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• W2079327750 abstract "NUB1 is a potent down-regulator of the ubiquitin-like protein NEDD8, because it targets NEDD8 to the proteasome for proteolytic degradation. From results in this study, we found that NUB1 physically interacts with synphilin-1 through its NEDD8-binding site, implying that NUB1 also targets synphilin-1 to the proteasome for degradation. Synphilin-1 is a major component of inclusion bodies found in the brains of patients with neurodegenerative α-synucleinopathies, including Parkinson's disease. In this study, we immunostained sections of brains from patients with Parkinson's disease and other α-synucleinopathies and demonstrated that NUB1, as well as synphilin-1, accumulates in the inclusion bodies. To define the role of NUB1 in the formation of these inclusion bodies, we performed a co-transfection assay using cultured HEK293 cells. This assay showed that NUB1 suppresses the formation of synphilin-1-positive inclusions. Further, biochemical assays revealed that NUB1 overexpression leads to the proteasomal degradation of synphilin-1. These results and our previous observations suggest that NUB1 indeed targets synphilin-1 to the proteasome for its efficient degradation, which, because of the resultant reduction in synphilin-1, suppresses the formation of synphilin-1-positive inclusions. NUB1 is a potent down-regulator of the ubiquitin-like protein NEDD8, because it targets NEDD8 to the proteasome for proteolytic degradation. From results in this study, we found that NUB1 physically interacts with synphilin-1 through its NEDD8-binding site, implying that NUB1 also targets synphilin-1 to the proteasome for degradation. Synphilin-1 is a major component of inclusion bodies found in the brains of patients with neurodegenerative α-synucleinopathies, including Parkinson's disease. In this study, we immunostained sections of brains from patients with Parkinson's disease and other α-synucleinopathies and demonstrated that NUB1, as well as synphilin-1, accumulates in the inclusion bodies. To define the role of NUB1 in the formation of these inclusion bodies, we performed a co-transfection assay using cultured HEK293 cells. This assay showed that NUB1 suppresses the formation of synphilin-1-positive inclusions. Further, biochemical assays revealed that NUB1 overexpression leads to the proteasomal degradation of synphilin-1. These results and our previous observations suggest that NUB1 indeed targets synphilin-1 to the proteasome for its efficient degradation, which, because of the resultant reduction in synphilin-1, suppresses the formation of synphilin-1-positive inclusions. NEDD8 is a ubiquitin-like protein that conjugates to a large number of target proteins in a manner analogous to ubiquitination.1Kamitani T Kito K Nguyen HP Yeh ETH Characterization of NEDD8, a developmentally down-regulated ubiquitin-like molecule.J Biol Chem. 1997; 272: 28557-28562Crossref PubMed Scopus (374) Google Scholar These target proteins include cullin family members, the von Hippel-Lindau tumor suppressor gene product, and p53.2Wada H Yeh ETH Kamitani T Identification of NEDD8-conjugation site in human cullin-2.Biochem Biophys Res Commun. 1999; 257: 100-105Crossref PubMed Scopus (75) Google Scholar, 3Stickle NH Chung J Klco JM Hill RP Kaelin Jr, WG Ohh M pVHL modification by NEDD8 is required for fibronectin matrix assembly and suppression of tumor development.Mol Cell Biol. 2004; 24: 3251-3261Crossref PubMed Scopus (147) Google Scholar, 4Xirodimas DP Saville MK Bourdon JC Hay RT Lane DP Mdm2-mediated NEDD8 conjugation of p53 inhibits its transcriptional activity.Cell. 2004; 118: 83-97Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar Because NEDD8 conjugation modifies the function of target