DJ-5 is a mouse monoclonal antibody specific for human DJ-1 protein [34
]. 691 is a rabbit polyclonal antibody raised against recombinant human DJ-1 protein but that reacts with DJ-1 from various species [34
Anti-Tim23 is a purified mouse monoclonal antibody to Tim23 (BD Transduction Laboratories, San Jose, CA). Anti-extracellular signal related kinase-1 (ERK-1) (C-16) is an affinity purified rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) that reacts with both ERK-1 and -2. Anti-Flag-tag mouse monoclonal antibody (GenScript Corporation, Piscataway, NJ) is a purified antibody that reacts with proteins with the amino acid sequence, DYKDDDDK. Anti-Actin is a purified mouse monoclonal antibody (Millipore Corporation, Billerica, MA 01821) that reacts with all six isoforms of vertebrate Actin.
Cloning of human DJ-1 constructs
Human full-length WT DJ-1 cDNA was cloned into the mammalian expression vector pZeoSV2 (Invitrogen, Carlsbad, CA) at the Not I and Hind III restriction sites to create the plasmid pZeoWThDJ. Using the pZeoWThDJ construct, the QuickChange® Site Directed Mutagenesis Kit (Stratagene, La Jolla, CA) was used in order to generate the E163K mutant form of human DJ-1 in the pZeoSV2 vector. The sequences of the oligonucleotides used for mutagenesis were as follows: Forward- 5′-GGG CCT GGG ACC AGC TTC AAG TTT GCG CTT GCA ATT GTT-3′ and Reverse- 5′- AAC AAT TGC AAG CGC AAA CTT GAA GCT GGT CCC AGG CCC-3′. The sequence of the plasmid with E163K mutant DJ-1 was verified by DNA sequencing as a service offered by the DNA Sequencing Facility of the University of Pennsylvania, and the construct was named pZeoEKhDJ.
Sequential restriction digestions with Not I and Apa I were performed on both pZeoWThDJ and pZeoEKhDJ. The DNA fragments containing the full-length DJ-1 sequences were ligated into the pcDNA3.1 (Invitrogen, Carlsbad, CA) mammalian expression vector in order to generate the human WT and E163K mutant DJ-1 constructs named pc3.1WThDJ1 and pc3.1EKhDJ1, respectively.
An N-terminal flag-tagged WT DJ-1 construct was generated by PCR, using the pZeoWTDJ-1 construct as a template. The sequences for the oligonucleotides used were as follows: Forward-5′- GAT CGC GGC CGC CAC CAT GGA TTA CAA GGA TGA CGA CGA TAA GGC TTC CAA AAG AGC TCT GGT CAT CCT -3′ and Reverse-5′-GAT CAA GCT TCT AGT CTT TAA GAA CAA GTG GAG CCT TC-3′. The tagged insert was cloned into the pCR 2.1 TOPO vector (Invitrogen, Carlsbad, CA) and subsequently cloned into the pcDNA3.1 (-) vector at the Hind III and Not I restriction sites. The construct was named pc3.1WThDJ1-Nflag. The sequence of the plasmid was verified by DNA sequencing as described above. The E163K N-terminal flag-tagged DJ-1 construct was generated by performing site directed mutagenesis using the pc3.1WThDJ1-Nflag construct as a template. The same oligonucleotides were used as described above for mutagenesis. The plasmid was named pc3.1E163KhDJ1-NFlag.
N2A and CHO cells were cultured in Dulbecco-modified Eagle medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Sigma, St.Louis, MO), 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA). Cells were incubated at 37°C and 95% air/5% CO2 atmosphere.
Generation of stable cell lines expressing WT and E163K DJ-1
The pc3.1WThDJ and pc3.1EKhDJ constructs were used to transfect N2A cells using FuGENE reagent following the manufacterer's protocol. Stably expressing clones were isolated and selected with G418 (Invitrogen, Carlsbad, CA) at 200-500 μg/mL and screened by Western blotting for the expression of DJ-1 using the antibody DJ5 that is specific for human DJ-1. Stably expressing clones expressing WT (clone #11 and clone #12) or E163K (clone #16, #47 and #52) human DJ-1 were used in the studies (See ).
The pZeoWThDJ and pZeoEKhDJ constructs were used to transfect CHO cells using FuGENE (Roche, Basel, Switzerland) transfection reagent according to the manufacturer's protocol. Stably expressing clones were isolated following selection with Zeocin (Invitrogen, Carlsbad, CA) at 50 ug/mL.
Native N2A and CHO cells or stable clonal lines expressing WT or E163K human DJ-1 were cultured in 10 cm dishes as described above. Cells were grown to confluency, rinsed and scraped in PBS, and harvested by centrifugation at 13,000 × g. Cells were vortexed vigorously in 2 pellet volumes of PBS/0.1% Triton, sedimented, and the supernatant was collected as the TX1 fraction. This was repeated on the remaining pellet and the supernatant was collected as the TX2 fraction. The pellet was resuspended in 2 volumes of RIPA buffer, vortexed vigorously, sedimented, and the supernatant was collected as the RIPA fraction. The remaining pellet was solubilized in 2%SDS/17mM Tris, pH 8.0 and kept as the SDS fraction.
