DJ-1 gene deletion reveals that DJ-1 is an atypical peroxiredoxin-like peroxidase

Andres-Mateos et 10.1073/pnas.0703219104.XXYYYYY103.

Supporting Information

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SI Figure 4
SI Figure 5
SI Figure 6
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SI Figure 8
SI Materials and Methods

SI Figure 4

Fig. 4. Targeted disruption of DJ-1 and the biochemical, anatomical, and behavioral characterization of the dopaminergic system in DJ-1 KO mice. (A) Schematic representation of the targeting strategy. (B) Southern blot analysis of genomic DNA from WT (+/+), heterozygous (+/-), and homozygous DJ-1 KO (-/-) mice. EcoRI-digested DNA was hybridized with the probe shown in (A) and PCR analysis of genomic DNA from WT (+/+), heterozygous (+/-), and homozygous DJ-1 KO (-/-) mice. (C) A cDNA fragment of DJ-1 mRNA open reading frame was used as the probe for Northern blot analysis of total RNA from WT (+/+), heterozygous (+/-), and homozygous DJ-1 KO (-/-) mice and immunoblot analysis of whole-brain lysate from WT (+/+), heterozygous (+/-), and homozygous DJ-1 KO (-/-) mice with anti-full length DJ-1 antibody. The blot was re-probed with anti-actin antibody as loading control. (D) Concentrations of catecholamines were determined by HPLC with electrochemical detection. Striatal levels of dopamine and its metabolites of WT and KO mice (n=5). (E-H) Immunohistochemical staining for TH with Nissl counter stain was performed on mesencephalic coronal brain sections from DJ-1 KO and WT mice. (E and F) striatum. (G and H) and ventral midbrain (scale bars,400 mm). (I) Stereological analysis of the number of TH-positive and Nissl-positive neurons in substantia nigra in young (2-3 month) and aged (18-24 month) mice (n=5). (J) Behavioral analysis: Total number of horizontal and vertical movements in the open field (n=8). Each mouse was placed in an open-field cage in the dark. Data were the average of data collected in 10-min trials for a 1-hr test period. Data are the mean ± SEM, n=X, * P < 0.05.

SI Figure 5

Fig. 5. Measurement of protein carbonyls and OxyBlot in DJ-1 mice. (A) Quantification of protein carbonyls in 2-3- and 18-24-month-old DJ-1 WT and KO mice after derivatization of brain mitochondrial fraction homogenates with 2,4-dinitrophenylhydrazine. Data are the mean ± SEM (n = 6). Significance was determined by Student's t-test, *, P ≤ 0.05. (B and C) Immunoblot detection of carbonyl groups in whole brain lysis (B) and mitochondrial fraction (C) with b-actin and VDAC as a loading control in 18- and 24-month-old mice.

SI Figure 6

Fig. 6. (A) No changes were found in mitochondrial oxygen consumption in the absence of presence of rotenone or succinate. Mitochondria (300 mg) were incubated (2.5 minutes) at 30ºC in 2-3 month mice. (B) Mitochondrial ATP measurements. Mitochondria from KO and WT littermates suspended in respiration buffer were incubated in the presence or absence of rotenone using the same incubation times as those of polarographical study. Light emitted from luciferase-mediate reaction was captured in a tube luminometer and calculated from a log-log plot of the standard curve of known ATP concentrations. (C) Complex I inhibition by MPP + (mM) or incubation with 100 nM recombinant Bid or with 100 mM calcium alone or in combination did not reveal differences in the release of cytochrome c from mitochondria isolated from young DJ-1 KO or WT mice. The control level at 1% is 100% of the level of the control. Data are expressed as mean ± SEM (n = 3), significance was determined by Student's t-test, *, P ≤ 0.05.

SI Figure 7

Fig. 7. Electrospray ionization mass spectrometry analysis of recombinant human DJ-1 (Upper) and recombinant human DJ-1 treated with 250 µM H₂O₂ (Lower). The spectra of multiply charged fragment ions with mass/charge ratio (m/z) values (Left) and corresponding deconvoluted spectra with calculated masses (Right) are shown.

SI Figure 8

Fig. 8. Specific recognition of DJ-1 oxidized at C106 in WT mice injected with MPTP or saline. (A) Immunoblot and densitometric analysis of the protein expression of DJ-1 oxidized at Cys106 and DJ-1 in whole brain lysate after immunoprecipitation with anti-DJ-1 antibody, total DJ-1 was used as loading control. Data are the mean ± SEM (n = 3). (B) immunoblot and quantitative analysis of DJ-1 oxidized at Cys106 and total DJ-1 in the mitochondrial fraction with VDAC as a loading control. Data are the mean ± SEM (n = 4). Significance was determined by Student's t-test, *, P ≤ 0.05.

