FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2010717647> ?p ?o ?g. }

• W2010717647 endingPage "707" @default.
• W2010717647 startingPage "696" @default.
• W2010717647 abstract "Copper metabolism Murr1 domain 1 (COMMD1) is a 21-kDa protein involved in copper export from the liver, NF-κB signaling, HIV infection, and sodium transport. The precise function of COMMD and the mechanism through which COMMD1 performs its multiple roles are not understood. Recombinant COMMD1 is a soluble protein, yet in cells COMMD1 is largely seen as targeted to cellular membranes. Using co-localization with organelle markers and cell fractionation, we determined that COMMD1 is located in the vesicles of the endocytic pathway, whereas little COMMD1 is detected in either the trans-Golgi network or lysosomes. The mechanism of COMMD1 recruitment to cell membranes was investigated using lipidspotted arrays and liposomes. COMMD1 specifically binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in the absence of other proteins and does not bind structural lipids; the phosphorylation of PtdIns at position 4 is essential for COMMD1 binding. Proteolytic sensitivity and molecular modeling experiments identified two distinct domains in the structure of COMMD1. The C-terminal domain appears sufficient for lipid binding, because both the full-length and C-terminal domain proteins bind to PtdIns(4,5)P2. In native conditions, endogenous COMMD1 forms large oligomeric complexes both in the cytosol and at the membrane; interaction with PtdIns(4,5)P2 increases the stability of oligomers. Altogether, our results suggest that COMMD1 is a scaffold protein in a distinct sub-compartment of endocytic pathway and offer first clues to its role as a regulator of structurally unrelated membrane transporters. Copper metabolism Murr1 domain 1 (COMMD1) is a 21-kDa protein involved in copper export from the liver, NF-κB signaling, HIV infection, and sodium transport. The precise function of COMMD and the mechanism through which COMMD1 performs its multiple roles are not understood. Recombinant COMMD1 is a soluble protein, yet in cells COMMD1 is largely seen as targeted to cellular membranes. Using co-localization with organelle markers and cell fractionation, we determined that COMMD1 is located in the vesicles of the endocytic pathway, whereas little COMMD1 is detected in either the trans-Golgi network or lysosomes. The mechanism of COMMD1 recruitment to cell membranes was investigated using lipidspotted arrays and liposomes. COMMD1 specifically binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in the absence of other proteins and does not bind structural lipids; the phosphorylation of PtdIns at position 4 is essential for COMMD1 binding. Proteolytic sensitivity and molecular modeling experiments identified two distinct domains in the structure of COMMD1. The C-terminal domain appears sufficient for lipid binding, because both the full-length and C-terminal domain proteins bind to PtdIns(4,5)P2. In native conditions, endogenous COMMD1 forms large oligomeric complexes both in the cytosol and at the membrane; interaction with PtdIns(4,5)P2 increases the stability of oligomers. Altogether, our results suggest that COMMD1 is a scaffold protein in a distinct sub-compartment of endocytic pathway and offer first clues to its role as a regulator of structurally unrelated membrane transporters. COMMD1 (Murr1) is the founding member of a recently discovered family of COMMD (Copper metabolism Murr1 domain) 3The abbreviations used are: COMMD, copper metabolism Murr1 domain; TGN, trans-Golgi network; HIV, human immunodeficiency virus; Ab, antibody; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; VARD, residues Met-1 through Gly-121; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; CAPS, 3-(cyclohexylamino)propanesulfonic acid; BCS, bathocuproine disulfonate; PA, phosphatidic acid; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; CTD, C-terminal domain; BN-PAGE, Blue-native PAGE.3The abbreviations used are: COMMD, copper metabolism Murr1 domain; TGN, trans-Golgi network; HIV, human immunodeficiency virus; Ab, antibody; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; VARD, residues Met-1 through Gly-121; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; CAPS, 3-(cyclohexylamino)propanesulfonic acid; BCS, bathocuproine disulfonate; PA, phosphatidic acid; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; CTD, C-terminal domain; BN-PAGE, Blue-native PAGE. proteins (1Maine G.N. Burstein E. Cell Mol. Life Sci. 2007; 64: 1997-2005Crossref PubMed Scopus (76) Google Scholar). COMMD1 was identified as the product of a gene mutated in dogs with severe hepatic copper toxicosis (2van De Sluis B. Rothuizen J. Pearson P.L. van Oost B.A. Wijmenga C. Hum. Mol. Genet. 