Gel filtration chromatography
Gel filtration chromatography was performed by calibrating a Superose 6 column (Amersham Biosciences) attached to a fast performance liquid chromatography (FPLC) system with standards of known molecular mass using 10 mM Tris, pH 7.5, 100 mM NaCl as the mobile phase. Molecular mass standards [BSA (66 kDa), ovalbumin (44 kDa), carbonic anhydrase (29 kDa) and cytochrome C (12 kDa)] were resolved separately to calibrate the column. The elution of the standards was monitored by protein absorbance at 280 nm. N2A or CHO cells and stable cell lines thereof were cultured in 10 cm dishes and grown to confluency. Cells were rinsed and scraped in phosphate buffered saline, pH 7.4. After recovery by centrifugation, cells were lysed in PBS/0.1% Triton and the cell debris was sedimented at 13,000 × g for 5 min. The extracts were filtered through a 0.45 μm filter and loaded onto the column. Fractions were analyzed by immunoblotting with anti-DJ-1 antibodies, DJ5 (1:1000) and 691 (1:1000).
Pulse-chase protein turnover experiments
N2A or CHO cells stably expressing WT or E163K DJ-1 were cultured in 6-well dishes. Cells were methionine-deprived for 15 minutes by incubation in methionine-free DMEM (Invitrogen, Carlsbad, CA)/ 10% dialyzed FBS before adding 100μCi [35S]-methionine (Invitrogen, Carlsbad, CA) per ml of methionine free DMEM/10% dialyzed FBS for 30 min. Chase experiments were conducted in quadruplicates with normal DMEM/FBS for 0, 3, 6, 12, and 25 hours. Cells were then rinsed with PBS and harvested in CSK buffer (100 mM NaCl, 50 mM Tris, pH 7.5, 2 mM EDTA, 1 % Triton X-100) containing 1 % SDS and boiled at 100 °C for 5 minutes. CSK buffer was added to the lysates in order to bring the final concentration of SDS to 0.25%. Lysates were frozen on dry ice and kept frozen at -20 °C until the last time point was harvested. The radiolabelled protein extracts were pre-cleared with a rabbit serum pre-incubated with protein A-agarose (Santa Cruz Biotechnologies, Santa Cruz, CA) for 3 hours at 4°C and radiolabeled extracts were then immunoprecipitated overnight at 4°C with anti-DJ-1 polyclonal antibody 691 pre-incubated with protein A-agarose (Santa Cruz Biotechnologies, Santa Cruz, CA). The antibody-protein complexes were washed 3 times with 10 volumes of CSK buffer, resuspended in 2 volumes of 2X SDS sample buffer and boiled at 100 °C for 5 minutes. The beads were removed by centrifugation and the samples were loaded on 12 % polyacrylamide gels. Following electrophoresis, gels were fixed with 50% methanol/5% glycerol, dried and exposed to a PhosphorImager plate and the signal was quantified using ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA).
The subcellular fractionation procedures used were similar to those previously described [36
] with some changes. Native N2A, or N2A cells stably expressing WT or E163K DJ-1 were cultured in 10 cm plates. Cells were rinsed and scraped in phosphate-buffered saline, pH 7.4 (Invitrogen, Carlsbad, CA) and pelleted at 13,000 × g. Cells were resuspended in 3 pellet volumes of subfractionation buffer (0.25 M sucrose, 10 mM HEPES/NaOH, pH 7.5, 1 mM DTT, and protease inhibitors). Cells were homogenized with 20 strokes of a Dounce homogenizer (Kontes Glass Co;Vineland, NJ). The nuclei and unlysed cells were pelleted by sedimentation at 489 × g for 10 minutes at room temperature (RT). The supernatants were further cleared at 1585 × g for 10 minutes. The supernatants (S1) were removed to fresh tubes and sedimented for 10 minutes at 1585 × g at RT. The supernatants (S2) were removed to fresh tubes and pelleted for 17 minutes at 22000 × g at 4° C. The supernatants (S3) were removed as the crude cytoplasmic fraction and incubated on ice. In the mean time, the pellet (mitochondrial enriched fraction) was rinsed with 3 pellet volumes of subfractionation buffer and centrifuged again at 22000 × g for 17 minutes at 4°C. The supernatant from this rinse was discarded and the mitochondrial pellet was solubilized in 3 pellet volumes of 2% SDS/17mM Tris and boiled at 100 °C for 5 minutes. The tube containing the crude cytoplasmic supernatant (S3) was then sedimented at 103,000 × g for 25 minutes at 4 °C in order to pellet small organelles and cell debris, and the resulting supernatant (S4) was collected as the cytosolic fraction. The protein concentrations were determined in each fraction using the bicinchoninic acid protein (BCA) Assay (Pierce, Rockford, IL) with and bovine serum albumin as a standard. Fractions were analyzed by immunobloting with antibodies DJ5 (1:1000), 691 (1:1000), Tim23 (0.5ug/mL), and ERK1 (0.2ug/mL).
