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Oxidative Damage of DJ-1 Is Linked to Sporadic Parkinson and Alzheimer Diseases *

Oxidative Damage of DJ-1 Is Linked to Sporadic Parkinson and Alzheimer Diseases * THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 16, pp. 10816 –10824, April 21, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Oxidative Damage of DJ-1 Is Linked to Sporadic Parkinson and Alzheimer Diseases Received for publication, August 17, 2005, and in revised form, February 13, 2006 Published, JBC Papers in Press, March 3, 2006, DOI 10.1074/jbc.M509079200 ‡ § ‡ ¶ Joungil Choi , M. Cameron Sullards , James A. Olzmann , Howard D. Rees , Susan T. Weintraub , § ¶ ‡ ‡1 David E. Bostwick , Marla Gearing**, Allan I. Levey , Lih-Shen Chin , and Lian Li ‡ ¶ From the Department of Pharmacology, Neurology, and **Pathology, Center for Neurodegenerative Disease, Emory University School of Medicine, Atlanta, Georgia 30322, Bioanalytical Mass Spectrometry Facility, Parker H. Petit Institute for Bioengineering and Biosciences, School of Chemistry and Biochemistry and School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332, and Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229 Mutations in DJ-1 cause an autosomal recessive, early onset AD and PD have been associated with increased production of reactive familial form of Parkinson disease (PD). However, little is presently oxygen species (ROS), which could result from a combination of aging, known about the role of DJ-1 in the more common sporadic form of genetic predisposition, and environmental factors (6). Epidemiological PD and in other age-related neurodegenerative diseases, such as studies suggest that exposure to pesticides, herbicides, and other envi- Alzheimer disease (AD). Here we report that DJ-1 is oxidatively ronmental toxins that inhibit mitochondrial complex I can lead to damaged in the brains of patients with idiopathic PD and AD. By excess production of ROS and increased incidence of sporadic PD (8). using a combination of two-dimensional gel electrophoresis and Furthermore, post-mortem analyses reveal that the overall levels of oxi- mass spectrometry, we have identified 10 different DJ-1 isoforms, of dative damage to proteins, lipids, and DNA are elevated in AD and PD which the acidic isoforms (pI 5.5 and 5.7) of DJ-1 monomer and the brains (4, 9). basic isoforms (pI 8.0 and 8.4) of SDS-resistant DJ-1 dimer are selec- The most widely used marker for oxidative damage to proteins is the tively accumulated in PD and AD frontal cortex tissues compared presence of carbonyl groups, which can be introduced into proteins by with age-matched controls. Quantitative Western blot analysis direct oxidation of Pro, Arg, Lys, or Thr side chains or by Michael shows that the total level of DJ-1 protein is significantly increased in addition reactions of Cys, His, or Lys residues with products of lipid PD and AD brains. Mass spectrometry analyses reveal that DJ-1 is peroxidation or glycooxidation (5, 10, 11). Elevation in the total level not only susceptible to cysteine oxidation but also to previously of protein carbonyls has been documented in both AD and PD (4, 9). unsuspected methionine oxidation. Furthermore, we show that Although it was initially thought that targets of oxidative damage by DJ-1 protein is irreversibly oxidized by carbonylation as well as by reactive oxygen species were random and indiscriminate, it has become methionine oxidation to methionine sulfone in PD and AD. Our increasingly clear that the susceptibility of proteins to oxidative damage study provides new insights into the oxidative modifications of DJ-1 is highly dependent on specific properties of individual proteins, such as and indicates association of oxidative damage to DJ-1 with sporadic unique sequence motifs, surface accessibility, protein folding, and sub- PD and AD. cellular localization (10–12). The identities of the oxidatively damaged proteins in AD and PD that have been modified by carbonylation or other types of oxidation remain largely unknown. Alzheimer disease (AD) and Parkinson disease (PD) are the two Mutations in DJ-1 has recently been linked to an autosomal reces- most common neurodegenerative disorders characterized by the selec- sive, early onset familial form of PD (13). DJ-1 is a ubiquitously tive loss of neurons in specific brain regions and the deposition of mis- expressed protein of the DJ-1/ThiJ/PfpI superfamily (14, 15). The folded proteins into aggregates or inclusions, such as neurofibrillary precise biochemical function of DJ-1 is unknown, although DJ-1 has tangles and amyloid plaques in AD and Lewy bodies in PD (1). The been proposed to act as a protease, chaperone, or antioxidant (15– majority of AD and PD cases are sporadic with hereditary familial cases 19). The identification of a homozygous DJ-1 deletion that prevents accounting for less than 10% (2, 3). The genetic defects underlying sev- the expression of DJ-1 protein in recessively transmitted PD (13) eral monogenic familial forms of AD and PD have recently been identi- strongly suggests that loss of DJ-1 function leads to neurodegenera- fied (3). However, the causes of other AD and PD cases, particularly tion. A loss-of-function pathogenic mechanism is also supported by sporadic cases, remain unclear. analysis of PD-linked DJ-1 L166P mutation (17, 20 –22) and DJ-1 Increasing evidence indicates that oxidative stress plays a crucial role knock-out studies in mice (23–25). Despite increasing genetic evi- in the pathogenesis of idiopathic AD and PD (4–7). For example, both dence indicating the importance of DJ-1 mutations in causing early onset familial PD (13, 26 –28), the role of DJ-1 in sporadic PD is * This work was supported by National Institutes of Health Grants NS047199, NS047575, unknown. Interestingly, previous studies have shown that DJ-1 is NS050650, AG021489, and AG025688. The costs of publication of this article were rarely present in Lewy bodies in PD (29, 30). However, DJ-1 has been defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to found to colocalize with -synuclein-immunoreactive glial inclu- indicate this fact. sions in multiple system atrophy and with a subset of pathological tau To whom correspondence should be addressed: Dept. of Pharmacology, Emory Uni- versity School of Medicine, 1510 Clifton Rd., Atlanta, GA 30322-3090. Tel.: 404-727- inclusions in a number of neurodegenerative tauopathies, including AD 5987; Fax: 404-727-0365; E-Mail: [email protected]. 2 (29, 30). Here, we show that DJ-1 is extensively and irreversibly oxidized The abbreviations used are: AD, Alzheimer disease; PD, Parkinson disease; DNP, 2,4- dinitrophenyl; ROS, reactive oxygen species; MALDI-TOF/MS, matrix-assisted laser in brains of patients with idiopathic PD and AD. The observed oxidative desorption ionization time-of-flight mass spectrometry; MALDI-TOF/TOF/MS/MS, damage to DJ-1 may have important implications for understanding the MALDI-TOF tandem mass spectrometry; HPLC, high performance liquid chromatog- raphy; ESI/MS/MS, electrospray ionization tandem mass spectrometry. pathogenesis of sporadic PD and AD. 10816 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 16 •APRIL 21, 2006 This is an Open Access article under the CC BY license. DJ-1 Oxidation in Neurodegenerative Diseases TABLE 1 RESULTS Demographic data of cases included in this study Accumulation of Acidic Isoforms of DJ-1 Monomer and Basic Isoforms Case no. Diagnosis Age Sex PMI of DJ-1 Dimer in PD and AD Brains—To investigate whether the years h expression and/or posttranslational modification(s) of DJ-1 is altered in 1 PD 79 Male 2 sporadic PD and AD, we performed comparative high resolution, two- 2 PD 69 Male 3 dimensional gel electrophoresis experiments on frontal cortex homo- 3 PD 74 Male 5 4 PD 74 Male 4.5 genates obtained from idiopathic PD and AD and age-matched con- 5 PD 66 Male 12 trols. The frontal cortex was chosen because of tissue availability and its 6 AD 76 Female 15 7 AD 91 Female 2.5 relevance to the pathology of both AD and PD. In AD the frontal cortex 8 AD 70 Male 11 exhibits neuronal loss and is rich in neurofibrillary tangles and amyloid 9 AD 85 Female 9.5 10 AD 50 Female 17 plaques. In PD, although neuronal loss mainly occurs in the substantia 11 Control 74 Female 3 nigra, the frontal cortex contains Lewy bodies, the pathological hall- 12 Control 68 Female 11 13 Control 65 Female 6 mark of PD. Analysis of DJ-1 expression and modifications in the sub- 14 Control 75 Female 6 stantia nigra or striatum might provide more PD-relevant information. 15 Control 87 Male 20.5 Unfortunately, we had difficulty in obtaining sufficient amounts of these PMI, post-mortem interval. tissues for biochemical analyses. MATERIALS AND METHODS Although DJ-1 exists as a homodimeric protein, DJ-1 migrates on Human Brain Samples—Brain tissues were obtained from the Emory SDS polyacrylamide gels as a 20-kDa monomer because of denaturation Center for Neurodegenerative Disease Brain Bank. For biochemical by SDS (17, 29). Immunoblot analysis of the two-dimensional SDS- studies, frontal cortex tissues from five PD cases, five AD cases, and five PAGE gels with anti-DJ-1 antibody revealed the presence of at least six healthy non-demented control subjects were used (Table 1). The neu- distinct isoforms of DJ-1 monomer that have the same apparent molec- ropathological diagnosis of PD was based on the presence of nigral ular mass of 20 kDa but different isoelectric points (5.5, 5.6, 5.7, 5.8, 6.1, degeneration and Lewy bodies. The neuropathological diagnosis of AD and 6.4, respectively; Fig. 1, A and B). The predominant form of DJ-1 is was made using Consortium to Establish a Registry for Alzheimer Dis- the 20-kDa/pI 6.4 isoform, which is in good agreement with the pre- ease criteria (31). dicted values of 19.9 kDa/pI 6.3 for DJ-1 monomer. Of the 6 identified 2,4-Dinitrophenyl (DNP) derivatization, Two-dimensional Gel Elec- DJ-1 monomer isoforms, the acidic isoforms (pI 5.5 and 5.7) of DJ-1 trophoresis, and Immunoblot Analysis—Protein extracts were prepared monomer were selectively accumulated in PD and AD compared with from human brain tissues as described (32). Protein samples (350 g) control brains (Fig. 1, B and C). The accumulation of these acidic DJ-1 were resolved by isoelectric focusing on 17-cm immobilized pH gradi- isoforms is likely due to specific modifications of DJ-1 in PD and AD ent strips (pH 3–10) followed by in-strip DNP derivatization (reacting instead of nonspecific changes in DJ-1 occurring post-mortem because with protein carbonyls) as described (32, 33). Second-dimensional sep- within each of the PD, AD, and control group, the relative levels of acidic aration was performed by electrophoresis on SDS-polyacrylamide gra- DJ-1 isoforms (pI 5.5 and 5.7) were similar among samples with different dient gels (10–20% porosity polyacrylamide) using the Ettan-DALT post-mortem intervals (Fig. 1F). slab gel SDS-PAGE system (Amersham Biosciences). After electro- Besides DJ-1 monomeric forms, we observed at least four different phoresis, the gels were analyzed by Sypro Ruby staining and by immu- isoforms of SDS-resistant DJ-1 dimer that migrated on the two-dimen- noblotting as described (32) with anti-DNP antibody (1:16,000, Molec- sional SDS-PAGE gels with an apparent molecular mass of 39 kDa and ular Probes) or anti-DJ-1 antibody (P7F, 1:5000) (17). The protein level pI values of 7.0, 7.4, 8.0, and 8.4, respectively (Fig. 1, A and D). Interest- and the carbonyl level of each DJ-1 isoform in PD, AD, and controls ingly, a previous study reported the presence of SDS-resistant DJ-1 were quantified using the two-dimensional gel analysis program PD dimers in the detergent-insoluble fraction of brains from patients with Quest (Bio-Rad). Statistical comparison of the data obtained from five multiple system atrophy (30). To determine whether the accumulation PD, five AD, and five control individuals were performed using analysis of SDS-resistant dimeric DJ-1 is related to PD and AD, we compared the of variance. Significant difference was accepted at p  0.05. levels of DJ-1 dimeric forms in PD, AD, and age-matched control brains. Mass Spectrometry—Spots of interest were excised from the gels and The results showed that the levels of the basic isoforms (pI 8.0 and 8.4) digested in situ with trypsin (modified; Promega) or Lys-C (Sigma-Al- of DJ-1 dimer were significantly increased in PD and AD brains com- drich). The Lys-C digests were derivatized with ProteoMass Guanidi- pared with controls (Fig. 1, D and E). nation kit (Sigma-Aldrich) to increase the mass spectral signal intensi- In addition to the monomeric and dimeric forms of DJ-1, weak DJ-1- ties of C-terminal lysine-containing peptides (34). The digests were immunoreactive spots at about 30 kDa were occasionally observed in subjected to analysis by matrix-assisted laser desorption ionization time-of- the control as well as in PD and AD brain samples (Fig. 1A). These flight mass spectrometry (MALDI-TOF/MS), matrix-assisted laser desorp- 30-kDa DJ-1 spots might represent modified forms of DJ-1 that have tion ionization time-of-flight tandem mass spectrometry (MALDI-TOF/ undergone some types of post-translational modification, such as phos- TOF/MS/MS), and capillary HPLC-electrospray ionization tandem mass phorylation or mono-ubiquitination. Of note, the mono-ubiquitinated spectrometry (HPLC-ESI/MS/MS). MALDI-TOF/MS and MALDI- form of recombinant DJ-1 protein with similar apparent molecular TOF-TOF/MS/MS mass spectra were acquired on an Applied Biosys- mass (30 kDa) has been shown to exist in transfected SH-SY5Y cells tems 4700 Proteomics discovery system. The peptide mass maps pro- (35). Furthermore, a 27-kDa DJ-1 species of unknown modification has duced by MALDI-TOF/MS and MALDI-TOF/TOF/MS/MS were TM been reported in the detergent-insoluble fraction of brains from searched against the published databases by means of 4700 Explorer patients with Pick’s disease as well as of AD brains (30). Unfortunately, software (Applied Biosystems). HPLC-ESI/MS/MS analysis was per- formed as described (32, 33). Assignment of the MS/MS fragments was despite repeated attempts, we were unable to further characterize these verified by comparison with the predicted ions generated in silico by 30-kDa DJ-1 species due to their extremely low abundance and the GPMAW (Lighthouse Data). variability of their appearance from sample to sample. APRIL 21, 2006• VOLUME 281 • NUMBER 16 JOURNAL OF BIOLOGICAL CHEMISTRY 10817 DJ-1 Oxidation in Neurodegenerative Diseases FIGURE 1. Altered expression of DJ-1 isoforms in PD and AD compared with control brains. A, two-dimensional immunoblot analysis with anti- DJ-1 antibody reveals the presence of multiple pI isoforms of DJ-1 monomer and dimer in human brain. The open arrowhead indicates the presence of weak DJ-1 immunoreactive spots at about 30 kDa (see “Results” for details). B and D, protein samples (350 g) of control, PD, or AD brains were subjected to two-dimensional gel electrophoresis followed by immunoblotting with anti-DJ-1 anti- body for detection of DJ-1 isoforms. C and E, the level of each DJ-1 isoform in PD or AD was quanti- fied using the two-dimensional gel analysis pro- gram PD Quest and normalized to the level of the same isoform in the control brains. Values repre- sent the mean  S.E. for five PD, five AD, and five control individuals. The asterisk indicates a statisti- cally significant ( p 0.05) increase in the level of the indicated DJ-1 isoform in PD or AD versus the corre- sponding control. F, the relative level of acidic DJ-1 isoforms was measured by quantification of the intensity of the DJ-1 pI 5.5 and pI 5.7 isoforms and expressed as a percentage of the total level of all DJ-1 monomeric isoforms in each human sample. A histogram plot of relative level of acidic DJ-1 iso- forms versus post-mortem interval (PMI) shows no correlation between accumulation of acidic DJ-1 iso- forms and post-mortem interval. The Total Level of DJ-1 Protein Is Increased in PD and AD Brains—Our revealed a significant increase in the total level of DJ-1 in PD and AD two-dimensional immunoblotting data (Fig. 1) suggest that the total brains compared with the controls (Fig. 2B). level of DJ-1 isoforms may be increased in PD and AD brains. To further DJ-1 Protein Is Oxidatively Damaged in PD and AD Brains—Protein investigate this possibility, we performed quantitative Western blot carbonylation is an irreversible oxidation that has been widely used as a analysis using protein samples obtained from PD and AD brains and biomarker of severe oxidative damage to proteins (5, 10, 11). Ample age-matched controls. The fact that all six DJ-1 monomer isoforms have evidence indicates that the total level of protein carbonyls is increased in the same molecular mass of 20 kDa and all four DJ-1 dimer isoforms are both AD and PD (4, 9). It has recently become clear that only a small of 39 kDa (Fig. 1) allowed us to use one-dimensional gel electrophoresis subset of total brain proteins in PD and AD is carbonylated (32, 33, 36). for direct comparison of the total DJ-1 levels of various brain samples on However, the identities of these oxidatively damaged proteins are the same gel. After electrophoresis the gels were subjected to immuno- largely unknown. Furthermore, it remains to be determined whether blotting with antibodies against DJ-1 and actin (Fig. 2A). Because the the protein targets of oxidative damage are the same or are different in amount of protein extract (50 g of protein/lane) loaded on one-dimen- AD and PD. sional gels were much less than the amount (350 g of protein/gel) on To determine whether DJ-1 protein is oxidatively damaged in PD two-dimensional gels, only a single DJ-1 band of 20 kDa was detected on or AD brain, we used a well established assay to detect protein car- the one-dimensional gels. The relative level of DJ-1 in each sample was bonyl groups by derivatization of the carbonyls with 2,4-dinitrophe- measured by quantification of the intensity of 20-kDa DJ-1 band and nylhydrazine and subsequent immuno-detection of the resulting normalized to the actin level in the corresponding sample. The results hydrazones using anti-DNP antibodies (32, 33, 37, 38). Protein 10818 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 16 •APRIL 21, 2006 DJ-1 Oxidation in Neurodegenerative Diseases FIGURE 2. Quantitative Western blot analysis of the total level of DJ-1 protein in PD, AD, and control brains. A, protein extracts (50 g of protein/lane) from PD, AD, or control brains were subjected to one-dimensional gel electrophoresis followed by immunoblotting with anti-DJ-1 antibody. Each lane represents a different individual from the PD, AD, or control group. B, the relative DJ-1 level was measured by quantifica- tion of the intensity of 20-kDa DJ-1 band and normalized to the actin level in the corre- sponding brain extract. The bar graph shows the results (mean S.E.) from five PD, five AD, and five control individuals. The asterisk indicates a statistically significant (p 0.05) increase in the total level of DJ-1 protein in PD or AD versus control. extracts from PD, AD, and control brains were resolved by isoelectric FIGURE 3. Increased oxidation of DJ-1 isoforms in PD and AD brains compared with focusing on an immobilized pH-gradient strip followed by in-strip controls. Protein samples (350 g) of control, PD, or AD brains were subjected to two- dimensional gel electrophoresis followed by immunoblotting (IB) with anti-DJ-1 anti- DNP derivatization of protein carbonyls and second-dimensional body for detection of DJ-1 isoforms or with anti-DNP antibody for detection of protein separation by SDS-PAGE and immunoblotting with anti-DJ-1 and carbonyls. The arrow indicates the isoform of DJ-1 monomer (A) and of DJ-1 dimer (B) anti-DNP antibodies (Fig. 3). We found that among the six identified that exhibits elevated oxidation in PD or AD. monomeric forms of DJ-1, only the 20-kDa/pI 6.4 isoform was oxi- datively modified, as evidenced by the significant increase in protein TABLE 2 carbonyl content of this isoform in PD and AD brains (Fig. 3A). Of Specific oxidation index of DJ-1 isoforms in PD, AD, the four dimeric forms of DJ-1, three isoforms (39 kDa/pI 7.4, 39 and control brains The specific oxidation index was obtained by normalization of the intensity of the kDa/8.0, and 39 kDa/pI 8.4) were found to exhibit elevated oxidative carbonyl level to the intensity of protein level of the indicated DJ-1 isoform. Values modification by carbonylation in PD and AD brains (Fig. 3B). No represent the mean  S.E. for five individuals of each of the PD, AD, or control DNP immunoreactivity was observed in the controls where the pri- group. mary anti-DNP antibody or the DNP derivatization procedure was DJ-1 isoform (M /pI) Control PD AD omitted (data not shown), confirming the specificity of our observa- n  5 n  5 n  5 a a 20/pI 6.4 0.30  0.03 1.17  0.23 1.07  0.30 tions. The lack of detection of carbonyls in the more acidic forms of a a 39/pI 7.4 0.43  0.06 1.93  0.50 2.06  0.60 DJ-1 monomers (pI 5.5– 6.1) could mean that these isoforms repre- a a 39/pI 8.0 0.73  0.17 1.63  0.26 1.67  0.30 a a sent un-carbonylated forms of DJ-1 or could be due to their lower 39/pI 8.4 0.97  0.13 4.00  0.40 3.66  0.60 p  0.05. abundance compared with the pI 6.4 DJ-1 isoform, making them undetectable because they might fall below the detection threshold required for anti-DNP immunoblotting. We favor the first interpre- tional modifications including oxidative modifications. However, despite tation, because the levels of these un-carbonylated DJ-1 monomeric recent advances in mass spectrometry technology, detection of modified forms were much higher than the dimeric forms of DJ-1 (pI 7.4 – 8.4) peptides, and localization of modification sites of an endogenous protein (Fig. 1A), which have been shown to be carbonylated by anti-DNP remain a major challenge. Therefore, we focused our mass spectrometry immunoblotting in the same experiments (Fig. 3B). study on PD and control samples. Individual DJ-1 protein spots were To quantitatively determine the differences in protein oxidation excised from the two-dimensional gels from five PD and five control between the disease brains and the controls, we quantified the protein brains, digested with a protease, and analyzed by mass spectrometry. level and the carbonyl level of each DJ-1 isoform in five PD, five AD, and Identification of the types and the sites of DJ-1 oxidative modifica- five control cases and obtained the specific oxidation index by normal- tions by mass spectrometry analysis is dependent on the ability to gen- ization of the intensity of carbonyl level to the intensity of protein level erate peptide fragments of