proteins, the conjugation system appears to regulate many important biological events.3Stickle NH Chung J Klco JM Hill RP Kaelin Jr, WG Ohh M pVHL modification by NEDD8 is required for fibronectin matrix assembly and suppression of tumor development.Mol Cell Biol. 2004; 24: 3251-3261Crossref PubMed Scopus (147) Google Scholar, 4Xirodimas DP Saville MK Bourdon JC Hay RT Lane DP Mdm2-mediated NEDD8 conjugation of p53 inhibits its transcriptional activity.Cell. 2004; 118: 83-97Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar, 5Yeh ETH Gong L Kamitani T Ubiquitin-like proteins: new wines in new bottles.Gene. 2000; 248: 1-14Crossref PubMed Scopus (421) Google Scholar Recently, we identified a novel down-regulator of the NEDD8 conjugation system, NUB1.6Kito K Yeh ETH Kamitani T NUB1, a NEDD8-interacting protein, is induced by interferon and downregulates the NEDD8 expression.J Biol Chem. 2001; 276: 20603-20609Crossref PubMed Scopus (84) Google Scholar NUB1 is a NEDD8-interacting protein composed of 601 amino acid residues with a calculated molecular mass of 69.1 kd. It possesses a ubiquitin-like (UBL) domain at the N-terminal region and two ubiquitin-associated (UBA) domains at the C-terminal region. In a biochemical analysis, we found that NUB1 recruits NEDD8 and its conjugates to the proteasome for degradation, making NUB1 a down-regulator in the NEDD8 conjugation system.6Kito K Yeh ETH Kamitani T NUB1, a NEDD8-interacting protein, is induced by interferon and downregulates the NEDD8 expression.J Biol Chem. 2001; 276: 20603-20609Crossref PubMed Scopus (84) Google Scholar, 7Kamitani T Kito K Fukuda-Kamitani T Yeh ETH Targeting of NEDD8 and its conjugates for proteasomal degradation by NUB1.J Biol Chem. 2001; 276: 46655-46660Crossref PubMed Scopus (118) Google Scholar Most recently, to elucidate the function of NUB1, we performed a yeast two-hybrid screening using NUB1 as bait and isolated the cDNA of synphilin-1 from a human cDNA library. Synphilin-1, a 919-amino acid protein, is an α-synuclein-interacting protein whose function is currently unknown. It is predominantly expressed in neurons, localized in the cytoplasm and presynaptic nerve terminals, and is thought to be involved in the pathogenesis of Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), collectively referred to as α-synucleinopathies.8Engelender S Kaminsky Z Guo X Sharp AH Amaravi RK Kleiderlein JJ Margolis RL Troncoso JC Lanahan AA Worley PF Dawson VL Dawson TM Ross CA Synphilin-1 associates with alpha-synuclein and promotes the formation of cytosolic inclusions.Nat Genet. 1999; 22: 110-114Crossref PubMed Scopus (441) Google Scholar, 9Wakabayashi K Hayashi S Kakita A Yamada M Toyoshima Y Yoshimoto M Takahashi H Accumulation of alpha-synuclein/NACP is a cytopathological feature common to Lewy body disease and multiple system atrophy.Acta Neuropathol (Berl). 1998; 96: 445-452Crossref PubMed Scopus (321) Google Scholar, 10Wakabayashi K Engelender S Yoshimoto M Tsuji S Ross CA Takahashi H Synphilin-1 is present in Lewy bodies in Parkinson's disease.Ann Neurol. 2000; 47: 521-523Crossref PubMed Scopus (243) Google Scholar, 11Wakabayashi K Engelender S Tanaka Y Yoshimoto M Mori F Tsuji S Ross CA Takahashi H Immunocytochemical localization of synphilin-1, an alpha-synuclein-associated protein, in neurodegenerative disorders.Acta Neuropathol (Berl). 2002; 103: 209-214Crossref PubMed Scopus (83) Google Scholar PD is a common neurodegenerative disorder that is characterized by the loss of midbrain dopamine neurons12Lang AE Lozano AM Parkinson's disease. First of two parts.N Engl J Med. 1998; 339: 1044-1053Crossref PubMed Scopus (1790) Google Scholar and the presence of Lewy bodies (LBs), proteinaceous cytoplasmic inclusions that contain ubiquitin, NEDD8, α-synuclein, synphilin-1, and parkin.9Wakabayashi K Hayashi S Kakita A Yamada M Toyoshima Y Yoshimoto M Takahashi H Accumulation of alpha-synuclein/NACP is a cytopathological feature common to Lewy body disease and multiple system atrophy.Acta Neuropathol (Berl). 