2002; 11: 165-173Crossref PubMed Scopus (303) Google Scholar). In several breeds of dogs, especially in Bedlington terriers, a mis-splicing event in the Murr1 gene results in the loss of protein expression (3Klomp A.E. van de Sluis B. Klomp L.W. Wijmenga C. J. Hepatol. 2003; 39: 703-709Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) and massive copper accumulation in the liver that can reach remarkably high concentrations of 10,000-12,000 μg/g and lead to acute organ failure (4Kawamura M. Takahashi I. Kaneko J.J. Vet. Pathol. 2002; 39: 747-750Crossref PubMed Scopus (4) Google Scholar). The identification of Murr1 (COMMD1) by van de Sluis and co-workers (2van De Sluis B. Rothuizen J. Pearson P.L. van Oost B.A. Wijmenga C. Hum. Mol. Genet. 2002; 11: 165-173Crossref PubMed Scopus (303) Google Scholar) generated a great deal of interest and helped to create tools for genetic screening. However, the molecular mechanism through which COMMD1 regulates copper metabolism remains unknown. COMMD1 was shown to bind Cu(II) in vitro (5Narindrasorasak S. Kulkarni P. Deschamps P. She Y.M. Sarkar B. Biochemistry. 2007; 46: 3116-3128Crossref PubMed Scopus (49) Google Scholar), but whether this binding represents an in vivo property is uncertain. The observation, that in dogs lacking COMMD1 hepatic copper uptake is unperturbed but copper accumulates in dense lysosomal granules, suggested a defect in biliary copper export (6Johnson G.F. Morell A.G. Stockert R.J. Sternlieb I. Hepatology. 1981; 1: 243-248Crossref PubMed Scopus (72) Google Scholar). The role of COMMD1 in copper export was further supported by the increased retention of copper in cultured cells in which COMMD1 was down-regulated with small interference RNA (7Burstein E. Ganesh L. Dick R.D. van De Sluis B. Wilkinson J.C. Klomp L.W. Wijmenga C. Brewer G.J. Nabel G.J. Duckett C.S. EMBO J. 2004; 23: 244-254Crossref PubMed Scopus (178) Google Scholar). However, COMMD1 is a small soluble protein (see below) and cannot directly mediate transmembrane copper transport. It was proposed that COMMD1 controls copper export by regulating the intracellular localization of the copper-transporting ATPase ATP7B, which is chiefly responsible for the removal of excess copper from the liver into the bile. In support of this hypothesis, recombinant COMMD1 was shown to interact with the N-terminal domain of ATP7B in vitro and in cell lysates (8Tao T.Y. Liu F. Klomp L. Wijmenga C. Gitlin J.D. J. Biol. Chem. 2003; 278: 41593-41596Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). However, down-regulation of COMMD1 has no apparent effect on the ability of ATP7B to traffic from the trans-Golgi network (TGN) to exocytic vesicles (Ref. 9de Bie P. van de Sluis B. Burstein E. van de Berghe P.V. Muller P. Berger R. Gitlin J.D. Wijmenga C. Klomp L.W. Gastroenterology. 2007; 133: 1316-1326Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar and our data, not shown), suggesting that the effect of COMMD1 is downstream of the TGN. More recently, the observed increase in interactions between ATP7B mutants and COMMD1 led to the alternative hypothesis that COMMD1 may be a part of a quality-control mechanism for ATP7B folding (9de Bie P. van de Sluis B. Burstein E. van de Berghe P.V. Muller P. Berger R. Gitlin J.D. Wijmenga C. Klomp L.W. Gastroenterology. 2007; 133: 1316-1326Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). A further layer of complexity (and uncertainly) about COMMD1 function was added by studies demonstrating that the genetic knockout of COMMD1 in mice leads to embryonic lethality between days 9.5 and 10.5 (10van de Sluis B. Muller P. Duran K. Chen A. Groot A.J. Klomp L.W. Liu P.P. Wijmenga C. Mol. Cell Biol. 2007; 27: 4142-4156Crossref PubMed Scopus (93) Google Scholar). Such a severe and early phenotype in rodents but not in dogs indicates that the functional significance of COMMD1 is species-dependent and may extend beyond hepatic copper export, a suggestion supported by the expression of Murr1 transcripts in all major tissues and cell types (3Klomp A.E. van de Sluis B. Klomp L.W. Wijmenga C. J. Hepatol. 