appropriate size and adequate abundance for each DJ-1 isoform. Statistic analysis of the results showed a signifi- that ideally should cover the entire length of DJ-1 amino acid sequence. cant increase in the specific oxidation levels of the pI 6.4 monomeric and To increase the sequence coverage, we tried digestion with different the dimeric forms (pI 7.4–8.4) of DJ-1 in PD as well as in AD compared protease (trypsin or Lys-C protease), used a guanidination procedure with the age-matched control brains (Table 2). for enhancing mass spectral signals of Lys-containing peptides (34), and Identification of the Types and the Sites of DJ-1 Oxidative Modifica- employed a combination of different types of mass spectrometric anal- tions by Mass Spectrometry—In addition to carbonylation, proteins can yses, including MALDI-TOF/MS, MALDI-TOF/TOF/MS/MS, and undergo several types of oxidative modifications in response to oxida- HPLC-ESI/MS/MS. Because of the low abundance of the SDS-resistant tive stress (39). Thus, we used mass spectrometry to further characterize dimeric forms of DJ-1 in PD and control brains (Fig. 1), we were unable to the oxidative modifications of DJ-1 in human brain samples. Mass spec- detect oxidative modifications associated with the DJ-1 dimer isoforms. trometry has emerged as a powerful tool for the analysis of post-transla- However, for each of the DJ-1 monomer isoforms in PD and control brains, APRIL 21, 2006• VOLUME 281 • NUMBER 16 JOURNAL OF BIOLOGICAL CHEMISTRY 10819 DJ-1 Oxidation in Neurodegenerative Diseases FIGURE 4. Characterization of DJ-1 oxidative modifications in PD and control brains by mass spectrometry. A, coverage map of DJ-1 pI 6.4 isoform from PD or control brains with underlined residues indicating peptides detected by the combination of MALDI-TOF/MS, HPLC-ESI/MS/MS, and MALDI-TOF/TOF/MS/MS. C*, cysteic acid; M*, methionine sulfoxide; M**, methionine sulfone. Similar coverage maps were obtained for all other 20-kDa DJ-1 pI isoforms in five PD and five control brains. B, MALDI-TOF/MS spectra of the Lys-C digest of DJ-1 isoforms from PD or control brains. *, peptides that match the theoretically predicted peptide masses in DJ-1. **, oxidatively modified peptides containing methionine sulfoxide and/or methionine sulfone. Note that the oxidized peptide fragments at m/z 1948.1, 1964.1, and 1990.1 and m/z 2249.1 in the spectrum of DJ-1 pI 6.4 isoform in PD are absent in the spectrum of DJ-1 pI 6.4 isoform in control brain. Similar results were obtained for five PD and five control brains. we obtained sequence coverage of 50%, which matched to the primary MALDI-TOF/MS analysis of the Lys-C digest of DJ-1 20-kDa/pI 6.4 TM sequence of DJ-1 (GenBank accession number NP_009193; Fig. 4 and isoform protein spot from PD brains suggested that six ions represented additional data not shown). by m/z 1948.1, 1964.1, 1990.1, 2233.3, 2249.3, and 2275.3, respectively, 10820 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 16 •APRIL 21, 2006 DJ-1 Oxidation in Neurodegenerative Diseases FIGURE 5. Identification of oxidation sites in DJ-1 by MALDI-TOF/TOF/MS/MS and HPLC-ESI/ MS/MS analyses. A, MALDI-TOF/TOF/MS/MS spectrum of the ion at m/z 1948.1 (1) from a Lys-C digest of DJ-1 pI 6.4 isoform in PD showing the identity as DJ-1 peptide 133–148 with oxida- tion of both Met-133 and Met-134 residues to methionine sulfoxide. The ion at m/z 1636.5 and 1654.4 are interpreted as b ion and b-H O ion, respectively, corresponding to the internal frag- ment M*NGGHYTYSENRVE. B, MALDI-TOF/TOF/ MS/MS spectrum of the ion at m/z 2249.3 (1) from a Lys-C digest of DJ-1 pI 6.4 isoform in PD showing the identity as DJ-1 peptide 13–32 with oxidation of Met-17 to methionine sulfone and Met-26 to methionine sulfoxide. C, HPLC-ESI/ MS/MS spectrum of the ion at m/z 761.8 (2) from a tryptic digest of DJ-1 pI 6.4 isoform in control brain, showing the identity as DJ-1 peptide 49 – 62 with oxidation of Cys-53 to cysteic acid. Peptide fragments are indicated using the nomenclature of Roepstorff and Fohlman (52). C*, cysteic acid; M*, methionine sulfoxide; M**, methionine sulfone. APRIL 21, 2006• VOLUME 281 • NUMBER 16 JOURNAL OF BIOLOGICAL CHEMISTRY 10821 DJ-1 Oxidation in Neurodegenerative Diseases could be peptides resulting from oxidative modification (Fig. 4). All six TABLE 3 suspected oxidized DJ-1 peptides were also present in the MALDI-TOF Oxidized peptides of DJ-1 detected by mass spectrometric analysis Oxidized fragments at m/z 1990.1 and 2275.3 were generated by guanidation (Gu) of mass spectra of DJ-1 20 kDa/pI 5.5, 20 kDa/pI 5.6, 20 kDa/pI 5.7, 20 lysine at the C terminus of 1948.1 and 2233.3 peptides, respectively. C*, cysteic acid; kDa/pI 5.8 isoforms from PD brains, and all except the m/z 2249.3 M*, methionine sulfoxide; M**, methionine sulfone. fragment were found in the spectrum of DJ-1 20 kDa/pI 6.1 isoform Group m/z Oxidized peptide sequence from PD brains (Fig. 4). In contrast, only two (m/z 2233.3 and 2275.3) of Control 761.8 (2) DVVIC*PDASLEDAK the six suspected oxidized DJ-1 peptides were detected in the MALDI- 2233.3 (1) GAEEM*ETVIPVDVM*RRAGIK 2275.3 (1) GAEEM*ETVIPVDVM*RRAGIK(Gu) TOF mass spectra of DJ-1 isoforms from control brains (Fig. 4). PD 761.8 (2) DVVIC*PDASLEDAK All putative oxidized DJ-1 peptides were further analyzed by using 1948.1 (1) M*M*NGGHYTYSENRVEK 1964.1 (1) M**M*NGGHYTYSENRVEK MALDI-TOF-TOF/MS/MS to identify oxidatively modified amino acid 1990.1 (1) M*M*NGGHYTYSENRVEK(Gu) residues. The results indicated that the three ions at m/z 1948.1, 1964.1, 2233.3 (1) GAEEM*ETVIPVDVM*RRAGIK and 1990.1 are oxidatively modified peptides corresponding to amino 2249.3 (1) GAEEM**ETVIPVDVM*RRAGIK 2275.3 (1) GAEEM*ETVIPVDVM*RRAGIK(Gu) acid residues 133–148 of DJ-1 (Fig. 5A and Table 3). The ion at m/z 1990.1 was generated from the m/z 1948.1 peptide by guanidination, a reaction that results in a 42-Da mass shift due to the conversion of found in AD brains, suggesting that increased oxidation of DJ-1 may C-terminal lysine residue to homoarginine (34). The MALDI-TOF- occur in AD as well. TOF tandem mass spectra of the m/z 1948.1 and 1990.1 peptides In addition to the monomeric forms of DJ-1, we found four different revealed the oxidative modification of both Met-133 and Met-134 res- isoforms of SDS-resistant DJ-1 dimer, of which the basic isoforms (pI idues to methionine sulfoxide (Fig. 5A and additional data not shown). 8.0 and 8.4) are selectively accumulated in PD and AD brains. Although In addition, we observed the oxidation of Met-133 to methionine sul- these dimeric DJ-1 isoforms have never been reported, SDS-resistant fone and of Met-134 to methionine sulfoxide in the spectrum of the m/z DJ-1 dimer has been seen in the detergent-insoluble fraction of multiple 1964.1 peptide (data not shown). These results together with the system atrophy brains (30). The nature of chemical modifications that MALDI-TOF/MS data showing the consistent presence of the m/z render these DJ-1 dimers to become SDS-resistant is unknown. The 1948.1, 1964.1, and 1990.1 peptides in 5 PD but not in 5 control brains observation that three of the four isoforms of SDS-resistant DJ-1 dimer (Fig. 4B; data not shown), suggest that the oxidation of DJ-1 Met-133 are irreversibly oxidized by carbonylation in PD and AD brains suggests and Met-134 may be a phenomenon unique to PD. that oxidative modifications may be involved in conferring the SDS MALDI-TOF-TOF/MS/MS analysis also showed that the three ions resistance. at m/z 2233.3, 2249.3, and 2275.3 are oxidatively modified peptides cor- Consistent with the accumulation of several DJ-1 isoforms, our quan- responding to amino acid residues 13–32 of DJ-1 (Fig. 5B and Table 3). titative one-dimensional immunoblot analysis has revealed a significant The spectra of the m/z 2233.3 peptide and its guanidinated product at increase in the total level of DJ-1 in PD and AD brains compared with m/z 2275.3 revealed the oxidative modification of both Met-17 and age-matched controls. Previous studies have reported mixed results Met-26 residues to methionine sulfoxide (data not shown). These oxi- regarding the DJ-1 levels in PD and AD brains (29, 35, 42). Bando- dation products were found in all 20-kDa DJ-1 pI isoforms from both PD padhyay et al. (42) failed to find any obvious difference in the total level and control brains (Fig. 4 and Table 3). However, the m/z 2249.3 frag- of DJ-1 between PD and controls (42), whereas Moore et al. (35) found ment, a more severely oxidized form of DJ-1 peptide 13–32 generated a5-fold increase in the DJ-1 level in the detergent-insoluble fraction of from the conversion of Met-17 into methionine sulfone and Met-26 PD brains compared with control and AD brains. In contrast, Rizzu et al. into methionine sulfoxide (Fig. 5B), was only found in the 20-kDa DJ-1 (29) observed a dramatic increase in the DJ-1 level in the detergent- pI 5.5–5.8 and pI 6.4 isoforms from 5 PD brains but not in the DJ-1 insoluble fraction of AD brains. The reason(s) for these discrepancies is isoforms from the 5 controls (Fig. 4 and Table 3). In addition, our unclear but could be due to differences in anti-DJ-1 antibodies, patient MALDI-TOF/MS and HPLC-ESI-MS/MS analyses of trypsin-digested samples, and extraction conditions used in the immunoblot analyses. DJ-1 revealed that Cys-53 residue was oxidized to cysteic acid (also Previous studies have shown that, in cultured mammalian cells, known as cysteine sulfonic acid) in all 20-kDa DJ-1 pI isoforms from Cys-53 and Cys-106 residues of DJ-1 can undergo reversible oxidation both PD and control brains (Fig. 5C and Table 3), suggesting the Cys-53 in response to oxidative stress induced by H O and PD-linked environ- 2 2 oxidation may occur under relatively mild oxidative stress conditions mental toxins, such as paraquat and MPTP (1-methyl-4-phenyl-1,2,3,6- related to aging. tetrahydropyridine) (19, 40, 41). The reversible oxidation of these resi- dues has been proposed to regulate the molecular chaperone function DISCUSSION (19, 45) or mitochondrial localization of DJ-1 (40). Our mass spectrom- Our study demonstrates for the first time the oxidative damage to etry analysis has shown that Cys-53 of DJ-1 is oxidized to cysteine sul- DJ-1 in idiopathic PD and AD brains. We showed that human DJ-1 fonic acid in PD brains as well as in the age-matched controls. As exists in 10 different isoforms; that is, 6 monomeric forms and 4 SDS- pointed out earlier, despite our repeated efforts we could only achieve resistant dimeric forms. Although DJ-1 adopts a homodimeric structure 50% sequence coverage. Because we did not recover any peptide con- in solution as well as in the crystal (14, 15, 17), DJ-1 usually appears as a taining Cys-106, we could not determine the oxidation status of this 20-kDa monomer on the SDS-PAGE gel (17, 19, 29, 40, 41). Consistent residue in human brain samples. Future studies to increase sequence with a recent report (42), we found that the acidic pI isoforms (pI 5.5 and coverage are needed to identify all oxidative modifications of endoge- 5.7) of DJ-1 monomer are selectively accumulated in PD brains. Because nous DJ-1 in PD and AD. the 20-kDa DJ-1 has been shown to undergo acidic pI shift upon expo- In addition to cysteine oxidation, our mass spectrometry analysis has sure of cells to oxidative stress (19, 40, 41, 43, 44), the observed accu- revealed that DJ-1 is susceptible to previously unsuspected methionine mulation of the acidic forms of DJ-1 monomer in PD provides a first clue oxidation. We identified four methionine residues (Met-17, Met-26, that the oxidative modifications of DJ-1 may be elevated in PD brains. A Met-133, and Met-134) as the sites of DJ-1 oxidation. We found that all similar accumulation of the acidic forms of DJ-1 monomer was also four methionine residues were oxidized to methionine sulfoxide in PD, 10822 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 16 •APRIL 21, 2006 DJ-1 Oxidation in Neurodegenerative Diseases 5. Levine, R. L., and Stadtman, E. R. (2001) Exp. Gerontol. 