RNA binding activity of the recessive parkinsonism
protein DJ-1 supports involvement in multiple
Marcel P. van der Brug*†, Jeff Blackinton*†‡, Jayanth Chandran§, Ling-Yang Hao¶, Ashish Lal?,
Krystyna Mazan-Mamczarz?, Jennifer Martindale?, Chengsong Xie§, Rili Ahmad*, Kelly J. Thomas*,
Alexandra Beilina*, J. Raphael Gibbs**, Jinhui Ding**, Amanda J. Myers*, Ming Zhan††, Huaibin Cai§,
Nancy M. Bonini¶, Myriam Gorospe?, and Mark R. Cookson*‡‡
*Cell Biology and Gene Expression Unit,§Transgenic Unit, and **Bioinformatics Core, Laboratory of Neurogenetics, National Institute on Aging, 35 Convent
Drive, Bethesda, MD 20892-3707;?Laboratory of Cellular and Molecular Biology and††Research Resources Branch, Gerontology Research Center, National
Institute on Aging, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825;¶Department of Biology, Howard Hughes Medical Institute, University of
Pennsylvania, Philadelphia, PA 19104; and‡Department of Neuroscience, Karolinska Institutet, Retzius vag 8, SE-171 77 Stockholm, Sweden
Edited by Gregory A. Petsko, Brandeis University, Waltham, MA, and approved May 8, 2008 (received for review September 7, 2007)
Parkinson’s disease (PD) is a major neurodegenerative condition with
several rare Mendelian forms. Oxidative stress and mitochondrial
function have been implicated in the pathogenesis of PD but the
molecular mechanisms involved in the degeneration of neurons
remain unclear. DJ-1 mutations are one cause of recessive parkinson-
ism, but this gene is also reported to be involved in cancer by
promoting Ras signaling and suppressing PTEN-induced apoptosis.
The specific function of DJ-1 is unknown, although it is responsive to
oxidative stress and may play a role in the maintenance of mitochon-
dria. Here, we show, using four independent methods, that DJ-1
associates with RNA targets in cells and the brain, including mito-
chondrial genes, genes involved in glutathione metabolism, and
deficient in this activity. We show that DJ-1 is sufficient for RNA
binding at nanomolar concentrations. Further, we show that DJ-1
binds RNA but dissociates after oxidative stress. These data implicate
a single mechanism for the pleiotropic effects of DJ-1 in different
an oxidation-dependent manner.
gene expression ? oxidative stress ? Parkinson’s disease ? translation
is a mitochondrial kinase (1). Results from Drosophila models
suggest that PINK1 and parkin define a single pathway that,
when disrupted, leads to mitochondrial damage (2, 3). The third,
and rarest, gene for recessive parkinsonism is DJ-1. The DJ-1
protein responds to oxidative stress evidenced by a pI shift in
sporadic PD (4, 5) and in cell (6, 7) and animal (8) models. DJ-1
knockout models also show increased sensitivity to toxins that
cause mitochondrial dysfunction or oxidative stress (9–13).
Cys-106 of DJ-1, which is oxidized to form a cysteine-sulfinic
acid, is critically required for DJ-1 to protect against these types
of damage both in vitro (6) and in vivo (12). However, the
molecular function of DJ-1 is unclear. DJ-1 is part of the
ThiJ/PfPI superfamily but the proteins most similar to DJ-1 form
a distinct clade away from other members with known function
(14, 15), implying a novel activity. As well as effects on oxidation
and mitochondrial function, DJ-1 enhances Ras-mediated on-
cogenesis (16), modulates the PTEN/Akt survival pathway (17,
18), suppresses Ask1-mediated apoptosis (19), and increases
glutathione (GSH) synthesis, Hsp70 (20) and tyrosine hydrox-
ylase (21, 22) expression. DJ-1 is a small, dimeric, single-domain
protein (23), so if all of these effects are true then either the
protein has multiple functions or there is a single biochemical
activity that explains all of them.
utations in any of three genes cause a recessively inherited
One of the original descriptions of the cloning of DJ-1 was as
RS, a regulatory component of an RNA binding complex (24).
Therefore, we examined whether DJ-1 associates with RNA in
vitro and in vivo and looked at the consequences of any inter-
action. We propose that the apparently pleiotropic effects of
DJ-1 relate to the common property of RNA binding.
