterol depletion induces autophagy.

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

Biochemical and biophysical research communications 351 ( 1 ) 246 - 52 2006.12 [International journal]

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.

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 2006.12 [Refereed]

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 2006.08 [Refereed] [International journal]

Authorship： Lead author

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.

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 2006.07 [Refereed] [International journal]

Authorship： Lead author

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.

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