From the Department of Neurology, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya 466-8550, Japan
Received for publication, March 18, 2003 , and in revised form, May 12, 2003.
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ABSTRACT |
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INTRODUCTION |
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-Synuclein is a 19-kDa presynaptic vesicular protein of unconfirmed
function and one of the major components of LBs
(17,
18). Mutations in
-synuclein (A30P and A53T) cause a rare autosomal dominant form of PD,
which shares many phenotypic characteristics with sporadic PD
(19,
20).
-Synuclein
aggregates deposit in LBs in both autosomal dominant and sporadic forms of PD
(21,
22). In addition, it has been
reported that transgenic flies and mice overexpressing human wild-type or
mutant
-synuclein have abnormal cellular accumulation of
-synuclein and neuronal dysfunction and degeneration
(2330),
indicating that
-synuclein has a role in the pathogenesis of both
familial and sporadic forms of PD.
Synphilin-1 was identified recently by yeast two-hybrid techniques as a
novel protein that interacts with -synuclein
(31).
-Synuclein amino
acids 165 are is sufficient for interaction, and the central portion of
synphilin-1 (amino acids 349555) is necessary and sufficient for
interaction with
-synuclein
(32). It has also been
reported that the C terminus of
-synuclein is closely associated with
the C terminus of synphilin-1 and that a weak interaction occurs between the N
terminus of
-synuclein and synphilin-1
(33). Synphilin-1 is highly
concentrated in presynaptic nerve terminals, and its association with synaptic
vesicles is modulated by
-synuclein
(34). Coexpression of
-synuclein and synphilin-1 in transfected cells results in the
formation of eosinophil cytoplasmic inclusions that resemble LBs
(31,
35), whereas transfection of
synphilin-1 alone without expression of
-synuclein or parkin can also
produce cytoplasmic inclusions in cultured cells
(36,
37). Furthermore, synphilin-1
is ubiquitylated and degraded by proteasomes in human embryonic kidney 293
(HEK293) cells (37) and is
localized as another major component of LBs in the brains of patients with PD
(38,
39). Thus, the process through
which aggregations are formed by synphilin-1 may be important in the
pathogenesis of PD.
Dorfin is a gene product (which we cloned from anterior horn tissues of human spinal cord) (40) that contains a RING finger/IBR (in between RING finger) motif (41) at its N terminus. It was reported that HHARI (human homologue of ariadne) and H7-AP1 (UbcH7-associated protein-1), both RING finger/IBR motif-containing proteins, interact with the ubiquitin carrier protein (E2) UbcH7 through the RING finger/IBR motif and that a distinct subclass of RING finger/IBR motif-containing proteins represents a new family of proteins that specifically interact with distinct E2 enzymes (42, 43). Dorfin is a juxtanuclearly located E3 ubiquityl ligase and may function in the microtubule-organizing centers (40). In the spinal cords of patients with sporadic and familial forms of amyotrophic lateral sclerosis (ALS) with an SOD1 mutation, Dorfin is colocalized with ubiquitin in hyaline inclusions (44). Dorfin physically binds and ubiquitylates various SOD1 mutants derived from familial ALS patients and enhances their degradation (44). Thus, an important and interesting question is whether Dorfin is colocalized with ubiquitin in LBs of PD.
In this study, we show that Dorfin is colocalized with ubiquitin in LBs of PD. We found that Dorfin ubiquitylates synphilin-1 and that overexpression of synphilin-1 leads to ubiquitylated inclusions resembling LBs in cultured cells.