36, 1495–1502 whereas only two of them (Met-17 and Met-26) were oxidized to methi- 6. Beal, M. F. (2002) Free Radic. Biol. Med. 32, 797–803 onine sulfoxide in age-matched controls. A variety of reactive oxygen 7. Ischiropoulos, H., and Beckman, J. S. (2003) J. Clin. Investig. 111, 163–169 species (e.g. O ,H O , OH, or peroxynitrite) can oxidize methionine resi- 2 2 2 8. Jenner, P. (2001) Trends Neurosci. 24, 245–247 dues to methionine sulfoxide (46), and this oxidation can be reversed by the 9. Giasson, B. I., Ischiropoulos, H., Lee, V. M., and Trojanowski, J. Q. (2002) Free Radic. Biol. Med. 32, 1264–1275 enzyme peptide methionine sulfoxide reductase (MsrA) in a thioredoxin- 10. Dalle-Donne, I., Giustarini, D., Colombo, R., Rossi, R., and Milzani, A. (2003) Trends dependent manner (47). The reversible methionine oxidation/reduction Mol. Med. 9, 169–176 has been suggested to act as a signaling device analogous to phospho- 11. Nystrom, T. (2005) EMBO J. 24, 1311–1317 rylation/dephosphorylation for regulating protein function and cellular 12. Gracy, R. W., Talent, J. M., Kong, Y., and Conrad, C. C. (1999) Mutat. Res. 428, 17–22 processes (46). It is, thus, tempting to speculate that the methionine 13. Bonifati, V., Rizzu, P., van Baren, M. J., Schaap, O., Breedveld, G. J., Krieger, E., oxidation of DJ-1 identified here represents a specific, reversible mech- Dekker, M. C., Squitieri, F., Ibanez, P., Joosse, M., van Dongen, J. W., Vanacore, N., van anism for regulation of DJ-1 activity by intracellular redox status. In Swieten, J. C., Brice, A., Meco, G., van Duijn, C. M., Oostra, B. A., and Heutink, P. addition, the reversible oxidation of methionine residues has been pro- (2003) Science 299, 256–259 14. Huai, Q., Sun, Y., Wang, H., Chin, L. S., Li, L., Robinson, H., and Ke, H. (2003) FEBS posed to serve as an important defense mechanism for scavenging ROS Lett. 549, 171–175 (48). Such a mechanism would support an antioxidant role of DJ-1 in 15. Tao, X., and Tong, L. (2003) J. Biol. Chem. 278, 31372–31379 protecting cells from oxidative damage (19, 24). Interestingly, Met-26, a 16. Lee, S. J., Kim, S. J., Kim, I. K., Ko, J., Jeong, C. S., Kim, G. H., Park, C., Kang, S. O., Suh, methionine residue of DJ-1 found to be oxidized in this study, is mutated P. G., Lee, H. S., and Cha, S. S. (2003) J. Biol. Chem. 278, 44552–44559 17. Olzmann, J. A., Brown, K., Wilkinson, K. D., Rees, H. D., Huai, Q., Ke, H., Levey, A. I., to isoleucine (M26I) in a rare, autosomal recessive form of familial PD Li, L., and Chin, L. S. (2004) J. Biol. Chem. 279, 8506–8515 (26). Thus, the reversible oxidation of Met-26 as well as of other iden- 18. Shendelman, S., Jonason, A., Martinat, C., Leete, T., and Abeliovich, A. (2004) PLoS tified methionine oxidation sites (Met-17, Met-133, and Met-134) may Biol. 2, 1764–1773 have a role in regulation of DJ-1 function and/or localization. Future 19. Taira, T., Saito, Y., Niki, T., Iguchi-Ariga, S. M., Takahashi, K., and Ariga, H. (2004) studies are needed to test these hypotheses and determine the func- EMBO Rep. 5, 213–218 20. Miller, D. W., Ahmad, R., Hague, S., Baptista, M. J., Canet-Aviles, R., McLendon, C., tional consequences of DJ-1 methionine oxidation. Carter, D. M., Zhu, P. P., Stadler, J., Chandran, J., Klinefelter, G. R., Blackstone, C., and In addition to the reversible methionine oxidation, we found that Cookson, M. R. (2003) J. Biol. Chem. 278, 36588–36595 Met-17 and Met-133 were irreversibly oxidized to methionine sulfone 21. Macedo, M. G., Anar, B., Bronner, I. F., Cannella, M., Squitieri, F., Bonifati, V., Hoo- geveen, A., Heutink, P., and Rizzu, P. (2003) Hum. Mol. Genet. 12, 2807–2816 in PD but not in controls. Unlike the reversible oxidation of methionine 22. Moore, D. J., Zhang, L., Dawson, T. M., and Dawson, V. L. (2003) J. Neurochem. 87, residues to methionine sulfoxide, which occurs under physiological 1558–1567 conditions, the irreversible oxidation to methionine sulfone is rare and 23. Goldberg, M. S., Pisani, A., Haburcak, M., Vortherms, T. A., Kitada, T., Costa, C., only takes place in the presence of strong oxidants (46). This irreversible Tong, Y., Martella, G., Tscherter, A., Martins, A., Bernardi, G., Roth, B. L., Pothos, oxidation is often associated with pathophysiological conditions and E. N., Calabresi, P., and Shen, J. (2005) Neuron 45, 489–496 24. Kim, R. H., Smith, P. D., Aleyasin, H., Hayley, S., Mount, M. P., Pownall, S., results in functional impairment of the oxidized proteins (46, 49). Wakeham, A., You-Ten, A. J., Kalia, S. K., Horne, P., Westaway, D., Lozano, A. M., Another important finding of the present study is the oxidative dam- Anisman, H., Park, D. S., and Mak, T. W. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, age to DJ-1 in PD and AD by the irreversible carbonylation. Protein 5215–5220 carbonylation occurs under stronger oxidative stress than that causing 25. Chen, L., Cagniard, B., Mathews, T., Jones, S., Koh, H. C., Ding, Y., Carvey, P. M., Ling, Z., Kang, U. J., and Zhuang, X. (2005) J. Biol. Chem. 280, 21418–21426 cysteine and methionine oxidation and has been associated with func- 26. Abou-Sleiman, P. M., Healy, D. G., Quinn, N., Lees, A. J., and Wood, N. W. (2003) tional impairment in a variety of structural proteins and enzymes (10, Ann. Neurol. 54, 283–286 11). For example, actin carbonylation occurs after its Cys and Met res- 27. Hedrich, K., Schafer, N., Hering, R., Hagenah, J., Lanthaler, A. J., Schwinger, E., Kramer, P. L., Ozelius, L. J., Bressman, S. B., Abbruzzese, G., Martinelli, P., Kostic, V., idues have already been oxidized, and the carbonylation leads to the Pramstaller, P. P., Vieregge, P., Riess, O., and Klein, C. (2004) Ann. Neurol. 55, 145 disruption of actin filaments and the inhibition of F-actin formation 28. Gorner, K., Holtorf, E., Odoy, S., Nuscher, B., Yamamoto, A., Regula, J. T., Beyer, K., (50). The lipid peroxidation product 4-hydroxynonenal induces car- Haass, C., and Kahle, P. J. (2004) J. Biol. Chem. 279, 6943–6951 bonylation of ubiquitin C-terminal hydrolase L1 (UCH-L1), resulting in 29. Rizzu, P., Hinkle, D. A., Zhukareva, V., Bonifati, V., Severijnen, L. A., Martinez, D., a dramatic reduction in the hydrolase activity of UCH-L1 (51). Thus, the Ravid, R., Kamphorst, W., Eberwine, J. H., Lee, V. M., Trojanowski, J. Q., and Heutink, P. (2004) Ann. Neurol. 55, 113–118 observed DJ-1 carbonylation together with irreversible methionine oxi- 30. Neumann, M., Muller, V., Gorner, K., Kretzschmar, H. A., Haass, C., and Kahle, P. J. dation may cause functional impairment of DJ-1 and contribute to the (2004) Acta Neuropathol. (Berl) 107, 489–496 pathophysiology and progression of sporadic PD and AD. Further inves- 31. Mirra, S. S., Heyman, A., McKeel, D., Sumi, S. M., Crain, B. J., Brownlee, L. M., Vogel, tigation of the relationship between oxidative modifications of DJ-1 as F. S., Hughes, J. P., van Belle, G., and Berg, L. (1991) Neurology 41, 479–486 32. Choi, J., Levey, A. I., Weintraub, S. T., Rees, H. D., Gearing, M., Chin, L. S., and Li, L. well as of other proteins and neuronal dysfunction should generate new (2004) J. Biol. Chem. 279, 13256–13264 insights into the mechanisms of oxidative damage in the pathogenesis of 33. Choi, J., Rees, H. D., Weintraub, S. T., Levey, A. I., Chin, L. S., and Li, L. (2005) J. Biol. PD and AD and provide new opportunities for developing therapeutic Chem. 280, 11648–11655 strategies for treating these devastating diseases. 34. Beardsley, R. L., and Reilly, J. P. (2002) Anal. Chem. 74, 1884–1890 35. Moore, D. J., Zhang, L., Troncoso, J., Lee, M. K., Hattori, N., Mizuno, Y., Dawson, T. M., and Dawson, V. L. (2005) Hum. Mol. Genet. 14, 71–84 Acknowledgments—We thank Christopher A. Carroll of the Institutional Mass 36. Castegna, A., Aksenov, M., Aksenova, M., Thongboonkerd, V., Klein, J. B., Pierce, Spectrometry Laboratory at the University of Texas Health Science Center at W. M., Booze, R., Markesbery, W. R., and Butterfield, D. A. (2002) Free Radic. Biol. San Antonio (supported in part by National Institutes of Health Grant Med. 33, 562–571 37. Levine, R. L. (2002) Free Radic. Biol. Med. 32, 790–796 CA54174) for mass spectrometric analyses. 38. Butterfield, D. A., and Castegna, A. (2003) Amino Acids 25, 419–425 39. Stadtman, E. R. (2001) Ann. N. Y. Acad. Sci. 928, 22–38 40. Canet-Aviles, R. M., Wilson, M. A., Miller, D. W., Ahmad, R., McLendon, C., REFERENCES Bandyopadhyay, S., Baptista, M. J., Ringe, D., Petsko, G. A., and Cookson, M. R. (2004) 1. Ross, C. A., and Poirier, M. A. (2004) Nat. Med. 10, (suppl.) 10–17 Proc. Natl. Acad. Sci. U. S. A. 101, 9103–9108 2. Cordato, D. J., and Chan, D. K. (2004) J. Clin. Neurosci. 11, 119–123 41. Kinumi, T., Kimata, J., Taira, T., Ariga, H., and Niki, E. (2004) Biochem. Biophys. Res. 3. Pardo, L. M., and van Duijn, C. M. (2005) Mutat. Res. 592, 89–101 Commun. 317, 722–728 4. Jenner, P. (2003) Ann. Neurol. 53, Suppl. 3, 26–36 42. Bandopadhyay, R., Kingsbury, A. E., Cookson, M. R., Reid, A. R., Evans, I. M., Hope, APRIL 21, 2006• VOLUME 281 • NUMBER 16 JOURNAL OF BIOLOGICAL CHEMISTRY 10823 DJ-1 Oxidation in Neurodegenerative Diseases A. D., Pittman, A. M., Lashley, T., Canet-Aviles, R., Miller, D. W., McLendon, C., 397, 172–178 Strand, C., Leonard, A. J., Abou-Sleiman, P. M., Healy, D. G., Ariga, H., Wood, N. W., 48. Levine, R. L., Mosoni, L., Berlett, B. S., and Stadtman, E. R. (1996) Proc. Natl. Acad. Sci. De Silva, R., Revesz, T., Hardy, J. A., and Lees, A. J. (2004) Brain 127, 1–11 U. S. A. 93, 15036–15040 43. Mitsumoto, A., and Nakagawa, Y. (2001) Free Radic. Res. 35, 885–893 49. Stadtman, E. R., Van Remmen, H., Richardson, A., Wehr, N. B., and Levine, R. L. 44. Mitsumoto, A., Nakagawa, Y., Takeuchi, A., Okawa, K., Iwamatsu, A., and Takan- (2005) Biochim. Biophys. Acta 1703, 135–140 ezawa, Y. (2001) Free Radic. Res. 35, 301–310 50. Dalle-Donne, I., Rossi, R., Giustarini, D., Gagliano, N., Di Simplicio, P., Colombo, R., 45. Martinat, C., Shendelman, S., Jonason, A., Leete, T., Beal, M. F., Yang, L., Floss, T., and and Milzani, A. (2002) Free Radic. Biol. Med. 32, 927–937 Abeliovich, A. (2004) PLoS Biol. 2, 1754–1763 51. Nishikawa, K., Li, H., Kawamura, R., Osaka, H., Wang, Y. L., Hara, Y., Hirokawa, T., 46. Hoshi, T., and Heinemann, S. (2001) J. Physiol. (Lond.) 531, 1–11 Manago, Y., Amano, T., Noda, M., Aoki, S., and Wada, K. (2003) Biochem. Biophys. 47. Weissbach, H., Etienne, F., Hoshi, T., Heinemann, S. H., Lowther, W. T., Res. Commun. 304, 176–183 Matthews, B., St John, G., Nathan, C., and Brot, N. (2002) Arch. Biochem. Biophys. 52. Roepstorff, P., and Fohlman, J. (1984) Biomed. Mass Spectrom. 11, 601 10824 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 16 •APRIL 21, 2006 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