1998; 96: 445-452Crossref PubMed Scopus (321) Google Scholar, 10Wakabayashi K Engelender S Yoshimoto M Tsuji S Ross CA Takahashi H Synphilin-1 is present in Lewy bodies in Parkinson's disease.Ann Neurol. 2000; 47: 521-523Crossref PubMed Scopus (243) Google Scholar, 13Shimura H Schlossmacher MG Hattori N Frosch MP Trockenbacher A Schneider R Mizuno Y Kosik KS Selkoe DJ Ubiquitination of a new form of alpha-synuclein by parkin from human brain: implications for Parkinson's disease.Science. 2001; 293: 263-269Crossref PubMed Scopus (964) Google Scholar, 14Mori F Nishie M Piao YS Kito K Kamitani T Takahashi H Wakabayashi K Accumulation of NEDD8 in neuronal and glial inclusions of neurodegenerative disorders.Neuropathol Appl Neurobiol. 2005; 31: 53-61Crossref PubMed Scopus (89) Google Scholar Importantly, the mutations of genes encoding α-synuclein, synphilin-1, and parkin have been linked to familial forms of PD, indicating that functional derangements of these proteins have prominent roles in the pathogenesis of PD.15Polymeropoulos MH Lavedan C Leroy E Ide SE Dehejia A Dutra A Pike B Root H Rubenstein J Boyer R Stenroos ES Chandrasekharappa S Athanassiadou A Papapetropoulos T Johnson WG Lazzarini AM Duvoisin RC Di Iorio G Golbe LI Nussbaum RL Mutation in the alpha-synuclein gene identified in families with Parkinson's disease.Science. 1997; 276: 2045-2047Crossref PubMed Scopus (6750) Google Scholar, 16Kruger R Kuhn W Muller T Woitalla D Graeber M Kosel S Przuntek H Epplen JT Schols L Riess O Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson's disease.Nat Genet. 1998; 18: 106-108Crossref PubMed Scopus (3349) Google Scholar, 17Kitada T Asakawa S Hattori N Matsumine H Yamamura Y Minoshima S Yokochi M Mizuno Y Shimizu N Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism.Nature. 1998; 392: 605-608Crossref PubMed Scopus (4244) Google Scholar, 18Marx FP Holzmann C Strauss KM Li L Eberhardt O Gerhardt E Cookson MR Hernandez D Farrer MJ Kachergus J Engelender S Ross CA Berger K Schols L Schulz JB Riess O Kruger R Identification and functional characterization of a novel R621C mutation in the synphilin-1 gene in Parkinson's disease.Hum Mol Genet. 2003; 12: 1223-1231Crossref PubMed Scopus (124) Google Scholar In the study described here, we demonstrated the interaction between NUB1 and synphilin-1 and investigated the possible role of NUB1 in the formation of inclusion bodies in the neurodegenerative disorders related to synphilin-1, such as PD and other α-synucleinopathies. Human embryonic kidney HEK293 cells (American Type Culture Collection, Manassas, VA) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and antibiotics. Mouse anti-HA antibody 16B12 was purchased from Covance (Richmond, CA). Mouse anti-RH antibody (specific for the amino acid sequence RGSHHHH) was purchased from Qiagen (Santa Clara, CA). Mouse anti-ubiquitin antibody 1B3 was purchased from MBL (Nagoya, Japan). GST-12, a mouse monoclonal antibody specific for glutathione S-transferase (GST), was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-synphilin-1 antibody (AB5450) and goat anti-synphilin-1 antibody (AB5838) were purchased from Chemicon (Temecula, CA) and used for Western blotting and tissue staining, respectively. Mouse anti-phosphorylated α-synuclein antibody no. 64 (specific for α-synuclein phosphorylated at Ser129) was purchased from Wako (Osaka, Japan).19Saito Y Kawashima A Ruberu NN Fujiwara H Koyama S Sawabe M Arai T Nagura H Yamanouchi H Hasegawa M Iwatsubo T Murayama S Accumulation of phosphorylated alpha-synuclein in aging human brain.J Neuropathol Exp Neurol. 