Purification of mRNA Associated with DJ-1 Protein. We used immu-
noprecipitation (IP; Fig. 1a) to isolate endogenous DJ-1 from
cultured human dopaminergic neuroblastoma cells (25). The
antibody we used was directed to the C-terminal region of DJ-1
that is solvent-accessible and was validated against knockdown
cell lines and knockout mouse brains [supporting information
(SI) Fig. S1]. We then isolated any associated mRNA and
candidate mRNA lists (Table S1; also available at GEO acces-
sion no. GSE8632). Using six DJ-1 IP samples and four control
IgG IP samples from multiple cell preparations, we identified
transcripts significantly enriched in the DJ-1 IP sample that were
reliably detected above background (P ? 0.001 for detection).
Clustering indicated a clear separation of the two groups across
all experiments (Fig. 1b). These results suggest that DJ-1 asso-
ciates with specific mRNA targets.
The transcripts that are associated with DJ-1 are potentially
involved in many cellular processes. We chose to examine three
groupings of transcripts associated with DJ-1 that might be
relevant to the survival of dopaminergic neurons: proteins that
are involved in the metabolism of selenocysteine and enzymes
that contain selenocysteine, particularly the GSH peroxidases;
mitochondrial transcripts, both nuclear and mitochondrially
encoded; and components of the PTEN/Akt survival pathway
(Tables S2–S4). We validated these interactions by RT-PCR and
saw preferential amplification of a series of candidate transcripts
Author contributions: M.P.v.d.B., J.B., A.L., N.M.B., M.G., and M.R.C. designed research;
M.P.v.d.B., J.B., J.C., L.-Y.H., A.L., K.M.-M., J.M., C.X., R.A., K.J.T., and A.B. performed
research; J.C., L.-Y.H., A.J.M., and H.C. contributed new reagents/analytic tools; M.P.v.d.B.,
M.R.C. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The data reported in this paper have been deposited in the Gene
Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE8632).
†M.P.v.d.B. and J.B. contributed equally to this work.
‡‡To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
July 22, 2008 ?
vol. 105 ?
(Fig. 1c). We used cross-linked immunoprecipitation (CLIP)
(26) as a second independent technique. UV-cross-linked com-
plexes containing DJ-1, and associated RNA were radiolabeled,
revealing smear of RNA complexes that were resolved to a
single, lower molecular weight band after RNase digestion (Fig.
1d). The apparent mobility of the UV-cross-linked complexes on
SDS/PAGE is consistent with a monomer of DJ-1 (22 kDa) plus
short RNA tags of 20–50 nt or 7–16 kDa (26). The DJ-1 dimer
is dissociated by SDS treatment, and we infer the 35-kDa bands
represent individual monomers with cross-linked radiolabeled
this material to identify the protected RNA site (see below).
We used a third, nonantibody-based technique and purified
6His-tagged DJ-1 from cells by using Ni-NTA affinity chroma-
tography (Fig. 1e). Pathogenic DJ-1 mutants showed lower
binding to mRNA compared with WT protein after correcting
all RT-PCR results to the amount of DJ-1 protein in the IP (Fig.
1f). Our estimates of fold enrichment are higher with this
approach (6- to 20-fold) than those predicted with arrays (2- to
underestimates strength of interaction of DJ-1 with mRNA. The
to represent cross-reactivity.
To address whether such interactions might occur in the brain,
we isolated complexes by IP with DJ-1 antibody from WT and
DJ-1 knockout (27) mouse brains (Fig. 2a). We validated the
interaction of DJ-1 with representative transcripts by using
cells. (b) Associated RNA was isolated, amplified, and hybridized to arrays.