Oxidative Damage of DJ-1 Is Linked to Sporadic Parkinson and Alzheimer Diseases *

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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 16, pp. 10816 –10824, April 21, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Oxidative Damage of DJ-1 Is Linked to Sporadic Parkinson and Alzheimer Diseases Received for publication, August 17, 2005, and in revised form, February 13, 2006 Published, JBC Papers in Press, March 3, 2006, DOI 10.1074/jbc.M509079200 ‡ § ‡ ¶ Joungil Choi , M. Cameron Sullards , James A. Olzmann , Howard D. Rees , Susan T. Weintraub , § ¶ ‡ ‡1 David E. Bostwick , Marla Gearing**, Allan I. Levey , Lih-Shen Chin , and Lian Li ‡ ¶ From the Department of Pharmacology, Neurology, and **Pathology, Center for Neurodegenerative Disease, Emory University School of Medicine, Atlanta, Georgia 30322, Bioanalytical Mass Spectrometry Facility, Parker H. Petit Institute for Bioengineering and Biosciences, School of Chemistry and Biochemistry and School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332, and Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229 Mutations in DJ-1 cause an autosomal recessive, early onset AD and PD have been associated with increased production of reactive familial form of Parkinson disease (PD). However, little is presently oxygen species (ROS), which could result from a combination of aging, known about the role of DJ-1 in the more common sporadic form of genetic predisposition, and environmental factors (6). Epidemiological PD and in other age-related neurodegenerative diseases, such as studies suggest that exposure to pesticides, herbicides, and other envi- Alzheimer disease (AD). Here we report that DJ-1 is oxidatively ronmental toxins that inhibit mitochondrial complex I can lead to damaged in the brains of patients with idiopathic PD and AD. By excess production of ROS and increased incidence of sporadic PD (8). using a combination of two-dimensional gel electrophoresis and Furthermore, post-mortem analyses reveal that the overall levels of oxi- mass spectrometry, we have identified 10 different DJ-1 isoforms, of dative damage to proteins, lipids, and DNA are elevated in AD and PD which the acidic isoforms (pI 5.5 and 5.7) of DJ-1 monomer and the brains (4, 9). basic isoforms (pI 8.0 and 8.4) of SDS-resistant DJ-1 dimer are selec- The most widely used marker for oxidative damage to proteins is the tively accumulated in PD and AD frontal cortex tissues compared presence of carbonyl groups, which can be introduced into proteins by with age-matched controls. Quantitative Western blot analysis direct oxidation of Pro, Arg, Lys, or Thr side chains or by Michael shows that the total level of DJ-1 protein is significantly increased in addition reactions of Cys, His, or Lys residues with products of lipid PD and AD brains. Mass spectrometry analyses reveal that DJ-1 is peroxidation or glycooxidation (5, 10, 11). Elevation in the total level not only susceptible to cysteine oxidation but also to previously of protein carbonyls has been documented in both AD and PD (4, 9). unsuspected methionine oxidation. Furthermore, we show that Although it was initially thought that targets of oxidative damage by DJ-1 protein is irreversibly oxidized by carbonylation as well as by reactive oxygen species were random and indiscriminate, it has become methionine oxidation to methionine sulfone in PD and AD. Our increasingly clear that the susceptibility of proteins to oxidative damage study provides new insights into the oxidative modifications of DJ-1 is highly dependent on specific properties of individual proteins, such as and indicates association of oxidative damage to DJ-1 with sporadic unique sequence motifs, surface accessibility, protein folding, and sub- PD and AD. cellular localization (10–12). The identities of the oxidatively damaged proteins in AD and PD that have been modified by carbonylation or other types of oxidation remain largely unknown. Alzheimer disease (AD) and Parkinson disease (PD) are the two Mutations in DJ-1 has recently been linked to an autosomal reces- most common neurodegenerative disorders characterized by the selec- sive, early onset familial form of PD (13). DJ-1 is a ubiquitously tive loss of neurons in specific brain regions and the deposition of mis- expressed protein of the DJ-1/ThiJ/PfpI superfamily (14, 15). The folded proteins into aggregates or inclusions, such as neurofibrillary precise biochemical function of DJ-1 is unknown, although DJ-1 has tangles and amyloid plaques in AD and Lewy bodies in PD (1). The been proposed to act as a protease, chaperone, or antioxidant (15– majority of AD and PD cases are sporadic with hereditary familial cases 19). The identification of a homozygous DJ-1 deletion that prevents accounting for less than 10% (2, 3). The genetic defects underlying sev- the expression of DJ-1 protein in recessively transmitted PD (13) eral monogenic familial forms of AD and PD have recently been identi- strongly suggests that loss of DJ-1 function leads to neurodegenera- fied (3). However, the causes of other AD and PD cases, particularly tion. A loss-of-function pathogenic mechanism is also supported by sporadic cases, remain unclear. analysis of PD-linked DJ-1 L166P mutation (17, 20 –22) and DJ-1 Increasing evidence indicates that oxidative stress plays a crucial role knock-out studies in mice (23–25). Despite increasing genetic evi- in the pathogenesis of idiopathic AD and PD (4–7). For example, both dence indicating the importance of DJ-1 mutations in causing early onset familial PD (13, 26 –28), the role of DJ-1 in sporadic PD is * This work was supported by National Institutes of Health Grants NS047199, NS047575, unknown. Interestingly, previous studies have shown that DJ-1 is NS050650, AG021489, and AG025688. The costs of publication of this article were rarely present in Lewy bodies in PD (29, 30). However, DJ-1 has been defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to found to colocalize with -synuclein-immunoreactive glial inclu- indicate this fact. sions in multiple system atrophy and with a subset of pathological tau To whom correspondence should be addressed: Dept. of Pharmacology, Emory Uni- versity School of Medicine, 1510 Clifton Rd., Atlanta, GA 30322-3090. Tel.: 404-727- inclusions in a number of neurodegenerative tauopathies, including AD 5987; Fax: 404-727-0365; E-Mail: [email protected]. 2 (29, 30). Here, we show that DJ-1 is extensively and irreversibly oxidized The abbreviations used are: AD, Alzheimer disease; PD, Parkinson disease; DNP, 2,4- dinitrophenyl; ROS, reactive oxygen species; MALDI-TOF/MS, matrix-assisted laser in brains of patients with idiopathic PD and AD. The observed oxidative desorption ionization time-of-flight mass spectrometry; MALDI-TOF/TOF/MS/MS, damage to DJ-1 may have important implications for understanding the MALDI-TOF tandem mass spectrometry; HPLC, high performance liquid chromatog- raphy; ESI/MS/MS, electrospray ionization tandem mass spectrometry. pathogenesis of sporadic PD and AD. 10816 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 16 •APRIL 21, 2006 This is an Open Access article under the CC BY license. DJ-1 Oxidation in Neurodegenerative Diseases TABLE 1 RESULTS Demographic data of cases included in this study Accumulation of Acidic Isoforms of DJ-1 Monomer and Basic Isoforms Case no. Diagnosis Age Sex PMI of DJ-1 Dimer in PD and AD Brains—To investigate whether the years h expression and/or posttranslational modification(s) of DJ-1 is altered in 1 PD 79 Male 2 sporadic PD and AD, we performed comparative high resolution, two- 2 PD 69 Male 3 dimensional gel electrophoresis experiments on frontal cortex homo- 3 PD 74 Male 5 4 PD 74 Male 4.5 genates obtained from idiopathic PD and AD and age-matched con- 5 PD 66 Male 12 trols. The frontal cortex was chosen because of tissue availability and its 6 AD 76 Female 15 7 AD 91 Female 2.5 relevance to the pathology of both AD and PD. In AD the frontal cortex 8 AD 70 Male 11 exhibits neuronal loss and is rich in neurofibrillary tangles and amyloid 9 AD 85 Female 9.5 10 AD 50 Female 17 plaques. In PD, although neuronal loss mainly occurs in the substantia 11 Control 74 Female 3 nigra, the frontal cortex contains Lewy bodies, the pathological hall- 12 Control 68 Female 11 13 Control 65 Female 6 mark of PD. Analysis of DJ-1 expression and modifications in the sub- 14 Control 75 Female 6 stantia nigra or striatum might provide more PD-relevant information. 15 Control 87 Male 20.5 Unfortunately, we had difficulty in obtaining sufficient amounts of these PMI, post-mortem interval. tissues for biochemical analyses. MATERIALS AND METHODS Although DJ-1 exists as a homodimeric protein, DJ-1 migrates on Human Brain Samples—Brain tissues were obtained from the Emory SDS polyacrylamide gels as a 20-kDa monomer because of denaturation Center for Neurodegenerative Disease Brain Bank. For biochemical by SDS (17, 29). Immunoblot analysis of the two-dimensional SDS- studies, frontal cortex tissues from five PD cases, five AD cases, and five PAGE gels with anti-DJ-1 antibody revealed the presence of at least six healthy non-demented control subjects were used (Table 1). The neu- distinct isoforms of DJ-1 monomer that have the same apparent molec- ropathological diagnosis of PD was based on the presence of nigral ular mass of 20 kDa but different isoelectric points (5.5, 5.6, 5.7, 5.8, 6.1, degeneration and Lewy bodies. The neuropathological diagnosis of AD and 6.4, respectively; Fig. 1, A and B). The predominant form of DJ-1 is was made using Consortium to Establish a Registry for Alzheimer Dis- the 20-kDa/pI 6.4 isoform, which is in good agreement with the pre- ease criteria (31). dicted values of 19.9 kDa/pI 6.3 for DJ-1 monomer. Of the 6 identified 2,4-Dinitrophenyl (DNP) derivatization, Two-dimensional Gel Elec- DJ-1 monomer isoforms, the acidic isoforms (pI 5.5 and 5.7) of DJ-1 trophoresis, and Immunoblot Analysis—Protein extracts were prepared monomer were selectively accumulated in PD and AD compared with from human brain tissues as described (32). Protein samples (350 g) control brains (Fig. 1, B and C). The accumulation of these acidic DJ-1 were resolved by isoelectric focusing on 17-cm immobilized pH gradi- isoforms is likely due to specific modifications of DJ-1 in PD and AD ent strips (pH 3–10) followed by in-strip DNP derivatization (reacting instead of nonspecific changes in DJ-1 occurring post-mortem because with protein carbonyls) as described (32, 33). Second-dimensional sep- within each of the PD, AD, and control group, the relative levels of acidic aration was performed by electrophoresis on SDS-polyacrylamide gra- DJ-1 isoforms (pI 5.5 and 5.7) were similar among samples with different dient gels (10–20% porosity polyacrylamide) using the Ettan-DALT post-mortem intervals (Fig. 1F). slab gel SDS-PAGE system (Amersham Biosciences). After electro- Besides DJ-1 monomeric forms, we observed at least four different phoresis, the gels were analyzed by Sypro Ruby staining and by immu- isoforms of SDS-resistant DJ-1 dimer that migrated on the two-dimen- noblotting as described (32) with anti-DNP antibody (1:16,000, Molec- sional SDS-PAGE gels with an apparent molecular mass of 39 kDa and ular Probes) or anti-DJ-1 antibody (P7F, 1:5000) (17). The protein level pI values of 7.0, 7.4, 8.0, and 