2003; 62: 644-654Crossref PubMed Scopus (306) Google Scholar Rabbit anti-actin antibody was purchased from Sigma (St. Louis, MO). Mouse monoclonal antibodies against the Rpt5 subunit of PA700 and the α-subunits of the 20S proteasome were purchased from Affiniti Research Products Ltd. (Mamhead, Exeter, UK). Mouse anti-FLAG antibody (M5) was purchased from Sigma. Rabbit anti-NEDD8 antibody was generated by immunization with a GST-fusion protein of human NEDD8, followed by affinity purification.7Kamitani T Kito K Fukuda-Kamitani T Yeh ETH Targeting of NEDD8 and its conjugates for proteasomal degradation by NUB1.J Biol Chem. 2001; 276: 46655-46660Crossref PubMed Scopus (118) Google Scholar Rabbit anti-human NUB1 antiserum was generated by immunization with a GST-fusion protein of NUB1 corresponding to amino acids 432 to 601, followed by affinity purification.6Kito K Yeh ETH Kamitani T NUB1, a NEDD8-interacting protein, is induced by interferon and downregulates the NEDD8 expression.J Biol Chem. 2001; 276: 20603-20609Crossref PubMed Scopus (84) Google Scholar Briefly, the IgG component was purified from the serum using a protein G-Sepharose column (Amersham Pharmacia Biotech, Piscataway, NJ). The IgG fraction was then passed over the GST column twice to remove antibodies to GST. The flow-through was subjected to the GST-NUB1 column for affinity purification. The purified anti-NUB1 antibody (100 μg/ml) was diluted and used. To express GST fusion proteins in Escherichia coli BL21 cells, cDNAs of HHR23B and NUB1 were subcloned into the pGEX-2TK plasmid (Amersham Pharmacia Biotech). To express RH-tagged synphilin-1 in E. coli BL21 cells, the cDNA was subcloned into the pTrcHis plasmid (Invitrogen, Carlsbad, CA). To experimentally form cytoplasmic inclusions, synphilin-1 was co-expressed with the NAC portion of α-synuclein in HEK293 cells. For this co-expression, we generated a plasmid to simultaneously express both EGFP-fused synphilin-1 and FLAG-tagged NAC. We constructed a pcDNA3 plasmid (Invitrogen) in which there are two sites for protein expression, a multiple cloning site (MCS) and a site for the neomycin-resistant gene (Neo). The Neo cDNA was first replaced with the cDNA of FLAG-NAC to generate a plasmid pNAC. Furthermore, the cDNA of synphilin-1 fused with EGFP at its C-terminus (Sph1-EGFP) was either inserted into the MCS of pcDNA3 to generate pSph1-EGFP or inserted into the MCS of pNAC to generate pNAC-Sph1 for the simultaneous expression of both synphilin-1-EGFP and FLAG-NAC. As a control vector, the cDNA of EGFP alone was either inserted into the MCS of pcDNA3 to generate pEGFP or inserted into the MCS of pNAC to generate pNAC-EGFP for the simultaneous expression of both EGFP and FLAG-NAC. These plasmids were transfected into HEK293 cells using FuGENE6 (BD Biosciences, San Jose, CA). To identify NUB1-interacting proteins, human testis cDNA library (Clontech, Mountain View, CA) was screened using the yeast two-hybrid assay system (Clontech) as described previously.20Tanaka T Yeh ET Kamitani T NUB1-mediated targeting of the ubiquitin precursor UbC1 for its C-terminal hydrolysis.Eur J Biochem. 2004; 271: 972-982Crossref PubMed Scopus (13) Google Scholar We performed a yeast two-hybrid assay to assess the interaction of synphilin-1 with truncated NUB1. First, by using a polymerase chain reaction (PCR) with appropriate primers, we prepared cDNAs of human synphilin-18Engelender S Kaminsky Z Guo X Sharp AH Amaravi RK Kleiderlein JJ Margolis RL Troncoso JC Lanahan AA Worley PF Dawson VL Dawson TM Ross CA Synphilin-1 associates with alpha-synuclein and promotes the formation of cytosolic inclusions.Nat Genet. 