Cluster analysis of replicate samples shows a strict separation of DJ-1 and
control IPs. (c) Using two control (lanes 1 and 2) and three DJ-1 (lanes 3–5) IP
samples, we performed RT-PCR for selenoproteins, mitochondrial transcripts,
and components of the PTEN/Akt pathway; gene symbols are listed on the
right of each gel. (d) Living M17 cells were subjected to UV cross-linking and
immunoprecipitated with a nonspecific IgG (lanes 1–4) or anti-DJ-1 (lanes
5–8). RNA was radiolabeled, showing a smear of label in the DJ-1 IP samples
(lane 5, bar on the left of the autoradiogram). A low concentration of RNase
(lanes 2, 3, 6, and 7) did not remove label, but a high concentration (lane 8)
on the right of the autoradiogram are in kDa. (e) M17 cells were transfected
V5-His tag. A vector containing GFP was used as a control (lane 1). DJ-1 was
purified by using NiNTA beads and subjected to Western blotting with an
anti-V5 antibody. (f) We performed qRT-PCR for selenoprotein genes, ex-
pressing these results as enrichment relative to GFP. As M26I is partially
unstable, the amount of DJ-1 protein in the input and IP is lower, and thus all
values were also corrected to amount of DJ-1 in the IP. Values are mean ?/?
SEM, n ? 3–4 replicates per construct.*, P ? 0.05;**, P ? 0.01;***, P ? 0.001
using one-way ANOVA for each gene with Bonferroni post hoc tests to
compare mutants with WT DJ-1.
Endogenous DJ-1 binds RNA. (a) DJ-1 was isolated by IP from M17
from knockout (lanes 1–3) or WT (lanes 4–6) mice. DJ-1 protein is absent from
the knockout mice. (b) Validation by qRT-PCR after IP of DJ-1 from WT (closed
bars) or knockout (open bars) mice. Error bars indicate the SEM (n ? 3).*, P ?
0.05;**, P ? 0.01;***, P ? 0.001 using t tests to compare genotypes.
van der Brug et al.
July 22, 2008 ?
vol. 105 ?
no. 29 ?
quantitative RT-PCR (Fig. 2b). These observations show that
DJ-1 can bind mRNA in vivo.
Identification of RNA Sequences That Can Bind DJ-1. We next used
several approaches to determine the region of the mRNAs to
which DJ-1 binds. Although the CLIP technique has lower
throughput than microarray analysis, it allows for cloning of
sequences directly bound to DJ-1 (26). Our CLIP tags included
selenoproteins and members of the PTEN/Akt1 pathway (Fig.
3a). The actual sequences all contained multiple copies of GG
or CC motifs, reflected in a GG/CC-rich consensus (Fig. 3b). We
also recovered many shorter tags (between 10 and 30 nt) that we
could not map to specific genes but that were also GG/CC-rich
(a candidate confirmed in Fig. 1 c and f) contains a short
GG-rich region (Fig. 3a). MAPK8IP1 contains a similar motif in
the 5? UTR but also sequences in the 3? UTR (Fig. 3a). To test
single-stranded RNA molecules were prepared by in vitro tran-
scription and incubated with nanomolar amounts of recombi-
nant DJ-1. We found that DJ-1 interacted specifically with the
5? UTR of GPx4 but not to the 3? UTR or the coding sequences
of two abundant mRNA species (UbC9 and GAPDH), which
showed only background binding (Fig. 3c). The binding of DJ-1
UTRs) was no greater than to the 5?UTR of GPx4 (Fig. 3d),
suggesting that additional sequences do not contribute to bind-
ing. These results indicate that there is an authentic RNA
binding sequence in the 5? UTR of GPx4.
We created stable dopaminergic neuroblastoma cell lines
expressing a nonsense shRNA or two different shRNA se-
quences to DJ-1 (Fig. S2). Nonsense shRNA did not affect DJ-1
expression levels but the shRNA constructs decreased expres-
sion by ?85% and ?95%, respectively. We made reporter
constructs for RNA binding by cloning UTR sequences from
GPx4 or MAPK8IP1 into vectors expressing GFP (Fig. 3e). A
construct with the 5? UTR of GPx4 placed 5? to the GFP coding
sequence gave higher protein levels when transfected into DJ-1
knockdown cell lines compared with control shRNA lines (Fig.
3 f and g). In contrast, the 3? UTR sequence, placed after GFP,
was translated as efficiently in both lines (Fig. 3 f and h). Both
the 5? and 3? UTRs of MAPK8IP1 were able to suppress
translation (Fig. 3 i and j). As a control, pEGFP alone without
of DJ-1 (Fig. 3i). These results imply that GG/CC-rich sequences
are sufficient for DJ-1 interaction in vitro but are also active in
vivo and that DJ-1 partially inhibits translation of its target
mRNAs while it is associated with them.