8.4, respectively (Fig. 1, A and D). Interest- and the carbonyl level of each DJ-1 isoform in PD, AD, and controls ingly, a previous study reported the presence of SDS-resistant DJ-1 were quantified using the two-dimensional gel analysis program PD dimers in the detergent-insoluble fraction of brains from patients with Quest (Bio-Rad). Statistical comparison of the data obtained from five multiple system atrophy (30). To determine whether the accumulation PD, five AD, and five control individuals were performed using analysis of SDS-resistant dimeric DJ-1 is related to PD and AD, we compared the of variance. Significant difference was accepted at p  0.05. levels of DJ-1 dimeric forms in PD, AD, and age-matched control brains. Mass Spectrometry—Spots of interest were excised from the gels and The results showed that the levels of the basic isoforms (pI 8.0 and 8.4) digested in situ with trypsin (modified; Promega) or Lys-C (Sigma-Al- of DJ-1 dimer were significantly increased in PD and AD brains com- drich). The Lys-C digests were derivatized with ProteoMass Guanidi- pared with controls (Fig. 1, D and E). nation kit (Sigma-Aldrich) to increase the mass spectral signal intensi- In addition to the monomeric and dimeric forms of DJ-1, weak DJ-1- ties of C-terminal lysine-containing peptides (34). The digests were immunoreactive spots at about 30 kDa were occasionally observed in subjected to analysis by matrix-assisted laser desorption ionization time-of- the control as well as in PD and AD brain samples (Fig. 1A). These flight mass spectrometry (MALDI-TOF/MS), matrix-assisted laser desorp- 30-kDa DJ-1 spots might represent modified forms of DJ-1 that have tion ionization time-of-flight tandem mass spectrometry (MALDI-TOF/ undergone some types of post-translational modification, such as phos- TOF/MS/MS), and capillary HPLC-electrospray ionization tandem mass phorylation or mono-ubiquitination. Of note, the mono-ubiquitinated spectrometry (HPLC-ESI/MS/MS). MALDI-TOF/MS and MALDI- form of recombinant DJ-1 protein with similar apparent molecular TOF-TOF/MS/MS mass spectra were acquired on an Applied Biosys- mass (30 kDa) has been shown to exist in transfected SH-SY5Y cells tems 4700 Proteomics discovery system. The peptide mass maps pro- (35). Furthermore, a 27-kDa DJ-1 species of unknown modification has duced by MALDI-TOF/MS and MALDI-TOF/TOF/MS/MS were TM been reported in the detergent-insoluble fraction of brains from searched against the published databases by means of 4700 Explorer patients with Pick’s disease as well as of AD brains (30). Unfortunately, software (Applied Biosystems). HPLC-ESI/MS/MS analysis was per- formed as described (32, 33). Assignment of the MS/MS fragments was despite repeated attempts, we were unable to further characterize these verified by comparison with the predicted ions generated in silico by 30-kDa DJ-1 species due to their extremely low abundance and the GPMAW (Lighthouse Data). variability of their appearance from sample to sample. APRIL 21, 2006• VOLUME 281 • NUMBER 16 JOURNAL OF BIOLOGICAL CHEMISTRY 10817 DJ-1 Oxidation in Neurodegenerative Diseases FIGURE 1. Altered expression of DJ-1 isoforms in PD and AD compared with control brains. A, two-dimensional immunoblot analysis with anti- DJ-1 antibody reveals the presence of multiple pI isoforms of DJ-1 monomer and dimer in human brain. The open arrowhead indicates the presence of weak DJ-1 immunoreactive spots at about 30 kDa (see “Results” for details). B and D, protein samples (350 g) of control, PD, or AD brains were subjected to two-dimensional gel electrophoresis followed by immunoblotting with anti-DJ-1 anti- body for detection of DJ-1 isoforms. C and E, the level of each DJ-1 isoform in PD or AD was quanti- fied using the two-dimensional gel analysis pro- gram PD Quest and normalized to the level of the same isoform in the control brains. Values repre- sent the mean  S.E. for five PD, five AD, and five control individuals. The asterisk indicates a statisti- cally significant ( p 0.05) increase in the level of the indicated DJ-1 isoform in PD or AD versus the corre- sponding control. F, the relative level of acidic DJ-1 isoforms was measured by quantification of the intensity of the DJ-1 pI 5.5 and pI 5.7 isoforms and expressed as a percentage of the total level of all DJ-1 monomeric isoforms in each human sample. A histogram plot of relative level of acidic DJ-1 iso- forms versus post-mortem interval (PMI) shows no correlation between accumulation of acidic DJ-1 iso- forms and post-mortem interval. The Total Level of DJ-1 Protein Is Increased in PD and AD Brains—Our revealed a significant increase in the total level of DJ-1 in PD and AD two-dimensional immunoblotting data (Fig. 1) suggest that the total brains compared with the controls (Fig. 2B). level of DJ-1 isoforms may be increased in PD and AD brains. To further DJ-1 Protein Is Oxidatively Damaged in PD and AD Brains—Protein investigate this possibility, we performed quantitative Western blot carbonylation is an irreversible oxidation that has been widely used as a analysis using protein samples obtained from PD and AD brains and biomarker of severe oxidative damage to proteins (5, 10, 11). Ample age-matched controls. The fact that all six DJ-1 monomer isoforms have evidence indicates that the total level of protein carbonyls is increased in the same molecular mass of 20 kDa and all four DJ-1 dimer isoforms are both AD and PD (4, 9). It has recently become clear that only a small of 39 kDa (Fig. 1) allowed us to use one-dimensional gel electrophoresis subset of total brain proteins in PD and AD is carbonylated (32, 33, 36). for direct comparison of the total DJ-1 levels of various brain samples on However, the identities of these oxidatively damaged proteins are the same gel. After electrophoresis the gels were subjected to immuno- largely unknown. Furthermore, it remains to be determined whether blotting with antibodies against DJ-1 and actin (Fig. 2A). Because the the protein targets of oxidative damage are the same or are different in amount of protein extract (50 g of protein/lane) loaded on one-dimen- AD and PD. sional gels were much less than the amount (350 g of protein/gel) on To determine whether DJ-1 protein is oxidatively damaged in PD two-dimensional gels, only a single DJ-1 band of 20 kDa was detected on or AD brain, we used a well established assay to detect protein car- the one-dimensional gels. The relative level of DJ-1 in each sample was bonyl groups by derivatization of the carbonyls with 2,4-dinitrophe- measured by quantification of the intensity of 20-kDa DJ-1 band and nylhydrazine and subsequent immuno-detection of the resulting normalized to the actin level in the corresponding sample. The results hydrazones using anti-DNP antibodies (32, 33, 37, 38). Protein 10818 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 16 •APRIL 21, 2006 DJ-1 Oxidation in Neurodegenerative Diseases FIGURE 2. Quantitative Western blot analysis of the total level of DJ-1 protein in PD, AD, and control brains. A, protein extracts (50 g of protein/lane) from PD, AD, or control brains were subjected to one-dimensional gel electrophoresis followed by immunoblotting with anti-DJ-1 antibody. Each lane represents a different individual from the PD, AD, or control group. B, the relative DJ-1 level was measured by quantifica- tion of the intensity of 20-kDa DJ-1 band and normalized to the actin level in the corre- sponding brain extract. The bar graph shows the results (mean S.E.) from five PD, five AD, and five control individuals. The asterisk indicates a statistically significant (p 0.05) increase in the total level of DJ-1 protein in PD or AD versus control. extracts from PD, AD, and control brains were resolved by isoelectric FIGURE 3. Increased oxidation of DJ-1 isoforms in PD and AD brains compared with focusing on an immobilized pH-gradient strip followed by in-strip controls. Protein samples (350 g) of control, PD, or AD brains were subjected to two- dimensional gel electrophoresis followed by immunoblotting (IB) with anti-DJ-1 anti- DNP derivatization of protein carbonyls and second-dimensional body for detection of DJ-1 isoforms or with anti-DNP antibody for detection of protein separation by SDS-PAGE and immunoblotting with anti-DJ-1 and carbonyls. The arrow indicates the isoform of DJ-1 monomer (A) and of DJ-1 dimer (B) anti-DNP antibodies (Fig. 3). We found that among the six identified that exhibits elevated oxidation in PD or AD. monomeric forms of DJ-1, only the 20-kDa/pI 6.4 isoform was oxi- datively modified, as evidenced by the significant increase in protein TABLE 2 carbonyl content of this isoform in PD and AD brains (Fig. 3A). Of Specific oxidation index of DJ-1 isoforms in PD, AD, the four dimeric forms of DJ-1, three isoforms (39 kDa/pI 7.4, 39 and control brains The specific oxidation index was obtained by normalization of the intensity of the kDa/8.0, and 39 kDa/pI 8.4) were found to exhibit elevated oxidative carbonyl level to the intensity of protein level of the indicated DJ-1 isoform. Values modification by carbonylation in PD and AD brains (Fig. 3B). No represent the mean  S.E. for five individuals of each of the PD, AD, or control DNP immunoreactivity was observed in the controls where the pri- group. mary anti-DNP antibody or the DNP derivatization procedure was DJ-1 isoform (M /pI) Control PD AD omitted (data not shown), confirming the specificity of our observa- n  5 n  5 n  5 a a 20/pI 6.4 0.30  0.03 1.17  0.23 1.07  0.30 tions. The lack of detection of carbonyls in the more acidic forms of a a 39/pI 7.4 0.43  0.06 1.93  0.50 2.06  0.60 DJ-1 monomers (pI 5.5– 6.1) could mean that these isoforms repre- a a 39/pI 8.0 0.73  0.17 1.63  0.26 1.67  0.30 a a sent un-carbonylated forms of DJ-1 or could be due to their lower 39/pI 8.4 0.97  0.13 4.00  0.40 3.66  0.60 p  0.05. abundance compared with the pI 6.4 DJ-1 isoform, making them undetectable because they might fall below the detection threshold required for anti-DNP immunoblotting. We favor the first interpre- tional modifications including oxidative modifications. However, despite tation, because the levels of these un-carbonylated DJ-1 monomeric recent advances in mass spectrometry technology, detection of modified forms were much higher than the dimeric forms of DJ-1 (pI 7.4 – 8.4) peptides, and localization of modification sites of an endogenous protein (Fig. 1A), which have been shown to be carbonylated by anti-DNP remain a major challenge. Therefore, we focused our mass spectrometry immunoblotting in the same experiments (Fig. 3B). study on PD and control samples. Individual DJ-1 protein spots were To quantitatively determine the differences in protein oxidation excised from the two-dimensional gels from five PD and five control between the disease brains and the controls, we quantified the protein brains, digested with a protease, and analyzed by mass spectrometry. level and the carbonyl level of each DJ-1 isoform in five PD, five AD, and Identification of the types and the sites of DJ-1 oxidative modifica- five control cases and obtained the specific oxidation index by normal- tions by mass spectrometry analysis is dependent on the ability to gen- ization of the intensity of carbonyl level to the intensity of protein level erate peptide fragments of appropriate size and adequate abundance for each DJ-1 isoform. Statistic