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EXPERIMENTAL PROCEDURES |
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Expression Plasmids, Cell Culture, and TransfectionHuman
synphilin-1 cDNA containing the entire coding region was amplified by
Pfu Turbo DNA polymerase (Stratagene) from human brain cDNAs using
5'-GTCAGGATCCACCACCATGGAAGCCCCTGAATACC-3' as the forward primer
and 5'-ATATCTCGAGTGCTGCCTTATTCTTTCCTTTG-3' as the reverse primer
and inserted in-frame into the BamHI and XhoI sites of the
pcDNA3.1/V5His vector (Invitrogen). A plasmid for DsRed-tagged synphilin-1 was
constructed by PCR amplification using
5'-ATATCTCGAGACCACCATGGAAGCCCCTGAATACC-3' as the forward primer
and 5'-GTCAGGATCCGCCTTTGCCTTATTCTTTCCTTTG-3' as the reverse primer
and inserted in-frame into the XhoI and BamHI sites of the
pDsRed-N1 vector (Clontech). A series of deletion mutants of synphilin-1 were
prepared as synphilin-1-N (amino acid 1348), synphilin-1-M (amino acid
349555), and synphilin-1-C (amino acid 556919). Synphilin-1-M is
the central portion of synphilin-1, containing the ankyrin-like repeat, the
coiled-coil domain, and the ATP/GTP-binding domain
(31). Primers pairs for each
deletion mutant were as follows:
5'-GTCAGGATCCACCACCATGGAAGCCCCTGAATACC-3' and
5'-ATATCTCGAGTTCGTCGTGAATTTTGTCT-3' for synphilin-1-N-V5,
5'-ATATCTCGAGACCACCATGGAAGCCCCTGAATACC-3' and
5'-GTCAGGATCCGCTTCGTCGTGAATTTTGTCTAG-3' for synphilin-1-N-DsRed,
5'-GTCAGGATCCACCATGAATGGAAACAATCTAT-3' and
5'-ATATCTCGAGCTTGCCCTCTGATTTCTGG-3' for synphilin-1-M-V5,
5'-ATATCTCGAGACCACCATGAATGGAAACAATCTAT-3' and
5'-GTCAGGATCCGCC TTGCCCTCTGATTTCTGGGC-3 for synphilin-1-M-DsRed,
5'-GTCAGGATCCACCACCATGTCACTCCCTTCTTCAC-3' and
5'-ATATCTCGAGTGCTGCCTTATTCTTTCCTTTG-3' for synphilin-1-C-V5, and
5'-ATATCTCGAGACCACCATGTCACTCCCTTCTTCAC-3' and
5'-GTCAGGATCCGCCTTTGCCTTATTCTTTCCTTTG-3' for synphilin-1-C-DsRed.
Construction of pcDNA4/His-Max-Dorfin, pcDNA3.1(+)FLAG-ubiquitin, and
pcDNA3.1/MycHis(+)-SOD1 vectors was described elsewhere
(40,
44). -Synuclein cDNA
was amplified by PCR from human brain cDNAs and cloned into the EcoRV
site of pcDNA3.1/MycHis(+) (Invitrogen). To generate the mutant
-synuclein expression vector, A30P and A53T mutations were introduced
into pcDNA3.1/MycHis(+)-
-synuclein with a QuikChange site-directed
mutagenesis kit (Stratagene) following the method of Lee et al.
(46). COS-7, HEK293, and
Neuro2a cells were maintained in Dulbecco's modified Eagle's medium with 10%
fetal calf serum. Transfections were performed using the Effectene
transfection reagent (QIAGEN Inc.) according to the manufacturer's
instructions. To inhibit cellular proteasome activity, cells were treated with
0.5 µM MG132
(carbobenzoxy-L-leucyl-L-leucyl-L-leucinal,
Sigma) for 16 h overnight after transfection.
Immunoprecipitation and Western Blot AnalysisCells were
lysed in lysis buffer (50 mM Tris, 150 mM NaCl, 1%
Nonidet P-40, and 0.1% SDS) with Complete protease inhibitor mixture (Roche
Diagnostics). Immunoprecipitation from transfected cell lysates was performed
with 2 µg of antibody and protein A/G Plus-agarose (Santa Cruz
Biotechnology), and the immunoprecipitate was then washed four times with
lysis buffer. Anti-V5 antibody (Invitrogen) for synphilin-1-V5 fusion proteins
and anti-Myc antibody (A-14, Santa Cruz Biotechnology) for
-synuclein-Myc or SOD1-Myc fusion proteins were used.