2003; 39: 703-709Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Furthermore, various protein-protein interaction experiments uncovered the ability of COMMD1 to dimerize with several members of COMMD family (11Burstein E. Hoberg J.E. Wilkinson A.S. Rumble J.M. Csomos R.A. Komarck C.M. Maine G.N. Wilkinson J.C. Mayo M.W. Duckett C.S. J. Biol. Chem. 2005; 280: 22222-22232Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 12de Bie P. van de Sluis B. Burstein E. Duran K.J. Berger R. Duckett C.S. Wijmenga C. Klomp L.W. Biochem. J. 2006; 398: 63-71Crossref PubMed Scopus (74) Google Scholar) and to form complexes with a growing number of other structurally and functionally unrelated proteins (3Klomp A.E. van de Sluis B. Klomp L.W. Wijmenga C. J. Hepatol. 2003; 39: 703-709Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Evaluations of these interactions revealed that COMMD1 levels influence HIV infection, NF-κB signaling, sodium transport, and proteasome-mediated protein degradation (11Burstein E. Hoberg J.E. Wilkinson A.S. Rumble J.M. Csomos R.A. Komarck C.M. Maine G.N. Wilkinson J.C. Mayo M.W. Duckett C.S. J. Biol. Chem. 2005; 280: 22222-22232Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 12de Bie P. van de Sluis B. Burstein E. Duran K.J. Berger R. Duckett C.S. Wijmenga C. Klomp L.W. Biochem. J. 2006; 398: 63-71Crossref PubMed Scopus (74) Google Scholar, 13Biasio W. Chang T. McIntosh C.J. McDonald F.J. J. Biol. Chem. 2004; 279: 5429-5434Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 14Maine G.N. Mao X. Komarck C.M. Burstein E. EMBO J. 2007; 26: 436-447Crossref PubMed Scopus (205) Google Scholar). Today, the importance of COMMD1 in mammalian physiology is well established, whereas the mechanism behind COMMD1 activity, particularly at a cell membrane, remains elusive. Human COMMD1, characterized in this study, is a soluble 21-kDa protein, which is 88% identical to its canine orthologue. Members of the COMMD family show only modest homology to each other, sharing a single invariant residue (Trp-124 in COMMD1). Structural studies of the full-length COMMD1 have been complicated by protein instability and aggregation. However, the structure of the N-terminal fragment of COMMD1 (residues 1-108) was recently solved using NMR (15Sommerhalter M. Zhang Y. Rosenzweig A.C. J. Mol. Biol. 2007; 365: 715-721Crossref PubMed Scopus (18) Google Scholar). These studies revealed a helical structure and two positive patches at the surface of the fragment, but structural motifs that might offer mechanistic clues to COMMD1 activity were not apparent. We were in trigued by the observation that several COMMD1-dependent effects involve membrane proteins (copper transporter ATP7B and sodium channel ENaC) or membrane-dependent events (HIV infection and viral escape). Although it is thought that COMMD1 is a cytosolic protein, available data on the staining of endogenous COMMD1 in cells shows vesicular rather than cytosolic pattern (3Klomp A.E. van de Sluis B. Klomp L.W. Wijmenga C. J. Hepatol. 2003; 39: 703-709Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Therefore, to better understand the biochemical function of COMMD1 we characterized in detail recombinant COMMD1, determined the localization of endogenous COMMD1 in hepatocytes, and generated a molecular model of this protein. We discovered that COMMD1 binds with high specificity to an important signaling and regulatory lipid, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), localizes to the endosomal compartment, and forms higher order oligomers, which are stabilized by interactions with lipid. These properties suggest a mechanism by which COMMD1 can be recruited to the endocytic membranes and act as a scaffold/adaptor protein contributing to protein/vesicle sorting. Antibodies for COMMD1 Detection—Mouse monoclonal anti-COMMD1 antibody (Ab) was obtained from Abnova (Taipei, Taiwan). Polyclonal anti-COMMD1 Abs were obtained after serial immunization of chickens with purified non-tagged COMMD1 and purification of immune IgY from egg yolk (Aves Laboratories, Tigard, OR). Monoclonal anti-COMMD1 and chicken polyclonal anti-COMMD1 detect equally the monomeric COMMD1, but recognize