1999; 22: 110-114Crossref PubMed Scopus (441) Google Scholar and the truncated NUB1 as described previously.21Tanaka T Kawashima H Yeh ETH Kamitani T Regulation of the NEDD8 conjugation system by a splicing variant, NUB1L.J Biol Chem. 2003; 278: 32905-32913Crossref PubMed Scopus (61) Google Scholar The yeast Matchmaker Two-Hybrid System 3 (Clontech) was used to examine the in vivo interaction of synphilin-1 with these mutants. To do so, the cDNA of synphilin-1 was subcloned into pGADT7 (a Gal4 DNA-activating domain vector for Gal4-AD fusion), and the cDNA of each mutant of NUB1 was subcloned into pGBKT7 (a Gal4 DNA-binding domain vector for Gal4-BD fusion). The plasmids of the two fusion constructs were then co-transfected into AH109 yeast cells using the lithium acetate method.22Okura T Gong L Kamitani T Wada T Okura I Wei C-F Chang H-M Yeh ETH Protection against Fas/APO-1- and tumor necrosis factor-mediated cell death by a novel protein, Sentrin.J Immunol. 1996; 157: 4277-4281PubMed Google Scholar Transformed yeast cells were grown on a His−/Trp−/Leu− synthetic agar plate for 3 days at 30°C. The specific protein-protein interaction was determined by the growth of the cells on the selection plate. We performed a GST pull-down assay to confirm the result of the yeast two-hybrid interaction between synphilin-1 and NUB1. RH (RGSHHHHHH)-tagged synphilin-1 and GST fusion proteins, including GST, GST-HHR23B, and GST-NUB1, were expressed in E. coli BL21 cells by transformation with the pTrcHis plasmid and pGEX-2TK plasmid, respectively. Cells were then resuspended in the lysis buffer (50 mmol/L Tris-HCl, pH 7.5, 100 mmol/L NaCl, and 0.1% Nonidet P-40) containing the protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN) and then lysed by brief sonication. The GST fusion proteins were purified using glutathione-Sepharose beads (Amersham Pharmacia Biotech) as described previously.23Kamitani T Nguyen HP Yeh ETH Activation-induced aggregation and processing of the human Fas antigen-detection with cytoplasmic domain specific antibodies.J Biol Chem. 1997; 272: 22307-22314Crossref PubMed Scopus (80) Google Scholar The bacterial crude lysate containing RH-synphilin-1 was centrifuged at 14,000 × g for 5 minutes, and the supernatant was incubated for 3 hours at room temperature with GST fusion proteins immobilized on glutathione-Sepharose beads. The beads were then washed four times with the lysis buffer. The precipitated RH-synphilin-1 on the beads was solubilized in 2% sodium dodecyl sulfate (SDS) treating solution (75 mmol/L Tris-HCl, pH 6.8, 2.0% SDS, 25% glycerol, 5% β-mercaptoethanol), followed by Western blot analysis using anti-RH antibody and anti-GST antibody. Protein samples were treated for 1 hour at 50°C in 2% SDS treating solution. After SDS-polyacrylamide gel electrophoresis, Western blot analysis was performed according to the protocol provided with the ECL detection system (Amersham Pharmacia Biotech). Horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG antibody (Santa Cruz Biotechnology) was used as a secondary antibody. Northern blotting was performed to show the mRNA expression of NUB1 in various human tissues. For this analysis, a human NUB1 cDNA fragment of 450 bp was amplified by PCR using pcDNA3/RH-NUB1 as the template6Kito K Yeh ETH Kamitani T NUB1, a NEDD8-interacting protein, is induced by interferon and downregulates the NEDD8 expression.J Biol Chem. 2001; 276: 20603-20609Crossref PubMed Scopus (84) Google Scholar, 20Tanaka T Yeh ET Kamitani T NUB1-mediated targeting of the ubiquitin precursor UbC1 for its C-terminal hydrolysis.Eur J Biochem. 