SI Materials and Methods

Measurements of mitochondrial H₂O₂. Brain mitochondria were suspended in respiration buffer (225 mM mannitol, 75 mM sucrose, 10 mM KCl, 5 mM HEPES, 5 mM K₂HPO₄, and freshly added 1 mg/ml defatted BSA) at 30°C. For each reading, 300 mg protein was used in a final 1-ml respiration buffer containing 10 mM glutamate and 5 mM malate as NADH-linked substrates. After 3 min of stabilization of the baseline, mitochondria were added, and, after an additional 5 min, the different inhibitors were added in the incubation medium for a total reading of 20 min. Hydrogen peroxide, converted from superoxide by manganese-superoxide dismutase, was measured using 5 μM Amplex red (Molecular Probes, Eugene, Oregon) and 5 units/ml HRP. Fluorescence was detected by a Perkin-Elmer (Boston, Massachusetts) LS55 spectrofluorometer with an excitation wavelength of 550 nm (slit 1.5 nm) and an emission wavelength of 585 nm (slit 3 nm). H₂O₂ production was calculated from a standard curve generated from known concentrations of H₂O₂.

Mitochondrial aconitase activity assay. For all the enzymatic assays, brain mitochondria were resuspended in Triton buffer (50 mM Tris, pH 7.4/1mM EDTA/0.05% Triton). Aconitase activity was measured by monitoring absorbance at 340 nm in a freshly prepared reaction mix containing 0.2 mM NADP⁺, 5 mM sodium citrate, and a 1.8 unit/ml concentration of isocitrate dehydrogenase in Tris·HCl pH 7.4 plus MnCl₂ buffer. To start the assay, freshly sonicated aliquots of mitochondria (50 mg of protein) were added to the 1.0-ml reaction mix preequilibrated at 25°C. Measurements at 340 nm were recorded in a 1-cm cuvette at intervals of 30 sec, and aconitase activity was calculated from the linear increase in absorbance at 340 nm. Activity was calculated by using an extinction coefficient for NADPH of 6.22 mM^-1 cm^-1 and assuming the conversion of one molecule of citrate to one molecule of NADPH via isocitrate dehydrogenase. The reactivation of aconitase was assessed according to Hausladen and Fridovich by incubation with the following reagents prepared fresh before use DTT 5 mM, Ferrous Ammonium Sulfate 0.2 mM, and Na₂S 0.2 mM. Activity was measured immediately, because aerobic inactivation by O₂ will lead to a time-dependent decrease in activity.

Mitochondrial a-ketoglutarate deshydrogenase activity assay. For a-ketoglutarate deshydrogenase (KGDHC) assay, all the reagents were prepared fresh (50 mM MOPS, pH 7.4/0.2 mM MgCl₂/0.01 mM CaCl₂/0.3 mM Thiamine pyropohosphate/0.166 mM CoA/2 mM NAD/0.3 mM DTT) and 100 mg of mitochondria were added. The incubation mixture was mixed by inversion, equilibrated at 30ºC and monitored at 340 nm. a-Ketoglutarate was added to the reaction and the reading was monitored for an additional 5 minutes.

DJ-1 consumptions of hydrogen peroxide. The assay was realized in phosphate buffer, pH 7.0, at 37ºC in presence of 10 nM of H₂O₂ with or without different concentrations of recombinant DJ-1. After 5 min of incubation, H₂O₂ decrease was measured using 5 µM Amplex red (Molecular Probes, Eugene, Oregon) and 5 U/ml HRP according to the manufacturers instructions.

ATP measurements. Mitochondrial samples were prepared under identical conditions to H₂O₂ measurements study. Mitochondria suspended in respiration buffer were incubated in the presence or absence of rotenone. Rotenone was added to the mitochondria 2.5 minutes before the addition of ADP 500 µM. When the reaction was stopped, mitochondrial suspension from the cuvette was lysed in an equal volume of lysis buffer from the ATP bioluminescence assay kit (Roche Molecular Biochemicals), and the content of ATP was measured with the light emitted from luciferase mediated reaction by capture in a tube luminometer and calculated from a log-log plot of the standard curve of known ATP concentrations.

Catalase activity. To ascertain whether recombinant DJ-1 has catalase-like activity, we measured O2 formation with a Clark electrode in phosphate buffer, pH 7.0, at 30ºC in presence of 10 mM of H₂O₂.

O₂ consumption measurements. Brain mitochondria were suspended in respiration buffer (225 mM mannitol/75 mM sucrose/10 mM KCl/5 mM HEPES/5 mM K₂HPO₄/freshly added 1 mg/ml defatted BSA) at 30°C. The oxygen-consumption rates were measured in a closed-chamber cuvette with a ministirring bar using a Clarktype electrode (Hansatech Instruments, Norfolk, U.K.). For each reading, 300 µg protein was used in a final 1-ml respiration buffer, and all mitochondria preparations had an average respiratory control ratio of at least 5 with 10 mM glutamate and 5 mM malate as NADH-linked substrates. ADP (500 µM) was added 2.5 minutes after the addition of mitochondria in the presence or absence of different concentrations of rotenone.

Measurement of protein oxidation and Oxyblot. Protein carbonyls were detected after derivatization of brain homogenates with 2,4-dinitrophenylhydrazine (DPNH) using methods previously described . The concentration of carbonyls were determined from the absorbance at 360 nm normalized to the protein concentration using an extinction coefficient of 21 nM^-1 cm^-1. Immunoblot detection of carbonyl groups into the proteins was carried out in brain homogenate containing 50 mM DTT using OxyBlot protein oxidation kit (Chemicon, Temecula, CA) as specified by the manufacturer.