various oligomeric forms of COMMD1 with different affinities (not shown). Immunofluorescence Detection of COMMD1 in HepG2 Cells—Cells were seeded onto lysine-coated coverslips and cultured in MEM+Glutamax, 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Coverslips were washed with phosphate-buffered saline (PBS), pH 7.4, and then cells were fixed in 4% paraformaldehyde in PBS. Blocking and permeabilization were done simultaneously with 3% bovine serum albumin, 1% gelatin, and 0.1% Tween 20. Primary Abs were diluted in PBS with 3% bovine serum albumin, and secondary antibodies were diluted in PBS with 0.1% Tween 20. Primary antibody dilutions were as follows: mouse anti-COMMD1 IgG2, 1:100, chicken anti-COMMD1 polyclonal, 1:500, mouse anti-EEA1 IgG1 (Abcam, Cambridge, MA) 1:100, anti-CHMP2B (Abcam), 1:100, rat anti-LAMP1 (DHSB, University of Iowa), 1:100, anti-Golgin-97 (Molecular Probes, Eugene, OR), 1:100, rat anti-ATP7B (16Tsivkovskii R. MacArthur B.C. Lutsenko S. J. Biol. Chem. 2001; 276: 2234-2242Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) 1:1000. Anti-Chicken HiLyte 555 (Anaspec, San Jose, CA) was used at 1 μg/ml. All other secondary Abs were from Invitrogen and used at 2 μg/ml and conjugated with either AlexaFluor 488 (shown in green) or AlexaFluor 555 (shown in red). Confocal microscopy was done utilizing a Zeiss LSM 500 laser-scanning microscope. Treatment of cells with 200 μm BCS or 50 μm CuCl2 was for 8 h in the media described above. The extent of co-localization, i.e. the proportion of overlapping pixels, was measured with the NIH ImageJ “Co-localization Test” plug-in using the Costes method of randomization for 25 iterations (17Costes S.V. Daelemans D. Cho E.H. Dobbin Z. Pavlakis G. Lockett S. Biophys. J. 2004; 86: 3993-4003Abstract Full Text Full Text PDF PubMed Scopus (958) Google Scholar). Immunodetection of COMMD1 in Fractionated HepG2 Cell Lysates—HepG2 cells were seeded on 10-cm Petri dishes and allowed to grow for 3 days in the growth medium composed of MEM+Glutamax, 10% fetal bovine serum, penicillin, and streptomycin. Four hours prior to harvest, growth medium was replaced with fresh media containing 200 μm BCS or 50 μm CuSO4. Cells were washed with PBS before harvest and scraped from the plate in a lysis buffer (20 mm HEPES, pH 7.5, 1.0 mm EDTA, 1.0 mm EGTA, 1.0 mm 4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride, and a Roche Complete protease inhibitor mixture). Cells were disrupted in a Dounce homogenizer with a loose-fitting pestle, incubated on ice for 5 min, made isotonic by addition of 0.1 volumes of 2.5 m sucrose, and homogenized by 25 strokes with a tight-fitting pestle. The cell lysates were then centrifuged at 6300 × g for 10 min to pellet nuclei and cell debris. The postnuclear supernatant was centrifuged for 30 min at 100,000 × g to separate the microsomal membranes from a soluble cytosolic fraction. The nuclei were purified from the initial 6300 × g pellet by twice re-suspending in TSE buffer (10 mm Tris-HCl, pH 7.6, 300 mm sucrose, 1.0 mm EDTA, 0.1% Igepal (Sigma-Aldrich)) and centrifuging for 5 min at 4000 × g. Membrane and nuclear fractions were then solubilized in TSE buffer containing 0.2% Triton X-100, and protein concentration was determined by Bradford assay (18Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar). Equal amounts of protein from nuclear, soluble, and membrane fractions were separated by SDS-PAGE on a 4-20% gradient gel (Pierce) under reducing conditions. Proteins were transferred to nitrocellulose by Western blot, and the membrane was blocked in Aquablock (EastCoastBio, North Berwick, ME) for 1 h at room temperature. Antibody incubations for immunodetection were used at following dilutions: mouse anti-COMMD1 (1:1000), chicken anti-COMMD1 (1:5000). Secondary antibodies, conjugated with either IRDye 700 or IRDye 800, were obtained from Rockland Immunochemicals (Gilbertsville, PA). Fluorescence detection of bound secondary Ab was performed with an Odyssey Scanner (Licor, Lincoln, NE). The same 21-kDa band corresponding to the endogenous COMMD1 was detected by different antibodies, immunodetection with chicken polyclonal anti-COMMD1 shown. Density Gradient Fractionation of Postnuclear Fraction—HepG2 cells were grown as described above. Fractionation was done essentially as described in a previous study (19Press