9 research outputs found
ORP8 interacts with Nup62.
<p><b>A</b> Bimolecular fluorescence complementation (BiFC) analysis of ORP8 interaction with Nup62. HuH7 cells were cotransformed for 24 h with plasmids encoding the fusion proteins Nup62/pVn-C1 and ORP8/pVc-C1 or ORP8pVc-N1 (indicated on the left) for 24 h, followed by 48 h incubation with 10 ”g/ml cycloheximide. ER-DsRed2 was contransfected as a transfection control and ER marker. BiFC (GFP channel) and DsRed fluorescence were imaged (identified at the top). Bar, 10 ”m. <b>B</b> Lysate of untransfected HuH7 cells was immunoprecipitated with anti-ORP8 (identified at the top) or an irrelevant control IgG, followed by Western blot analysis with anti-Nup62 (top panel) or anti-ORP8 (bottom panel). H, IgG heavy chain.</p
The ORP8 ligand-binding domain binds cholesterol.
<p><b>A</b> The purified His<sub>6</sub>-ORP8 ORD preparation, Coomassie blue stained SDS-PAGE gel. The two major bands (indicated, ORP8) represent the ORP8 ORD fusion protein. <b>B</b> The ability of His<sub>6</sub>-ORP8 ORD to extract [<sup>3</sup>H]cholesterol from unilamellar PCâ¶cholesterol (99â¶1 mol%) vesicles was assayed. Purified GST was used as a negative control. Each assay contained 9.9 nmol PC and 100 pmol cholesterol; The protein amounts used (25â100 pmol) are indicated at the bottom. The data represents % of total DPM extracted from the vesicles, a mean ± s.e.m. (GST, nâ=â4; His<sub>6</sub>-ORP8 ORD, nâ=â5).</p
Primers used for mRNA quantification by real-time RT-PCR.
<p>The abbreviations are: LDL-R, low-density lipoprotein receptor; HMG-CR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; HMG-CS, 3-hydroxy-3-methylglutaryl coenzyme A synthetase; FAS, fatty acid synthetase; ACS, acetyl-CoA synthetase; SCD-1, stearoyl-CoA desaturase 1. The prefix m indicates mouse and h human sequence.</p
ORP8 co-localizes with Nup62 at the nuclear envelope: Confocal microscopy analysis.
<p>HuH7 cells were transfected with ORP8, ORP1L, ORP3, or ORP10 cDNA for 24 h using Lipofectamine 2000, followed by processing for confocal immunofluorescence microscopy double staining with anti-Nup62 (green) and anti-ORP (red) antibodies. <b>AâC</b> Nup62 and ORP8 localization in transfected Huh7 cells. Co-localization of ORP8 and Nup62 at the nuclear envelope is indicated with arrows in the channel merge panel. No Nup62 colocalization was observed with ORP1L (<b>D</b>), ORP3 (<b>E</b>), or ORP10 (<b>F</b>). Bars, 10 ”m. <b>G</b> Analysis of ORP8, 1L, 3, or 10 (identified in the panels) colocalization with Nup62 at the nuclear envelope in representative cells. Fluorescence intensity (on an arbitrary scale) of the nuclear circumference at the Nup62 (green) and ORP (red) channels was quantified by using the Leica LCS software.</p
ORP8 overexpression reduces plasma and hepatic lipids in mice.
<p><b>A</b> Western analysis of adenovirus (Ad)-mediated ORP8 overexpression in mouse liver. Liver total protein (20 ”g/lane) from mice at day 5 after infection with AdGFP and AdORP8 was Western blotted with ORP8 antibody. <b>B</b> Plasma Cholesterol (Chol), choline phospholipids (PL), and triglycerides (TG) measured from animals at 5 days after infection with AdGFP or AdORP8. <b>C</b> Hepatic lipid levels of the mice at day 5. <b>D</b> Mouse liver nuclear (1st and 3rd panel) or total (2nd and 4th panel) protein fractions (40 ”g/lane) at day 5 after infection were Western blotted with antibodies against SREBP-1 or SREBP-2. nSREBP, nuclear SREBP; pSREBP, precursor SBEBP. (E) Analysis of SREBP target gene mRNAs (identified at the bottom) in the liver of mice transduced with AdGFP (open bars) or AdORP8 (closed bars) by qPCR. The data represents mean ± s.e.m. (*p<0.05; **p<0.01; nâ=â5; t-test).</p
Identification of the interacting domains in Nup62 and ORP8.