For subcellular fractionation experiments that followed oxidative stress, N2A cells or cells stably expressing human WT or E163K mutant DJ-1 were cultured in 10 cm plates and were treated for 1.5 hours with DMEM/FBS or DMEM/FBS containing 350uM H2O2. Cells were treated with H2O2 in duplicate. Cells were rinsed and scraped in PBS. Subcellular fractionation by differential centrifugation was performed as described above.
Cell viability assay
Native N2A or N2A cell lines expressing WT or E163K DJ-1 were cultured separately into 48 well plates. Each cell type was treated for 20 hours in sextuplicates with DMEM/FBS containing either 10, 20 or 30 uM H2O2, 7.5, 10, or 20 uM MPP dihydrochloride (Sigma, St.Louis, MO), 10, 20, or 30 uM MG-132 (Sigma, St.Louis, MO), or fresh DMEM/FBS. In separate experiments, these cell lines were treated for 96 hours in sextuplicates with either 50, 100, or 200 nM Antimycin A (Sigma, St.Louis, MO) or 5, 10, or 15 uM 3-nitropropionic acid (Sigma, St.Louis, MO). After treatment, the media from each well was collected into separate 1.5 mL microfuge tubes. The cells remaining in the wells were trypsinized, and harvested in corresponding microfuge tubes. Cells were pelleted and resuspended in fresh DMEM/FBS, and then 3-fold volumes of Trypan Blue solution (Sigma, St.Louis, MO) was added. Live and dead cells were counted manually using a hemacytometer and an Olympus CKX41 microscope. The percentage of live cells relative to the total number in each well was calculated.
Co-immunoprecitipation and heterodimer studies
N2A cells were cultured in 10 cm plates. Cells were mock transfected or transiently transfected with pc3.1E163KhDJ1-NFlag, or pc3.1WThDJ1-NFlag constructs using Lipofectamine reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. After 24 hours, cells were rinsed and scraped in PBS and lysed into 500 uL of 1X CSK buffer by vortexing. Cell debris was sedimented at 13,000 × g for 5 minutes and supernatants were removed to fresh tubes. 30 uL of each supernatant was saved as the “Start” fraction. The remainders of the supernatants were incubated at 4 °C with a mouse anti-flag antibody preabsorbed to protein A/G PLUS Agarose beads (Santa Cruz Biotechonogy, Inc., Santa Cruz, CA). The beads were sedimented, repeatedly rinsed with 1X CSK buffer, resuspended in sample buffer, heated to 100 °C and saved as the “IP” fractions. The supernatants remaining after the immunoprecipitation were saved as the “Unbound” fractions. Sample buffer was added to the “Start” and “Unbound” fractions and then they were heated to 100 °C for 5 minutes.
Immunofluorescence and confocal microscopy
Native N2A, or N2A cells stably expressing WT or E163K DJ-1 were treated with DMEM/FBS or DMEM/FBS containing 20 uM H2O2 for 3 hours. The media was removed and replaced with warmed DMEM/FBS containing 100 nM Mitotracker ® Red CMXRos (Invitrogen, Carlsbad, CA). The cells were allowed to respire for 20 minutes and then were fixed with neutral buffered formalin according to the manufacturer's protocol. The cells were rinsed with PBS for 5 minutes and then blocked for 30 minutes at room temperature in PBS/2% FBS/0.1% Triton. Cells were then incubated at 4°C with DJ5 antibody diluted into PBS/2% FBS at a concentration of 1:500 overnight, washed 3 times with PBS at 10 minutes each, and then incubated at room temperature with a goat anti-mouse secondary antibody conjugated to Alexa Fluor ® 488 (Invitrogen) diluted into PBS/2% FBS at a concentration of 1:500 for 2 hours. Cells were rinsed for 10 minutes with PBS, incubated at room temperature with DAPI (Pierce, Rockford, IL) diluted into PBS at a concentration of 1ug/mL for 5 minutes, and then rinsed 3 times with PBS at 10 minutes each. Cells were coversliped with Cytoseal™ 60 mounting media (Richard-Allen Scientific, Kalamazoo, MI). The images were visualized with a Zeiss LSM-510 Meta confocal microscope. For each sample, five 143 × 143 μm images were taken from a single plane using the 63× /1.4 oil objective. The images were then quantified using MetaMorph 6.0 software (Molecular Devices, Sunnyvale, CA). The relative integrated staining for DJ-1 per mitochondrial area was calculated by measuring the integrated pixel intensity for DJ5 signal that overlapped with pixels positive for Mitotracker Red CMXRos divided by total mitochondrial area. Integrated pixel intensity is defined as pixel intensity times the area of pixels positive for the signal. The average values for the replicate images were calculated.