B. Feng Y. Hoflack B. Wandinger-Ness A. J. Cell Biol. 1998; 140: 1075-1089Crossref PubMed Scopus (224) Google Scholar). Cells were washed once on the plate with PBS containing 2.0 mm CaCl2 and 0.9 mm MgCl2. Cells were scraped into PBS and centrifuged at 200 × g for 10 min. The cell pellet was resuspended in 3 volumes of 20 mm HEPES, pH 7.4, 150 mm NaCl, and protease inhibitors (Complete, Roche Applied Science). HepG2 cells were lysed by 15 passes through a 27-guage needle. Nuclei and unbroken cells were pelleted by two centrifugation steps at 1000 × g for 10 min. The postnuclear supernatant was loaded onto the top of a 20% Percoll cushion made isotonic with Tris-buffered saline and including protease inhibitors. Centrifugation was carried out in a Beckman Type 60 Ti ultracentrifuge rotor at 20,000 × g for 55 min. Fractions were collected from the bottom of the tube (∼950 μl each), to which CHAPS was added to 10 mm to solubilize membranes. After 1 h on ice with CHAPS, Percoll was sedimented by centrifugation at 100,000 × g for 60 min. Proteins were precipitated with 15% trichloroacetic acid in the presence of 0.12% deoxycholic acid. Denatured proteins were sedimented at 20,000 × g for 20 min, washed with 90% acetone, and air-dried. Denatured proteins were resuspended in 62.5 mm Tris, pH 6.8, 5% SDS, and 10% glycerol, then heated to 95 °C for 5 min. Urea was added to 2.5 m, and solubilized proteins were loaded onto a 12.5% gel for SDS-PAGE. Cloning, Expression, and Purification of Recombinant COMMD1 and Its N-terminal and C-terminal Domains—Human COMMD1 cDNA (American Type Culture Collection, Manassas, VA) was cloned into pTYB12 (New England Biolabs, Ipswich, MA) to generate a fusion protein with the chitin-binding domain/intein moiety or into the pET28b vector (Novagen/EMD Biosciences, Darmstadt, Germany) to add an N-terminal His6 tag (construct pET28b-COMMD1). In either case the cloning sites, NdeI (5′) and SalI (3′), were introduced by PCR using proofreading Pfu DNA polymerase (Roche Applied Science) and the forward 5′-CATATGGCAGCAGGCGAGCTTGAGGGTGGGCAAA and reverse 5′-GTCGACCTATCAGTTAGGCTGGCTGATCAGTGT primers, respectively. The N-terminal domain of COMMD1 (residues Met-1 through Gly-121, VARD) was cloned into pET28b utilizing pET28b-COMMD1 as a template, the same forward primer as for cloning of the full-length COMMD1, and the reverse primer 5′-GCCATCAAGTCTCGCGCTCAGGCCCCG to generate an SalI restriction site. The C-terminal domain, CTD (COMMD) of COMMD1 was cloned into pET28b from pET28b-COMMD1 template. The forward primer 5′-CATATGAGAGTTGATGGCAAG containing the NdeI restriction site was used along with the reverse primer described for cloning the full-length COMMD1. Expression and Purification—Initially, recombinant COMMD1 was expressed as a fusion with an intein and a chitin-binding domain in Escherichia coli ER2566 cells and purified in a nontagged form following dithiothreitol-induced excision from the fusion protein. In this purification system COMMD1 showed unusual properties. Specifically, despite good expression and solubility of the fusion protein, the yield of purified untagged COMMD1 was low because COMMD1 remained tightly bound to chitin (a polymer of N-acetylglucosamine) even after cleavage from the fusion portion. The protein could only be eluted from the resin using 4-6 m guanidine chloride. The small amount of non-tagged COMMD1 that was eluted from chitin beads showed rapid proteolysis to a stable ∼13-kDa fragment. Consequently, the non-tagged COMMD1 was only used to produce a polyclonal antibody (see above) and to confirm the identity of the 13-kDa product. For all functional studies, the His6-tagged variant was utilized. To produce recombinant His-tagged proteins (COMMD1 or individual domains), overnight cultures of E. coli BL21(DE3) cells transformed with the appropriate plasmid were diluted 1:200 to into 1-liter volumes of LB in baffled Erlenmeyer flasks and grown at 37 °C to an A600 of 0.8-1.0. Cultures were cooled to 16 °C before induction with 1 mm isopropyl 1-thio-β-d-galactopyranoside. Cells were harvested after 4 h of growth at 16 °C. Cell pellets were resuspended in Buffer A (50 mm sodium phosphate, pH 7.8, 300 mm NaCl, 10 mm imidazole, 1 mm 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, and a mixture of protease inhibitors (Complete Protease Inhibitors, Roche Applied