2004; 271: 972-982Crossref PubMed Scopus (13) Google Scholar and subcloned into the pGEM-T plasmid (Promega, Madison, WI). The insert was then excised and labeled with [α-32P]-dCTP by the Ready-To-Go DNA labeling kit (Amersham Pharmacia Biotech). The radioactive probe was then hybridized with two human multiple-tissue Northern blots (Clontech) in ExpressHyb solution (Clontech). After washing, the blot membrane was exposed to film for 5 days. As a control, the radioactive probe of β-actin was hybridized with the Northern blots, followed by exposure to film for 2 days. Immunohistochemical studies were performed to determine the presence of NUB1 in samples of brains from patients with PD (n = 5), DLB (n = 5), and MSA (n = 5), as well as brain samples from normal subjects (n = 5), which were obtained from the Department of Pathology, Brain Research Institute, University of Niigata, Niigata, Japan, and the Department of Neuropathology, Hirosaki University School of Medicine, Hirosaki, Japan. Brains were fixed with 10% buffered formalin for 3 weeks and then embedded in paraffin. For this study, serial 4-μm-thick sections were prepared. We routinely deparaffinized and rehydrated sections from the midbrain and upper pons of cases of PD, the temporal lobe of cases of DLB, the upper pons of cases of MSA, and the temporal lobe and brainstem of normal controls. The sections were immunostained using the avidin-biotin-peroxidase complex method with diaminobenzidine as described previously.11Wakabayashi K Engelender S Tanaka Y Yoshimoto M Mori F Tsuji S Ross CA Takahashi H Immunocytochemical localization of synphilin-1, an alpha-synuclein-associated protein, in neurodegenerative disorders.Acta Neuropathol (Berl). 2002; 103: 209-214Crossref PubMed Scopus (83) Google Scholar, 24Kamitani T Suzuki H Yano S An antibody reacting with splenic red pulp macrophages in the sera of patients with rheumatic diseases.Clin Immunol Immunopathol. 1991; 58: 217-235Crossref PubMed Scopus (8) Google Scholar The antibodies used for the immunostaining were polyclonal anti-NUB1 (2 μg/ml), polyclonal anti-synphilin-1 (1:500), and monoclonal anti-phosphorylated α-synuclein (1:5000). The sections were then counterstained with hematoxylin. The total number of inclusions immunostained with anti-NUB1 and anti-phosphorylated α-synuclein was quantified in contiguous sections. Selected sections from the brainstem of PD, the temporal lobe of DLB, and the pons of MSA were double-immunolabeled with anti-NUB1 (10 μg/ml) and anti-phosphorylated α-synuclein antibodies (1:500). The secondary antibodies used were fluorescein isothiocyanate-conjugated anti-rabbit IgG (Vector Laboratories, Burlingame, CA) and Texas Red-conjugated anti-mouse IgG (Vector Laboratories). The sections were examined with an Olympus Provis fluorescence microscope (Olympus, Tokyo, Japan). To investigate synphilin-1-positive inclusions in cultured cells, we performed immunocytochemical studies. HEK293 cells were cultured on a coverslip in a 3.5-cm dish and transfected with 2 μg of pNAC-Sph1. After 24 hours, the cells were fixed with a 4% paraformaldehyde solution, pH 7.5, for 30 minutes and permeabilized with 0.1% Triton X-100 for 15 minutes at room temperature. The cells were first labeled with one of the following primary antibodies: mouse anti-FLAG (1:1800) for FLAG-NAC, rabbit anti-NUB1 (1:2000), mouse anti-Rpt5/PA700 (1:20,000), mouse anti-20S proteasome core subunits (1:4000),25Ito T Niwa J Hishikawa N Ishigaki S Doyu M Sobue G Dorfin localizes to Lewy bodies and ubiquitylates synphilin-1.J Biol Chem. 2003; 278: 29106-29114Crossref PubMed Scopus (69) Google Scholar mouse anti-ubiquitin (1:200), or rabbit anti-NEDD8 (1:2000). After washing, the cells were labeled with Texas Red-conjugated anti-mouse IgG (1:400) or anti-rabbit IgG (1:400) secondary antibody (Santa Cruz Biotechnology). The cells were then analyzed under a fluorescence microscope (Axioplan 2 Imaging; Carl Zeiss, Thornwood, NY). The localization of EGFP-fused synphilin-1 was shown by the green fluorescence of EGFP, and the localization of other proteins