Because DJ-1 is protective under oxidative conditions, we
exposed cells to paraquat, which induced a shift in DJ-1 to more
acidic isoforms (data not shown) as reported (6). Using NiNTA
purification of V5his-tagged WT DJ-1 (Fig. 4a), we found that
paraquat treatment significantly decreased the amount of GPx4
(Fig. 4b) and MAPK8IP1 (Fig. 4c) mRNA bound to DJ-1. This
decrease in interaction corresponded to an increased expression
of the GPx4 5? UTR-GFP construct in control cell lines (Fig. 4d).
This observation suggests DJ-1 releases from target transcripts
under oxidative stress.
DJ-1 Effects on RNA and Protein. Next, we examined the effects of
DJ-1 deficiency and oxidative stress on the RNA and protein
levels for two candidate targets. We chose GPx4 as a represen-
tative selenoprotein and MAPK8IP1 as a representative mem-
ber of the Akt pathway. In both cases, we were able to obtain
high-quality antibodies to the endogenous proteins, although the
GPx4 antibody was human-specific (data not shown). In shRNA-
mediated knockdown stable cell lines, DJ-1 deficiency was
associated with increased GPx4 (Fig. S3 a and b) or MAPK8IP1
(Fig. S3 d and e) protein levels. After oxidative stress, both
proteins were induced in control cells but decreased in the
knockdown cells. In contrast to the changes in protein levels,
mRNA levels for GPx4 or MAPK8IP1 were similar in all cell
lines and did not change after paraquat (PQ) treatment (Fig. S3
c and f). To extend this comparison to the in vivo situation, we
examined aging DJ-1 knockout mice. We showed that aging is
associated with increased oxidation of DJ-1, comparing young
(?1 month) and old (24 months old) WT animals (Fig. S3 g and
h). MAPK8IP3 protein levels were higher in young knockout
animals compared with WT littermates but in aged samples
MAPK8IP3 was higher in the WT animals and decreased in the
knockouts (Fig. S3 i and j) such that the relative expression levels
in knockouts and controls were reversed. Similar patterns were
seen with MAPK8IP1 (Fig. S3k). Quantitation of protein levels,
correcting for ?-actin, revealed a significant effect of age (P ?
0.0012 for MAPK8IP3; P ? 0.026 for MAPK8IP1) and an
interaction between age and genotype (P ? 0.0006 for
MAPK8IP3; P ? 0.0039 for MAPK8IP1) by two-way ANOVA
(n ? 3–4 animals per group). The steady-state levels of each
mRNA did not vary between conditions (data not shown). These
data therefore support the hypothesis that DJ-1 binds to RNA
for GPx4 and MAPK8IP1/3 in vivo and have a mild suppressive
effect on translation.
DJ-1 Knockout Flies Are More Sensitive to Disrupted GSH Peroxidase
and PTEN/Akt-related (18) DJ-1 targets are important in main-
tenance of viability in vivo. However, the hypothesis that GSH
peroxidases are also targets is novel and led us test the idea in
vivo by exposing DJ-1-deficient flies to GSH inhibitors. Flies
deficient in DJ-1 showed an increase in sensitivity to the GSH
synthesis inhibitor buthionine-S,R-sulfoximine that was similar
in the ability of DJ-1 to protect organisms in vivo.
Overall, our results indicate that DJ-1 associates with specific
several transcripts that interact may help explain previous ob-
servations about the biological roles of DJ-1. By allowing for a
simultaneous control of the mRNA for GSH peroxidases, and
selenoproteins needed to synthesize functional GPx, DJ-1 may
control antioxidant defenses. Targets of DJ-1 within the PTEN/
Akt1 pathways may be related to the ability of the protein to
suppress cell death, as has been reported in many previous
studies, including those demonstrating a specific connection to
this pathway (18). The mitochondrial transcripts are also of
interest because of the emerging evidence of a mitochondrial
basis of recessive parkinsonism (2, 3). DJ-1 can be found on the
surface of mitochondria (6) and within the mitochondrial matrix
(28), supporting the idea that a small pool of DJ-1 may authen-
tically associate with RNA in the mitochondria. However, this
hypothesis requires additional confirmation.