<p><b>A</b> Schematic presentation of the Nup62 prey constructs used in yeast two-hybrid assays. The numbers indicate amino acid positions. <b>B</b> Interaction of the Nup62 constructs and ORP8m (lacking the C-terminal trans-membrane segment). Yeast colonies grown on SD/2- plates (left), SD/4- plates (middle) and x-gal assay (right) are shown. <b>C</b> Schematic presentation of the ORP8 bait constructs used. <b>D</b> Interaction of the ORP8 constructs with Nup62 in the yeast two-hybrid assay. Yeast colonies grown on SD/2- plates (top), SD/4- plates (middle) and x-gal assay (bottom) are shown. pGADT7 is the empty prey vector and pGBKT7 the empty bait vector.</p
Nup62 is involved in reduction of nuclear SREBPs by excess ORP8.
<p><b>A</b> HuH7 cells were transfected with empty vector plasmid (Mock), <i>ORP8</i> cDNA (ORP8), non-targeting siRNA (siNT) or siNup62 as indicated, followed by preparation of total protein specimens and nuclear fractions, and Western blot analysis thereof with antibodies against SREBP-1 and SREBP-2. Nuclear SREBPs (nSREBPs) decreased in cells overexpressing ORP8 (the top panels), while no change in nSREBPs was seen in cells transfected with siNup62 (the two bottom panels). <b>B</b> HuH7 cells were transfected with combinations of siRNAs and plasmids as indicated at the top. Nuclear fractions Western blotted for SREBP-1 and SREBP-2, and total protein specimens (40 ”g/lane) blotted for Nup62, ORP8, and ÎČ-actin are shown at the bottom. <b>C,D</b> Quantification of the relative levels of nSREBP-1 and nSREBP-2 after the indicated combined transfections. <b>E</b> Effect of the combined transfections on the mRNA levels of SREBP target genes (identified at the bottom); qPCR analysis. The results represent mean ± s.e.m. (nâ=â4, *p<0.05, **p<0.01, t-test; difference to values of the siNT and Mock-transfected control, which was set at 1).</p
Silencing and overexpression of ORP8 have opposite effects on SREBP target gene expression in HuH7 cells.
<p><b>A</b> Western analysis of HuH7 cells subjected to ORP8 silencing and overexpression. Stable expression of scrambled control shRNA (shNT) or shORP8, or overexpression by plasmid transfection for 36 h using Neonâą electroporation (ORP8; Mockâ=âtransfection with empty vector). <b>B</b> Quantification of selected SREBP target gene mRNAs (identified at the bottom) in cells expressing shNT or shORP8. <b>C</b> Quantification of selected mRNAs (identified at the bottom) in cells transfected with the empty vector (Mock) or <i>ORP8</i> cDNA (ORP8). <b>D</b> Cholesterol biosynthesis as measured by [<sup>3</sup>H]acetic acid incorporation (30 min pulse, 90 min chase), in cells expressing shNT or shORP8. The result is given as % of the biosynthesis in shNT-expressing cells. <b>E</b> Cholesterol biosynthesis in cells transfected with the plain vector (Mock) or <i>ORP8</i> cDNA (ORP8) for 36 h. The data represents mean ± s.e.m. (*p<0.05, **p<0.01, nâ=â4â6; t-test).</p
Aster proteins facilitate nonvesicular plasma membrane to ER cholesterol transport in mammalian cells
The mechanisms underlying sterol transport in mammalian cells are poorly understood. In particular, how cholesterol internalized from HDL is made available to the cell for storage or modification is unknown. Here, we describe three ER-resident proteins (Aster-A, -B, -C) that bind cholesterol and facilitate its removal from the plasma membrane. The crystal structure of the central domain of Aster-A broadly resembles the sterol-binding fold of mammalian StARD proteins, but sequence differences in the Aster pocket result in a distinct mode of ligand binding. The Aster N-terminal GRAM domain binds phosphatidylserine and mediates Aster recruitment to plasma membrane-ER contact sites in response to cholesterol accumulation in the plasma membrane. Mice lacking Aster-B are deficient in adrenal cholesterol ester storage and steroidogenesis because of an inability to transport cholesterol from SR-BI to the ER. These findings identify a nonvesicular pathway for plasma membrane to ER sterol trafficking in mammals