Science). Cells were lysed in a French press, centrifuged at 20,000 × g for 30 min, and the soluble fraction was passed through a 27-guage needle to reduce viscosity. The soluble lysate was applied to a column of either nickel-nitrilotriacetic acid (Qiagen) or Talon Cobalt resin (Clontech, Mountain View, CA) equilibrated with buffer A. The column was washed by gravity flow with 20 column bed volumes of buffer A, then 20 volumes buffer A containing 0.1% Triton X-100, and another 20 volumes of buffer A. COMMD1 was eluted with steps of buffer B (50 mm sodium phosphate, pH 7.8, 300 mm NaCl) containing 50 mm, 100 mm, 150 mm, and 500 mm imidazole. Most of the full-length COMMD1 elutes with 150 mm imidazole, whereas degradation products elute at 50-100 mm imidazole (supplemental Fig. S1A). The VARD and CTD were purified using the same methods and eluted with 150 mm imidazole. For the liposome floatation assay, purified proteins were dialyzed to liposome buffer (20 mm HEPES, 50 mm KAc, 1 mm EDTA, pH 7.4). Mass spectrometry of purified COMMD1 and of the stable degradation fragment was performed at the Oregon Health & Science University Metal Ion Core by Dr. M. Ralle. N-terminal sequencing was performed at the Microchemical Facility at Emory University by Dr. J. Pohl. PIPstrip Assays—PIPstrips (Echelon, Salt Lake City, UT) were blocked in SuperBlock T-20 PBS (Pierce) for 2 h at room temperature and incubated with 1.0 μg/ml COMMD1, CTD, VARD, or the His6-tagged ATP-binding domain of ATP7B ATP-BD (16Tsivkovskii R. MacArthur B.C. Lutsenko S. J. Biol. Chem. 2001; 276: 2234-2242Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) (used as a negative control) for 2 h in SuperBlock (Pierce). After washing with PBS-T, the strips were incubated with anti-6His monoclonal antibody (Abcam, Cambridge, UK) or chicken anti-COMMD1 Ab and visualized with appropriate secondary IRDye 800 Ab (Rockland) using an Odyssey scanner. Both primary Abs gave similar results. Liposome Binding/Enzyme-linked Immunosorbent Assay Plate Assay—COMMD1, ATP-BD, alcohol dehydrogenase (Sigma-Aldrich), and PH domain of phospholipase Cδ (PIP2grip, Echelon Inc., Salt Lake City, UT) were bound to a Corning Costar 3591 polystyrene (medium binding) plate by incubating 1.0, 0.5, and 0.1 μg of each protein in 100 μl of PBS overnight at 4 °C. Unbound protein was removed, and wells were then blocked for 2 h at room temperature with 200 μl of blocking buffer (3% bovine serum albumin in PBS). Liposomes (PIPosome, Echelon), containing 1% of phosphorylated or non-phosphorylated phosphatidylinositol, were added at a total lipid concentration of 1 mm in 50 μl of blocking buffer and incubated for 1 h at room temperature with gentle rocking. The liposome solution was removed, and the wells were washed twice with 200 μl of PBS, then 4 times 10 min with blocking buffer. Bound liposomes were detected with Alexa-fluor 680-streptavidin (Molecular Probes, Eugene, OR) using the Odyssey scanner. Fluorescence was quantified with Odyssey 2.0 software. Data are shown for 0.5 μg of protein bound to the plate. Testing Specificity of Lipid Binding in Solution Using a Liposome Floatation Assay—Lipids were obtained as chloroform solutions from Avanti Polar Lipids (Alabaster, AL). Phosphatidylcholine was mixed with target lipids in glass tubes at indicated percentages (w/w) for 2 mg of total lipid. Chloroform was removed under a stream of nitrogen gas. The dried lipids were dissolved in 200 μl of hexane, dried under nitrogen, and then dried for 45 min under vacuum. The dried lipid film was resuspended in liposome buffer (20 mm HEPES, 50 mm KAc, 1 mm EDTA, pH 7.4) as described by Kno¨dler and Mayinger (20Knodler A. Mayinger P. BioTechniques. 2005; 38: 858Crossref PubMed Scopus (16) Google Scholar) and sonicated in a cup-horn sonicator for 15 min at 130-140 watts. For liposome binding, 100 μl of 2.0 mg/ml liposomes was mixed with purified COMMD1, VARD, or CTD at a final volume of 150 μl and allowed to bind at room temperature for 15 min. 450 μl of 40% sucrose in liposome buffer was added to a final sucrose concentration of 30%, and the mixture was layered onto a 700-μl 45% sucrose cushion. The liposome-protein-30% sucrose step was overlaid with 600 μl of 15% sucrose in liposome buffer, then with 150 μl of liposome buffer. The resultant sucrose step gradients were centrifuged at 40,000 rpm in a Beckman TLS-55 rotor for 20 min. Liposomes and bound protein were removed from the buffer phase (100 μl), and the protein was precipitated by addition of 400 μl of methanol. 