was shown by the red fluorescence of Texas Red. Their co-localization with synphilin-1 was shown by the merging of both fluorescences. HEK293 cells were cultured on a coverslip in a 3.5-cm dish and transfected with the following plasmids: 1) pEGFP (1 μg) and pcDNA3 (1 μg), 2) pNAC-EGFP (1 μg) and pcDNA3 (1 μg), 3) pSph1-EGFP (1 μg) and pcDNA3 (1 μg), 4) pNAC-Sph1 (1 μg) and pcDNA3 (1 μg), and 5) pNAC-Sph1 (1 μg) and pcDNA3/FLAG-NUB1 (1 μg). Twenty-four hours after transfection, the cells were fixed in a 4% paraformaldehyde solution, pH 7.5, and examined under a fluorescence microscope. Cells expressing EGFP or synphilin-1-EGFP were counted to determine the number of transfected cells. The transfected cells containing cytoplasmic inclusions were also counted. The value of percent cells with inclusions was calculated as the ratio of the number of transfected cells containing inclusions to the total number of transfected cells. All values were calculated from three independent experiments. To inhibit the expression of endogenous NUB1 in HEK293 cells, RNAi was used. Four short interfering RNAs (siRNAs) were designed and synthesized by Dharmacon (Lafayette, CO). The effect of these siRNAs was tested in our lab. Because one of the siRNAs showed complete inhibition of NUB1 expression, we used this siRNA for RNAi of NUB1. The NUB1 siRNA sequences, corresponding to nucleotides 458 to 476 after the start codon, were as follows: 5′-CGAUGGUGCUUGAACUAAAUU-3′ and 5′-UUUAGUUCAAGCACCAUCGUU-3′. The NUB1 siRNA or control siRNA was transfected for RNAi. Briefly, the siRNA and a plasmid DNA (pNAC-SphI) were co-transfected into HEK293 cells by Lipofectamine 2000 (Invitrogen). We performed a filter-trap assay to determine the experimental conditions under which inclusions were solubilized for the purpose of performing biochemical studies of inclusion formation. We modified the filter-trap assay method described previously.26van der Spuy J Cheetham ME The Leber congenital amaurosis protein AIPL1 modulates the nuclear translocation of NUB1 and suppresses inclusion formation by NUB1 fragments.J Biol Chem. 2004; 279: 48038-48047Crossref PubMed Scopus (27) Google Scholar In brief, cells were lysed in a lysis buffer [20 mmol/L Tris-HCl, pH 8.0, 100 mmol/L NaCl, 0.01% SDS, protease inhibitor cocktail (Roche)] and sonicated for 10 seconds. The samples were boiled for 2 minutes and immediately applied to a 0.22-μm cellulose acetate membrane (Osmonics Inc., Minnetonka, MN) on a dot-blot apparatus (Millipore, Billerica, MA) using a vacuum manifold. After a 20-minute incubation at room temperature, the membrane was washed three times with a wash buffer (20 mmol/L Tris-HCl, pH 8.0, 100 mmol/L NaCl, 0.08% SDS). The membrane was removed and Western blotted as described above. To solubilize all derivatives of RH-synphilin-1 in 6 mol/L guanidine HCl and biochemically analyze them, we performed TALON-bead precipitation of RH-synphilin-1 as described previously.27Wada H Yeh ETH Kamitani T A dominant negative Ubc12 mutant sequesters NEDD8 and inhibits NEDD8-conjugation in vivo.J Biol Chem. 2000; 275: 17008-17015Crossref PubMed Scopus (69) Google Scholar Briefly, 1 × 106 HEK293 cells were co-transfected by FuGENE6 (Roche) to express RH-synphilin-1, HA-ubiquitin, NAC, and FLAG-NUB1. Twenty-four hours after transfection, the culture medium was replaced with fresh medium or medium containing 20 μmol/L MG132 (Calbiochem, San Diego, CA) and cultured at 37°C for 4 hours. The cells were then harvested and lysed in lysis buffer (20 mmol/L Tris-HCl, pH 8.0, 6 mol/L guanidine-HCl, 100 mmol/L NaCl). In this lysis buffer, all proteins, including deubiquitinating enzymes, were denatured by 6 mol/L guanidine HCl. Therefore, the ubiquitinated synphilin-1 was stable during the procedure. DNA in the lysate sample was sheared with a 22-gauge needle. The lysate was then incubated with cobalt-immobilized TALON beads (Clontech) for 1 hour at room temperature. Because the sequence of the RH tag was RGSHHHHHH, RH-synphilin-1 could be purified by TALON beads.2Wada H Yeh ETH Kamitani T Identification of NEDD8-conjugation site in human cullin-2.Biochem Biophys Res Commun. 