analysis of the results showed a signifi- that ideally should cover the entire length of DJ-1 amino acid sequence. cant increase in the specific oxidation levels of the pI 6.4 monomeric and To increase the sequence coverage, we tried digestion with different the dimeric forms (pI 7.4–8.4) of DJ-1 in PD as well as in AD compared protease (trypsin or Lys-C protease), used a guanidination procedure with the age-matched control brains (Table 2). for enhancing mass spectral signals of Lys-containing peptides (34), and Identification of the Types and the Sites of DJ-1 Oxidative Modifica- employed a combination of different types of mass spectrometric anal- tions by Mass Spectrometry—In addition to carbonylation, proteins can yses, including MALDI-TOF/MS, MALDI-TOF/TOF/MS/MS, and undergo several types of oxidative modifications in response to oxida- HPLC-ESI/MS/MS. Because of the low abundance of the SDS-resistant tive stress (39). Thus, we used mass spectrometry to further characterize dimeric forms of DJ-1 in PD and control brains (Fig. 1), we were unable to the oxidative modifications of DJ-1 in human brain samples. Mass spec- detect oxidative modifications associated with the DJ-1 dimer isoforms. trometry has emerged as a powerful tool for the analysis of post-transla- However, for each of the DJ-1 monomer isoforms in PD and control brains, APRIL 21, 2006• VOLUME 281 • NUMBER 16 JOURNAL OF BIOLOGICAL CHEMISTRY 10819 DJ-1 Oxidation in Neurodegenerative Diseases FIGURE 4. Characterization of DJ-1 oxidative modifications in PD and control brains by mass spectrometry. A, coverage map of DJ-1 pI 6.4 isoform from PD or control brains with underlined residues indicating peptides detected by the combination of MALDI-TOF/MS, HPLC-ESI/MS/MS, and MALDI-TOF/TOF/MS/MS. C*, cysteic acid; M*, methionine sulfoxide; M**, methionine sulfone. Similar coverage maps were obtained for all other 20-kDa DJ-1 pI isoforms in five PD and five control brains. B, MALDI-TOF/MS spectra of the Lys-C digest of DJ-1 isoforms from PD or control brains. *, peptides that match the theoretically predicted peptide masses in DJ-1. **, oxidatively modified peptides containing methionine sulfoxide and/or methionine sulfone. Note that the oxidized peptide fragments at m/z 1948.1, 1964.1, and 1990.1 and m/z 2249.1 in the spectrum of DJ-1 pI 6.4 isoform in PD are absent in the spectrum of DJ-1 pI 6.4 isoform in control brain. Similar results were obtained for five PD and five control brains. we obtained sequence coverage of 50%, which matched to the primary MALDI-TOF/MS analysis of the Lys-C digest of DJ-1 20-kDa/pI 6.4 TM sequence of DJ-1 (GenBank accession number NP_009193; Fig. 4 and isoform protein spot from PD brains suggested that six ions represented additional data not shown). by m/z 1948.1, 1964.1, 1990.1, 2233.3, 2249.3, and 2275.3, respectively, 10820 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 16 •APRIL 21, 2006 DJ-1 Oxidation in Neurodegenerative Diseases FIGURE 5. Identification of oxidation sites in DJ-1 by MALDI-TOF/TOF/MS/MS and HPLC-ESI/ MS/MS analyses. A, MALDI-TOF/TOF/MS/MS spectrum of the ion at m/z 1948.1 (1) from a Lys-C digest of DJ-1 pI 6.4 isoform in PD showing the identity as DJ-1 peptide 133–148 with oxida- tion of both Met-133 and Met-134 residues to methionine sulfoxide. The ion at m/z 1636.5 and 1654.4 are interpreted as b ion and b-H O ion, respectively, corresponding to the internal frag- ment M*NGGHYTYSENRVE. B, MALDI-TOF/TOF/ MS/MS spectrum of the ion at m/z 2249.3 (1) from a Lys-C digest of DJ-1 pI 6.4 isoform in PD showing the identity as DJ-1 peptide 13–32 with oxidation of Met-17 to methionine sulfone and Met-26 to methionine sulfoxide. C, HPLC-ESI/ MS/MS spectrum of the ion at m/z 761.8 (2) from a tryptic digest of DJ-1 pI 6.4 isoform in control brain, showing the identity as DJ-1 peptide 49 – 62 with oxidation of Cys-53 to cysteic acid. Peptide fragments are indicated using the nomenclature of Roepstorff and Fohlman (52). C*, cysteic acid; M*, methionine sulfoxide; M**, methionine sulfone. APRIL 21, 2006• VOLUME 281 • NUMBER 16 JOURNAL OF BIOLOGICAL CHEMISTRY 10821 DJ-1 Oxidation in Neurodegenerative Diseases could be peptides resulting from oxidative modification (Fig. 4). All six TABLE 3 suspected oxidized DJ-1 peptides were also present in the MALDI-TOF Oxidized peptides of DJ-1 detected by mass spectrometric analysis Oxidized fragments at m/z 1990.1 and 2275.3 were generated by guanidation (Gu) of mass spectra of DJ-1 20 kDa/pI 5.5, 20 kDa/pI 5.6, 20 kDa/pI 5.7, 20 lysine at the C terminus of 1948.1 and 2233.3 peptides, respectively. C*, cysteic acid; kDa/pI 5.8 isoforms from PD brains, and all except the m/z 2249.3 M*, methionine sulfoxide; M**, methionine sulfone. fragment were found in the spectrum of DJ-1 20 kDa/pI 6.1 isoform Group m/z Oxidized peptide sequence from PD brains (Fig. 4). In contrast, only two (m/z 2233.3 and 2275.3) of Control 761.8 (2) DVVIC*PDASLEDAK the six suspected oxidized DJ-1 peptides were detected in the MALDI- 2233.3 (1) GAEEM*ETVIPVDVM*RRAGIK 2275.3 (1) GAEEM*ETVIPVDVM*RRAGIK(Gu) TOF mass spectra of DJ-1 isoforms from control brains (Fig. 4). PD 761.8 (2) DVVIC*PDASLEDAK All putative oxidized DJ-1 peptides were further analyzed by using 1948.1 (1) M*M*NGGHYTYSENRVEK 1964.1 (1) M**M*NGGHYTYSENRVEK MALDI-TOF-TOF/MS/MS to identify oxidatively modified amino acid 1990.1 (1) M*M*NGGHYTYSENRVEK(Gu) residues. The results indicated that the three ions at m/z 1948.1, 1964.1, 2233.3 (1) GAEEM*ETVIPVDVM*RRAGIK and 1990.1 are oxidatively modified peptides corresponding to amino 2249.3 (1) GAEEM**ETVIPVDVM*RRAGIK 2275.3 (1) GAEEM*ETVIPVDVM*RRAGIK(Gu) acid residues 133–148 of DJ-1 (Fig. 5A and Table 3). The ion at m/z 1990.1 was generated from the m/z 1948.1 peptide by guanidination, a reaction that results in a 42-Da mass shift due to the conversion of found in AD brains, suggesting that increased oxidation of DJ-1 may C-terminal lysine residue to homoarginine (34). The MALDI-TOF- occur in AD as well. TOF tandem mass spectra of the m/z 1948.1 and 1990.1 peptides In addition to the monomeric forms of DJ-1, we found four different revealed the oxidative modification of both Met-133 and Met-134 res- isoforms of SDS-resistant DJ-1 dimer, of which the basic isoforms (pI idues to methionine sulfoxide (Fig. 5A and additional data not shown). 8.0 and 8.4) are selectively accumulated in PD and AD brains. Although In addition, we observed the oxidation of Met-133 to methionine sul- these dimeric DJ-1 isoforms have never been reported, SDS-resistant fone and of Met-134 to methionine sulfoxide in the spectrum of the m/z DJ-1 dimer has been seen in the detergent-insoluble fraction of multiple 1964.1 peptide (data not shown). These results together with the system atrophy brains (30). The nature of chemical modifications that MALDI-TOF/MS data showing the consistent presence of the m/z render these DJ-1 dimers to become SDS-resistant is unknown. The 1948.1, 1964.1, and 1990.1 peptides in 5 PD but not in 5 control brains observation that three of the four isoforms of SDS-resistant DJ-1 dimer (Fig. 4B; data not shown), suggest that the oxidation of DJ-1 Met-133 are irreversibly oxidized by carbonylation in PD and AD brains suggests and Met-134 may be a phenomenon unique to PD. that oxidative modifications may be involved in conferring the SDS MALDI-TOF-TOF/MS/MS analysis also showed that the three ions resistance. at m/z 2233.3, 2249.3, and 2275.3 are oxidatively modified peptides cor- Consistent with the accumulation of several DJ-1 isoforms, our quan- responding to amino acid residues 13–32 of DJ-1 (Fig. 5B and Table 3). titative one-dimensional immunoblot analysis has revealed a significant The spectra of the m/z 2233.3 peptide and its guanidinated product at increase in the total level of DJ-1 in PD and AD brains compared with m/z 2275.3 revealed the oxidative modification of both Met-17 and age-matched controls. Previous studies have reported mixed results Met-26 residues to methionine sulfoxide (data not shown). These oxi- regarding the DJ-1 levels in PD and AD brains (29, 35, 42). Bando- dation products were found in all 20-kDa DJ-1 pI isoforms from both PD padhyay et al. (42) failed to find any obvious difference in the total level and control brains (Fig. 4 and Table 3). However, the m/z 2249.3 frag- of DJ-1 between PD and controls (42), whereas Moore et al. (35) found ment, a more severely oxidized form of DJ-1 peptide 13–32 generated a5-fold increase in the DJ-1 level in the detergent-insoluble fraction of from the conversion of Met-17 into methionine sulfone and Met-26 PD brains compared with control and AD brains. In contrast, Rizzu et al. into methionine sulfoxide (Fig. 5B), was only found in the 20-kDa DJ-1 (29) observed a dramatic increase in the DJ-1 level in the detergent- pI 5.5–5.8 and pI 6.4 isoforms from 5 PD brains but not in the DJ-1 insoluble fraction of AD brains. The reason(s) for these discrepancies is isoforms from the 5 controls (Fig. 4 and Table 3). In addition, our unclear but could be due to differences in anti-DJ-1 antibodies, patient MALDI-TOF/MS and HPLC-ESI-MS/MS analyses of trypsin-digested samples, and extraction conditions used in the immunoblot analyses. DJ-1 revealed that Cys-53 residue was oxidized to cysteic acid (also Previous studies have shown that, in cultured mammalian cells, known as cysteine sulfonic acid) in all 20-kDa DJ-1 pI isoforms from Cys-53 and Cys-106 residues of DJ-1 can undergo reversible oxidation both PD and control brains (Fig. 5C and Table 3), suggesting the Cys-53 in response to oxidative stress induced by H O and PD-linked environ- 2 2 oxidation may occur under relatively mild oxidative stress conditions mental toxins, such as paraquat and MPTP (1-methyl-4-phenyl-1,2,3,6- related to aging. tetrahydropyridine) (19, 40, 41). The reversible oxidation of these resi- dues has been proposed to regulate the molecular chaperone function DISCUSSION (19, 45) or mitochondrial localization of DJ-1 (40). Our mass spectrom- Our study demonstrates for the first time the oxidative damage to etry analysis has shown that Cys-53 of DJ-1 is oxidized to cysteine sul- DJ-1 in idiopathic PD and AD brains. We showed that human DJ-1 fonic acid in PD brains as well as in the age-matched controls. As exists in 10 different isoforms; that is, 6 monomeric forms and 4 SDS- pointed out earlier, despite our repeated efforts we could only achieve resistant dimeric forms. Although DJ-1 adopts a homodimeric structure 50% sequence coverage. Because we did not recover any peptide con- in solution as well as in the crystal (14, 15, 17), DJ-1 usually appears as a taining Cys-106, we could not determine the oxidation status of this 20-kDa monomer on the SDS-PAGE gel (17, 19, 29, 40, 41). Consistent residue in human brain samples. Future studies to increase sequence with a recent report (42), we found that the acidic pI isoforms (pI 5.5 and coverage are needed to identify all oxidative modifications of endoge- 5.7) of DJ-1 monomer are selectively accumulated in PD brains. Because nous DJ-1 in PD and AD. the 20-kDa DJ-1 has been shown to undergo acidic pI shift upon expo- In addition to cysteine oxidation, our mass spectrometry analysis has sure of cells to oxidative stress (19, 40, 41, 43, 44), the observed accu- revealed that DJ-1 is susceptible to previously unsuspected methionine mulation of the acidic forms of DJ-1 monomer in PD provides a first clue oxidation. We identified four methionine residues (Met-17, Met-26, that the oxidative modifications of DJ-1 may be elevated in PD brains. A Met-133, and Met-134) as the sites of DJ-1 oxidation. We found that all similar accumulation of the acidic forms of DJ-1 monomer was also four methionine residues were oxidized to methionine sulfoxide in PD, 10822 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 16 •APRIL 21, 2006 DJ-1 Oxidation in Neurodegenerative Diseases 5. Levine, R. L., and Stadtman, E. R. (2001) Exp. Gerontol. 