Further work is also required to define the nature of the
DJ-1–mRNA interaction. The structural basis of the interaction
between the DJ-1 and RNA is unclear as DJ-1 lacks classical
RNA binding motifs. However, our data found using in vitro
approaches suggests that DJ-1 alone is sufficient to GG/CC-rich
sequences at nanomolar concentrations. We have evidence that
the RNA targets that DJ-1 binds are distinct from other RNA–
protein complexes. In previous large-scale studies of RNA–
protein interactions in the brain, FMR-1, associated with mental
retardation, binds a distinct set of targets from those discussed
here (29), and binding to the splicing factors Nova1/2 are also
different (30). Other RNA binding proteins, such as HuR (31),
also gave different targets in our studies using the same exper-
www.pnas.org?cgi?doi?10.1073?pnas.0708518105van der Brug et al.
sequenced in duplicate. Similar sequences were seen in other sequences from the array dataset including GPx4 (5? UTR) and MAPK8IP1 (both 5? and 3? UTR). (b)
(50 nM). DJ-1 was coprecipitated with the 5?-UTR of GPx4 (lane 2) but not the 3?-UTR (lane 3) or the coding sequence from UbC9 (lane 1). Data are representative of
four independent experiments. (d) The 5? UTR (lane 1) of GPx4 gives equivalent amounts of pull down compared with the full-length RNA (lane 2) and higher than
the coding sequence of GAPDH (lane 3). (e) Diagrams of 5? and 3? reporter constructs, which were driven by a CMV promoter (PCMV) and contained an SV40 poly
adenylation [poly(A)] sequence. EGFP alone was used as a control for transfection. (f–j) M17 cell lines stably expressing nonspecific shRNA controls (lanes 1 and 2) or
cell lines and each graph shows the average of at least three independent transfections per construct.*, P ? 0.05;**, P ? 0.01 by t test comparing lines with control
shRNA and lines with DJ-1 shRNA.
DJ-1 interacts directly with GG/CC-rich mRNA sequences. (a) Sequences recovered from CLIP tags for targets also present on the array; each of these was
van der Brug et al.
July 22, 2008 ?
vol. 105 ?
no. 29 ?
imental procedures (data not shown). Therefore, DJ-1 is specific
in the sense of binding only a subset of targets.
Our data are consistent with a model whereby DJ-1 interacts
with mRNA and dissociates under conditions of oxidative stress.
but would allow efficient translation under appropriate local or
environmental cues. Rapid translational processes are important
in control of stress responses and apoptosis (32), and local
translation at synapses is critical for neuronal function. If this
interpretation is correct then the increased levels of MAPK8IP1
and MAPKIP3 in the mouse brains with aging would represent
successful responses to oxidative stress but the failure of the
DJ-1-deficient animals would represent an inadequate ability to
do so. The steady-state levels of RNA for these targets are not
different in the absence of DJ-1, suggesting that the protein does
not modify RNA turnover. However, we cannot exclude that the
of another function of the protein. For example, RNA is
transported throughout the cell and translation is repressed
during transport (33). Future studies will be required to clarify
whether DJ-1 has additional activities on its RNA targets.
In summary, we have shown both in vitro and in vivo that DJ-1
is capable of binding a series of target mRNA molecules,
identifying a GG/CC-rich sequence that is sufficient for DJ-1
recruitment. The functional classification of these transcripts
suggests how DJ-1 may play roles in coordinating responses to
oxidative damage and suppression of cell death. Our data
support the hypothesis that the apparently pleiotropic roles of
DJ-1 may be related to the single function of binding to multiple
DJ-1 Knockout Mice and Cell Lines. DJ-1 knockout mice lacking exon 2 have
been described in detail (27). DJ-1 expression constructs have been described
(6, 34, 35). Stable M17 human neuroblastoma cell lines were cultured as
described (6). Clonal M17 cell lines stably expressing different DJ-1 siRNAs
the manufacturer’s instructions. Two target sequences (GGAAGTAAAGTTA-
(GCCTAGACGCGATAGTATGGA) were used.
Precipitation of DJ-1 RNA Complexes. Anti-DJ-1 antibody C-16 (Santa Cruz
Biotechnology) had no detectable RNA-degrading activity and was used for IPs.