1999; 257: 100-105Crossref PubMed Scopus (75) Google Scholar, 27Wada H Yeh ETH Kamitani T A dominant negative Ubc12 mutant sequesters NEDD8 and inhibits NEDD8-conjugation in vivo.J Biol Chem. 2000; 275: 17008-17015Crossref PubMed Scopus (69) Google Scholar The beads were washed once with the lysis buffer and then washed twice with washing buffer (20 mmol/L Tris-HCl, pH 7.0, 15 mmol/L imidazole, 8 mol/L urea, 100 mmol/L NaCl). Finally, the beads were washed twice with phosphate-buffered saline and treated for 1 hour at 50°C in 2% SDS treating solution. The solubilized proteins were analyzed by Western blotting using anti-HA antibody and anti-RH antibody. All values were presented as means ± SE. Statistical significance of the data were evaluated using analysis of variance, followed by post hoc tests using the Fisher's adjustment or the Student's t-test when comparing two conditions. Probability values less than 0.05 (P < 0.05) were considered significant and probability values less than 0.01 (P < 0.01) were considered highly significant. We have previously identified the novel NEDD8-interacting protein NUB1. To investigate the molecular function of NUB1, we searched NUB1-interacting proteins by yeast two-hybrid screening using NUB1 as bait. Because the mRNA of NUB1 is highly enriched in the testis,6Kito K Yeh ETH Kamitani T NUB1, a NEDD8-interacting protein, is induced by interferon and downregulates the NEDD8 expression.J Biol Chem. 2001; 276: 20603-20609Crossref PubMed Scopus (84) Google Scholar a human testis cDNA library was used for the screening. Approximately 2 × 106 primary library transformants were inoculated onto selection plates. A total of 84 colonies grew on the selection plates, 40 of which stained positive when tested for β-galactosidase expression. Subsequent DNA sequencing of the positive clones showed that two clones encoded synphilin-1. The rest of the positive clones included 2 clones of NEDD8,6Kito K Yeh ETH Kamitani T NUB1, a NEDD8-interacting protein, is induced by interferon and downregulates the NEDD8 expression.J Biol Chem. 2001; 276: 20603-20609Crossref PubMed Scopus (84) Google Scholar 25 clones of UbC1,20Tanaka T Yeh ET Kamitani T NUB1-mediated targeting of the ubiquitin precursor UbC1 for its C-terminal hydrolysis.Eur J Biochem. 2004; 271: 972-982Crossref PubMed Scopus (13) Google Scholar and the other 11 clones that were not investigated yet. The result mentioned above indicated that synphilin-1 interacts with NUB1 directly or indirectly in vivo. To further investigate the interaction, an in vitro interaction assay was performed. RH-tagged synphilin-1 was expressed in bacteria. The bacterial lysate containing RH-synphilin-1 was then incubated with GST alone (negative control), GST-fused HHR23B (negative control), or GST-fused NUB1" @default.
• W2079327750 created "2016-06-24" @default.
• W2079327750 creator A5000409879 @default.
• W2079327750 creator A5000880725 @default.
• W2079327750 creator A5006691819 @default.
• W2079327750 creator A5008524978 @default.
• W2079327750 creator A5046502423 @default.
• W2079327750 creator A5067901270 @default.
• W2079327750 creator A5088439484 @default.
• W2079327750 date "2006-08-01" @default.
• W2079327750 modified "2023-10-14" @default.
• W2079327750 title "NUB1 Suppresses the Formation of Lewy Body-Like Inclusions by Proteasomal Degradation of Synphilin-1" @default.
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