100 μl of chloroform was added, then 300 μl of water. The mixture was centrifuged at 13,000 rpm for 3 min in a tabletop microcentrifuge, and the aqueous upper phase was removed, leaving the precipitated protein at the interphase. 400 μl of methanol was added to reduce density, and proteins were pelleted by centrifugation at 13,000 rpm for 5 min. Protein pellets were air-dried and dissolved in SDS-PAGE sample buffer. Proteins were separated by reducing SDS-PAGE and visualized by Coomassie Brilliant Blue staining or by Western blot as described above. BN-PAGE—HepG2 cells were cultured in 10-cm plates as described above and harvested by scraping into BN buffer (50 mm Bis-Tris, 500 mm ε-amino caproic acid, 2.5% glycerol, Roche Complete protease inhibitors, pH 7.0). Cells were lysed by 25 passages through a 27-gauge needle. Nuclei and cell debris were pelleted by centrifugation at 5,000 × g. The postnuclear supernatant was centrifuged at 100,000 × g for 30 min to separate soluble and membrane fractions. Membrane pellets were dissolved in BN buffer with 2% dodecyl maltoside DDM and incubated on ice for 1 h. Solubilized membrane fractions were centrifuged at 20,000 × g for 20 min, and the supernatants were used for BN-PAGE. Soluble and membrane fractions were loaded onto a 4-20% NativePage gel (Invitrogen) in BN buffer containing 2% dodecyl maltoside and 0.5% Coomassie G-250. For Western blot analysis, the gel was equilibrated in 10 mm CAPS, pH 11.0, and transferred to a polyvinylidene difluoride membrane in the same buffer for 3 h at 180 mA. Immunodetection was performed as described above utilizing polyclonal chicken anti-COMMD1 and secondary detection with horse-radish peroxidase-conjugated donkey anti-Chicken (Aves Labs, Tigard, OR). Molecular Modeling of COMMD1 Structure—Ab initio structure prediction was carried out on a locally installed Rosetta ab initio software version 2.0 licensed through the University of Washington (the web-based version of this program is known as “Robetta”); the fragment libraries were generated using the web version of the Rosetta fragment server. In the fragment selection, the “homs” option was included while all remaining parameters were set as defaults. Using “ab initio” mode after decoy population filtering 1,000 structures had been obtained. In the case of the “constrained” mode, the distances between the Cα atoms for the N-terminal part of COMMD1 (2H2M) were calculated using a Shell script. Cα distances >8 but <12 Å were used to create a constraint file, which was used for decoy population filtering. The 1000 structures thus obtained were clustered using the Rosetta clustering program. The center of the most populated cluster was selected and minimized using CharmM and then validated as previously described (21Subbian E. Yabuta Y. Shinde U. Biochemistry. 2004; 43: 14348-14360Crossref PubMed Scopus (27) Google Scholar). COMMD1 Is Localized to Endosomal Vesicles in Hepatic Cells—The original genetics experiments revealed the important physiological role of COMMD1 in copper export from the liver, however the intracellular localization of COMMD1 in polarized hepatocytes has not been characterized. Consequently, we first examined the distribution of endogenous COMMD1 in cell compartments using subcellular fractionation. HepG2 cells were allowe" @default.
• W2010717647 created "2016-06-24" @default.
• W2010717647 creator A5046814999 @default.
• W2010717647 creator A5046871960 @default.
• W2010717647 creator A5066661471 @default.
• W2010717647 creator A5078466345 @default.
• W2010717647 creator A5079179187 @default.
• W2010717647 date "2009-01-01" @default.
• W2010717647 modified "2023-09-30" @default.
• W2010717647 title "COMMD1 Forms Oligomeric Complexes Targeted to the Endocytic Membranes via Specific Interactions with Phosphatidylinositol 4,5-Bisphosphate" @default.
• W2010717647 cites W1945261349 @default.
• W2010717647 cites W1965762453 @default.
• W2010717647 cites W1968921354 @default.
• W2010717647 cites W1972204116 @default.
• W2010717647 cites W1985980245 @default.
• W2010717647 cites W1990741894 @default.
• W2010717647 cites W1996633441 @default.
• W2010717647 cites W2019364864 @default.