36, 1495–1502 whereas only two of them (Met-17 and Met-26) were oxidized to methi- 6. Beal, M. F. (2002) Free Radic. Biol. Med. 32, 797–803 onine sulfoxide in age-matched controls. A variety of reactive oxygen 7. Ischiropoulos, H., and Beckman, J. S. (2003) J. Clin. Investig. 111, 163–169 species (e.g. O ,H O , OH, or peroxynitrite) can oxidize methionine resi- 2 2 2 8. Jenner, P. (2001) Trends Neurosci. 24, 245–247 dues to methionine sulfoxide (46), and this oxidation can be reversed by the 9. Giasson, B. I., Ischiropoulos, H., Lee, V. M., and Trojanowski, J. Q. (2002) Free Radic. Biol. Med. 32, 1264–1275 enzyme peptide methionine sulfoxide reductase (MsrA) in a thioredoxin- 10. Dalle-Donne, I., Giustarini, D., Colombo, R., Rossi, R., and Milzani, A. (2003) Trends dependent manner (47). The reversible methionine oxidation/reduction Mol. Med. 9, 169–176 has been suggested to act as a signaling device analogous to phospho- 11. Nystrom, T. (2005) EMBO J. 24, 1311–1317 rylation/dephosphorylation for regulating protein function and cellular 12. Gracy, R. W., Talent, J. M., Kong, Y., and Conrad, C. C. (1999) Mutat. Res. 428, 17–22 processes (46). It is, thus, tempting to speculate that the methionine 13. Bonifati, V., Rizzu, P., van Baren, M. J., Schaap, O., Breedveld, G. J., Krieger, E., oxidation of DJ-1 identified here represents a specific, reversible mech- Dekker, M. C., Squitieri, F., Ibanez, P., Joosse, M., van Dongen, J. W., Vanacore, N., van anism for regulation of DJ-1 activity by intracellular redox status. In Swieten, J. C., Brice, A., Meco, G., van Duijn, C. M., Oostra, B. A., and Heutink, P. addition, the reversible oxidation of methionine residues has been pro- (2003) Science 299, 256–259 14. Huai, Q., Sun, Y., Wang, H., Chin, L. S., Li, L., Robinson, H., and Ke, H. (2003) FEBS posed to serve as an important defense mechanism for scavenging ROS Lett. 549, 171–175 (48). Such a mechanism would support an antioxidant role of DJ-1 in 15. Tao, X., and Tong, L. (2003) J. Biol. Chem. 278, 31372–31379 protecting cells from oxidative damage (19, 24). Interestingly, Met-26, a 16. Lee, S. J., Kim, S. J., Kim, I. K., Ko, J., Jeong, C. S., Kim, G. H., Park, C., Kang, S. O., Suh, methionine residue of DJ-1 found to be oxidized in this study, is mutated P. G., Lee, H. S., and Cha, S. S. (2003) J. Biol. Chem. 278, 44552–44559 17. Olzmann, J. A., Brown, K., Wilkinson, K. D., Rees, H. D., Huai, Q., Ke, H., Levey, A. I., to isoleucine (M26I) in a rare, autosomal recessive form of familial PD Li, L., and Chin, L. S. (2004) J. Biol. Chem. 279, 8506–8515 (26). Thus, the reversible oxidation of Met-26 as well as of other iden- 18. Shendelman, S., Jonason, A., Martinat, C., Leete, T., and Abeliovich, A. (2004) PLoS tified methionine oxidation sites (Met-17, Met-133, and Met-134) may Biol. 2, 1764–1773 have a role in regulation of DJ-1 function and/or localization. Future 19. Taira, T., Saito, Y., Niki, T., Iguchi-Ariga, S. M., Takahashi, K., and Ariga, H. (2004) studies are needed to test these hypotheses and determine the func- EMBO Rep. 5, 213–218 20. Miller, D. W., Ahmad, R., Hague, S., Baptista, M. J., Canet-Aviles, R., McLendon, C., tional consequences of DJ-1 methionine oxidation. Carter, D. M., Zhu, P. P., Stadler, J., Chandran, J., Klinefelter, G. R., Blackstone, C., and In addition to the reversible methionine oxidation, we found that Cookson, M. R. (2003) J. Biol. Chem. 278, 36588–36595 Met-17 and Met-133 were irreversibly oxidized to methionine sulfone 21. Macedo, M. G., Anar, B., Bronner, I. F., Cannella, M., Squitieri, F., Bonifati, V., Hoo- geveen, A., Heutink, P., and Rizzu, P. (2003) Hum. Mol. Genet. 12, 2807–2816 in PD but not in controls. Unlike the reversible oxidation of methionine 22. Moore, D. J., Zhang, L., Dawson, T. M., and Dawson, V. L. (2003) J. Neurochem. 87, residues to methionine sulfoxide, which occurs under physiological 1558–1567 conditions, the irreversible oxidation to methionine sulfone is rare and 23. Goldberg, M. S., Pisani, A., Haburcak, M., Vortherms, T. A., Kitada, T., Costa, C., only takes place in the presence of strong oxidants (46). This irreversible Tong, Y., Martella, G., Tscherter, A., Martins, A., Bernardi, G., Roth, B. L., Pothos, oxidation is often associated with pathophysiological conditions and E. N., Calabresi, P., and Shen, J. (2005) Neuron 45, 489–496 24. Kim, R. H., Smith, P. D., Aleyasin, H., Hayley, S., Mount, M. P., Pownall, S., results in functional impairment of the oxidized proteins (46, 49). Wakeham, A., You-Ten, A. J., Kalia, S. K., Horne, P., Westaway, D., Lozano, A. M., Another important finding of the present study is the oxidative dam- Anisman, H., Park, D. S., and Mak, T. W. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, age to DJ-1 in PD and AD by the irreversible carbonylation. Protein 5215–5220 carbonylation occurs under stronger oxidative stress than that causing 25. Chen, L., Cagniard, B., Mathews, T., Jones, S., Koh, H. C., Ding, Y., Carvey, P. M., Ling, Z., Kang, U. J., and Zhuang, X. (2005) J. Biol. Chem. 280, 21418–21426 cysteine and methionine oxidation and has been associated with func- 26. Abou-Sleiman, P. M., Healy, D. G., Quinn, N., Lees, A. J., and Wood, N. W. (2003) tional impairment in a variety of structural proteins and enzymes (10, Ann. Neurol. 54, 283–286 11). For example, actin carbonylation occurs after its Cys and Met res- 27. Hedrich, K., Schafer, N., Hering, R., Hagenah, J., Lanthaler, A. J., Schwinger, E., Kramer, P. L., Ozelius, L. J., Bressman, S. B., Abbruzzese, G., Martinelli, P., Kostic, V., idues have already been oxidized, and the carbonylation leads to the Pramstaller, P. P., Vieregge, P., Riess, O., and Klein, C. (2004) Ann. Neurol. 55, 145 disruption of actin filaments and the inhibition of F-actin formation 28. Gorner, K., Holtorf, E., Odoy, S., Nuscher, B., Yamamoto, A., Regula, J. T., Beyer, K., (50). The lipid peroxidation product 4-hydroxynonenal induces car- Haass, C., and Kahle, P. J. (2004) J. Biol. Chem. 279, 6943–6951 bonylation of ubiquitin C-terminal hydrolase L1 (UCH-L1), resulting in 29. Rizzu, P., Hinkle, D. A., Zhukareva, V., Bonifati, V., Severijnen, L. A., Martinez, D., a dramatic reduction in the hydrolase activity of UCH-L1 (51). Thus, the Ravid, R., Kamphorst, W., Eberwine, J. H., Lee, V. M., Trojanowski, J. Q., and Heutink, P. (2004) Ann. Neurol. 55, 113–118 observed DJ-1 carbonylation together with irreversible methionine oxi- 30. Neumann, M., Muller, V., Gorner, K., Kretzschmar, H. A., Haass, C., and Kahle, P. J. dation may cause functional impairment of DJ-1 and contribute to the (2004) Acta Neuropathol. (Berl) 107, 489–496 pathophysiology and progression of sporadic PD and AD. Further inves- 31. Mirra, S. S., Heyman, A., McKeel, D., Sumi, S. M., Crain, B. J., Brownlee, L. M., Vogel, tigation of the relationship between oxidative modifications of DJ-1 as F. S., Hughes, J. P., van Belle, G., and Berg, L. (1991) Neurology 41, 479–486 32. Choi, J., Levey, A. I., Weintraub, S. T., Rees, H. D., Gearing, M., Chin, L. S., and Li, L. well as of other proteins and neuronal dysfunction should generate new (2004) J. Biol. Chem. 279, 13256–13264 insights into the mechanisms of oxidative damage in the pathogenesis of 33. Choi, J., Rees, H. D., Weintraub, S. T., Levey, A. I., Chin, L. S., and Li, L. (2005) J. Biol. PD and AD and provide new opportunities for developing therapeutic Chem. 280, 11648–11655 strategies for treating these devastating diseases. 34. Beardsley, R. L., and Reilly, J. P. (2002) Anal. Chem. 74, 1884–1890 35. Moore, D. J., Zhang, L., Troncoso, J., Lee, M. K., Hattori, N., Mizuno, Y., Dawson, T. M., and Dawson, V. L. (2005) Hum. Mol. Genet. 14, 71–84 Acknowledgments—We thank Christopher A. Carroll of the Institutional Mass 36. Castegna, A., Aksenov, M., Aksenova, M., Thongboonkerd, V., Klein, J. B., Pierce, Spectrometry Laboratory at the University of Texas Health Science Center at W. M., Booze, R., Markesbery, W. R., and Butterfield, D. A. (2002) Free Radic. Biol. San Antonio (supported in part by National Institutes of Health Grant Med. 33, 562–571 37. Levine, R. L. (2002) Free Radic. Biol. Med. 32, 790–796 CA54174) for mass spectrometric analyses. 38. Butterfield, D. A., and Castegna, A. (2003) Amino Acids 25, 419–425 39. Stadtman, E. R. (2001) Ann. N. Y. Acad. Sci. 928, 22–38 40. Canet-Aviles, R. M., Wilson, M. A., Miller, D. W., Ahmad, R., McLendon, C., REFERENCES Bandyopadhyay, S., Baptista, M. J., Ringe, D., Petsko, G. A., and Cookson, M. R. (2004) 1. Ross, C. A., and Poirier, M. A. (2004) Nat. Med. 10, (suppl.) 10–17 Proc. Natl. Acad. Sci. U. S. A. 101, 9103–9108 2. Cordato, D. J., and Chan, D. K. (2004) J. Clin. Neurosci. 11, 119–123 41. Kinumi, T., Kimata, J., Taira, T., Ariga, H., and Niki, E. (2004) Biochem. Biophys. Res. 3. Pardo, L. M., and van Duijn, C. M. (2005) Mutat. Res. 592, 89–101 Commun. 317, 722–728 4. Jenner, P. (2003) Ann. Neurol. 53, Suppl. 3, 26–36 42. Bandopadhyay, R., Kingsbury, A. E., Cookson, M. R., Reid, A. R., Evans, I. M., Hope, APRIL 21, 2006• VOLUME 281 • NUMBER 16 JOURNAL OF BIOLOGICAL CHEMISTRY 10823 DJ-1 Oxidation in Neurodegenerative Diseases A. D., Pittman, A. M., Lashley, T., Canet-Aviles, R., Miller, D. W., McLendon, C., 397, 172–178 Strand, C., Leonard, A. J., Abou-Sleiman, P. M., Healy, D. G., Ariga, H., Wood, N. W., 48. Levine, R. L., Mosoni, L., Berlett, B. S., and Stadtman, E. R. (1996) Proc. Natl. Acad. Sci. De Silva, R., Revesz, T., Hardy, J. A., and Lees, A. J. (2004) Brain 127, 1–11 U. S. A. 93, 15036–15040 43. Mitsumoto, A., and Nakagawa, Y. (2001) Free Radic. Res. 35, 885–893 49. Stadtman, E. R., Van Remmen, H., Richardson, A., Wehr, N. B., and Levine, R. L. 44. Mitsumoto, A., Nakagawa, Y., Takeuchi, A., Okawa, K., Iwamatsu, A., and Takan- (2005) Biochim. Biophys. Acta 1703, 135–140 ezawa, Y. (2001) Free Radic. Res. 35, 301–310 50. Dalle-Donne, I., Rossi, R., Giustarini, D., Gagliano, N., Di Simplicio, P., Colombo, R., 45. Martinat, C., Shendelman, S., Jonason, A., Leete, T., Beal, M. F., Yang, L., Floss, T., and and Milzani, A. (2002) Free Radic. Biol. Med. 32, 927–937 Abeliovich, A. (2004) PLoS Biol. 2, 1754–1763 51. Nishikawa, K., Li, H., Kawamura, R., Osaka, H., Wang, Y. L., Hara, Y., Hirokawa, T., 46. Hoshi, T., and Heinemann, S. (2001) J. Physiol. (Lond.) 531, 1–11 Manago, Y., Amano, T., Noda, M., Aoki, S., and Wada, K. (2003) Biochem. Biophys. 47. Weissbach, H., Etienne, F., Hoshi, T., Heinemann, S. H., Lowther, W. T., Res. Commun. 304, 176–183 Matthews, B., St John, G., Nathan, C., and Brot, N. (2002) Arch. Biochem. Biophys. 52. Roepstorff, P., and Fohlman, J. (1984) Biomed. Mass Spectrom. 11, 601 10824 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 16 •APRIL 21, 2006

Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Apr 21, 2006

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