Negative control IPs were performed by using normal goat IgG from the same
manufacturer (catalog no. SC-2028). Cell or brain samples were harvested in PLB
buffer (0.5% Nonidet P-40, 10 mM Hepes, 100 mM KCl, 5 mM MgCl2, 1 mM DTT,
RNase OUT and protease inhibitors) and prepared for RNA-IP as described (31).
beads (Qiagen) were incubated with precleared lysate in NT2 buffer (0.05%
Nonidet P-40, 50 mM Tris, 150 mM NaCl, 1 mM MgCl2), and then washed four
the supernatant, then eluting after treatment with Proteinase K. RNA was
washed with acid phenol-chloroform and precipitated with 100% ethanol con-
and cDNA was generated by using the SuperScript III kit (Invitrogen). CLIP was
performed as described (26, 30).
Expression Arrays and Analysis. Illumina human oligonucleotide arrays were
used according to the manufacturer’s instructions, starting with 500 ng of
total RNA for each sample. Arrays were read on an Illumina Bead array reader
Illumina Custom algorithm within the Illumina BeadStudio software suite.
Biotin-Labeled RNA Pull-Down Assays. Biotin-labeled RNAs corresponding to
the UTR sequences for GPx4 were synthesized with T7 polymerase, biotin-
ylated, and used in pull-down assays with recombinant GST-tagged DJ-1 as
Quantitative RT-PCR (qRT-PCR). Primers were designed against potential DJ-1
binding targets and validated for use in comparative qRT-PCR with ?-actin
in gene expression was calculated from reactions performed in quadruplicate
with four biological replicates per sample.
Protein Analyses. Analysis of the oxidation state of DJ-1 was performed as
described (6) using monoclonal anti-DJ-1 (Stressgen). Antibodies for
MAPK8IP1 (JIP1) and MAPK8IP3 (JIP3) were obtained from Santa Cruz Bio-
technologies. Quantitation of signal intensities was performed by using a
Storm fluorescence scanner, and protein loading was normalized by reprob-
constructs, forward and reverse primers were designed to amplify the 5? and
3? UTRs of human GPx4 or MAPK8IP1. Sequences were cloned into the
pEGFP-N1 and pEGFP-C1, respectively (Clontech). Constructs were transiently
transfected into cell lines by using Lipofectamine 2000 (Invitrogen), and
protein was measured by blotting for GFP.
Drosophila melanogaster Viability Experiments. Drosophila DJ-1b-deleted (DJ-
1b?93) and double knockout (DKO, DJ-1b?93with DJ-1a?72) and exposure to
were isolated from untreated (lane 2) or PQ-treated (lane 3) cells stably
transfected with V5–6His-tagged WT DJ-1, or empty vector (lane 1) as a
control, using NiNTA beads, then blotted for DJ-1. Arrows show V5–6His-
tagged DJ-1, and arrowheads show endogenous DJ-1. (b and c) Associated
RNA was analyzed by qRT-PCR (bars show mean signal, error bars indicate the
SEM, n ? 3 independent PQ treatments and pull downs per construct). PQ
treatment decreases the binding of DJ-1 to the RNA for GPx4 and MAPK8IP1.
Differences in qRT-PCR were analyzed by one-way ANOVA with Bonferroni’s
post hoc tests.*, P ? 0.05;**, P ? 0.01;***, P ? 0.001. (d) PQ increases the
protein levels of the GPx4 5? UTR GFP construct in cells containing DJ-1. M17
(see Fig. 3) placed 5? to EGFP and were either untreated (Control) or exposed
of n ? 4 experiments is shown in the bar graph.*, P ? 0.05 by t test.
www.pnas.org?cgi?doi?10.1073?pnas.0708518105 van der Brug et al.
drugs (5% buthionine-S,R-sulfoximine or 1 mM selenium, in 5% sucrose, 1% Download full-text
agar medium) have been described (11).
ACKNOWLEDGMENTS. This research was supported in part by the Intramural
Research Program of the National Institute on Aging, National Institutes of
Health. Computational approaches used the high-performance computa-
tional capabilities of the Biowulf Linux cluster at the National Institutes of
Health (http://biowulf.nih.gov). L.-Y.H. and N.M.B. were supported by Na-
tional Institute on Aging Grant P01-AG09215. N.M.B. is an Investigator of the
Howard Hughes Medical Institute. A.J.M. received grant support from
Access for Disabled or Elderly Members of the community) project.
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