• W2010717647 cites W2024004845 @default.
• W2010717647 cites W2026564074 @default.
• W2010717647 cites W2037753219 @default.
• W2010717647 cites W2038070265 @default.
• W2010717647 cites W2040734911 @default.
• W2010717647 cites W2049881028 @default.
• W2010717647 cites W2060733266 @default.
• W2010717647 cites W2065389632 @default.
• W2010717647 cites W2078748790 @default.
• W2010717647 cites W2085777790 @default.
• W2010717647 cites W2088757838 @default.
• W2010717647 cites W2114387248 @default.
• W2010717647 cites W2115167726 @default.
• W2010717647 cites W2116224862 @default.
• W2010717647 cites W2118864057 @default.
• W2010717647 cites W2122866334 @default.
• W2010717647 cites W2123298030 @default.
• W2010717647 cites W2125782175 @default.
• W2010717647 cites W2134224836 @default.
• W2010717647 cites W2139842719 @default.
• W2010717647 cites W2150698399 @default.
• W2010717647 cites W2155824745 @default.
• W2010717647 cites W2168318070 @default.
• W2010717647 cites W4293247451 @default.
• W2010717647 doi "https://doi.org/10.1074/jbc.m804766200" @default.
• W2010717647 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2610505" @default.
• W2010717647 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18940794" @default.
• W2010717647 hasPublicationYear "2009" @default.
• W2010717647 type Work @default.
• W2010717647 sameAs 2010717647 @default.
• W2010717647 citedByCount "39" @default.
• W2010717647 countsByYear W20107176472012 @default.
• W2010717647 countsByYear W20107176472013 @default.
• W2010717647 countsByYear W20107176472014 @default.
• W2010717647 countsByYear W20107176472015 @default.
• W2010717647 countsByYear W20107176472016 @default.
• W2010717647 countsByYear W20107176472017 @default.
• W2010717647 countsByYear W20107176472018 @default.
• W2010717647 countsByYear W20107176472019 @default.
• W2010717647 countsByYear W20107176472020 @default.
• W2010717647 countsByYear W20107176472021 @default.
• W2010717647 countsByYear W20107176472023 @default.
• W2010717647 crossrefType "journal-article" @default.
• W2010717647 hasAuthorship W2010717647A5046814999 @default.
• W2010717647 hasAuthorship W2010717647A5046871960 @default.
• W2010717647 hasAuthorship W2010717647A5066661471 @default.
• W2010717647 hasAuthorship W2010717647A5078466345 @default.
• W2010717647 hasAuthorship W2010717647A5079179187 @default.
• W2010717647 hasBestOaLocation W20107176471 @default.
• W2010717647 hasConcept C170493617 @default.
• W2010717647 hasConcept C185592680 @default.
• W2010717647 hasConcept C2777853560 @default.
• W2010717647 hasConcept C2780610907 @default.
• W2010717647 hasConcept C28005876 @default.
• W2010717647 hasConcept C41625074 @default.
• W2010717647 hasConcept C55493867 @default.
• W2010717647 hasConcept C62478195 @default.
• W2010717647 hasConcept C79747257 @default.
• W2010717647 hasConcept C86803240 @default.
• W2010717647 hasConcept C95444343 @default.
• W2010717647 hasConceptScore W2010717647C170493617 @default.
• W2010717647 hasConceptScore W2010717647C185592680 @default.
• W2010717647 hasConceptScore W2010717647C2777853560 @default.
• W2010717647 hasConceptScore W2010717647C2780610907 @default.
• W2010717647 hasConceptScore W2010717647C28005876 @default.
• W2010717647 hasConceptScore W2010717647C41625074 @default.
• W2010717647 hasConceptScore W2010717647C55493867 @default.
• W2010717647 hasConceptScore W2010717647C62478195 @default.
• W2010717647 hasConceptScore W2010717647C79747257 @default.
• W2010717647 hasConceptScore W2010717647C86803240 @default.
• W2010717647 hasConceptScore W2010717647C95444343 @default.
• W2010717647 hasIssue "1" @default.
• W2010717647 hasLocation W20107176471 @default.
• W2010717647 hasLocation W20107176472 @default.
• W2010717647 hasLocation W20107176473 @default.
• W2010717647 hasOpenAccess W2010717647 @default.
• W2010717647 hasPrimaryLocation W20107176471 @default.
• W2010717647 hasRelatedWork W1503078246 @default.
• W2010717647 hasRelatedWork W2028679396 @default.
• W2010717647 hasRelatedWork W2055248999 @default.

• next