Immunoprecipitates were subjected to SDS-PAGE and analyzed by Western blotting
with ECL detection reagents (Amersham Biosciences).
In Vitro Ubiquitylation AssayImmunopurified (IP)
Xpress-Dorfin bound to anti-Xpress antibody (Invitrogen) with protein A/G
Plus-agarose (Santa Cruz Biotechnology) was prepared from lysates of HEK293
cells transfected with pcDNA4/HisMax-Dorfin. IP-synphilin-1-V5 was prepared
with anti-V5 antibody bound to protein A/G Plus-agarose from lysates of HEK293
cells transfected with pcDNA3.1/V5His-synphilin-1. IP--synuclein-Myc
and IP-SOD1-Myc were prepared with anti-Myc antibodies from lysates of
pcDNA3.1/MycHis(+)-
-synuclein- and pcDNA3.1/MycHis(+)-SOD1-transfected
HEK293 cells, respectively. Slurries of IP-Xpress-Dorfin were mixed with
IP-synphilin-1-V5, IP-
-synuclein-Myc, or IP-SOD1-Myc and incubated at
30 °C for 90 min in 50 µl of reaction buffer containing ATP (4
mM ATP in 50 mM Tris-HCl (pH 7.5), 2 mM
MgCl2, and 2 mM dithiothreitol), 100 ng of rabbit E1
(Calbiochem), 2 µg of UbcH7 (Affiniti), and 2 µg of His-ubiquitin
(Calbiochem). The reaction was terminated by adding 20 µl of 4x
sample buffer, and 20-µl aliquots of the reaction mixtures were subjected
to SDS-PAGE, followed by Western blotting with anti-His antibody
(Novagen).
Neurotoxicity Analysis and Quantification of Synphilin-1 AggregatesCOS-7 cells (1 x 104) were grown overnight on collagen-coated 4-chamber well slides. They were transfected with 0.2 µg of pDsRed-N1-synphilin-1 or its deletion mutants. To inhibit cellular proteasome activity, cells were treated with 0.5 µM MG132 for 16 h overnight after transfection. The number of inclusions was counted in >100 cells randomly selected, and data were averaged from three independent experiments. For cell viability assay, 5 x 103 Neuro2a cells were grown in collagen-coated 96-well plates overnight. They were then transfected with 0.1 µg of pcDNA3.1/V5His-synphilin-1 or deletion mutants of synphilin-1. pcDNA3.1/V5His-LacZ was used as a control. Next, an MTS-based cell proliferation assay was performed using CellTiter 96 (Promega) at 24 h after serum deprivation. The assay was carried out in triplicate. Absorbance at 490 nm was measured in a multiple plate reader.
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RESULTS |
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Expression of Synphilin-1 Induces LB-like Large Juxtanuclear
Inclusions, and Dorfin Localizes to These Inclusions To
investigate the relationships of Dorfin to components of LBs other than
ubiquitin, we first examined the subcellular localization of -synuclein
and synphilin-1 in cultured cells. We created wild-type and mutant
-synuclein-green fluorescent protein and
-synuclein-Myc fusion
constructs, but there was no evidence of
-synuclein aggregation in
transfected COS-7 cells in the presence or absence of the proteasome inhibitor
(data not shown). We created a synphilin-1-DsRed fusion construct by fusing
the red fluorescent protein DsRed to the C terminus of synphilin-1 and carried
out transient transfection in COS-7 cells with this construct. Large
juxtanuclear inclusions were spontaneously formed in the transfected COS-7
cells in the absence of the proteasome inhibitor
(Fig. 2, AO).
We also constructed synphilin-1 fusion proteins with a smaller
V5/His6 tag, which formed identical inclusions when overexpressed
in COS-7 cells, although to a lesser extent than synphilin-1-DsRed fusion
proteins (data not shown). Immunostaining with anti-ubiquitin and anti-Dorfin
antibodies revealed that most of the large juxtanuclear inclusions of
synphilin-1 contained ubiquitin (Fig. 2,
AC) and Dorfin (DF).
Immunohistochemical studies of human LBs have previously shown that LBs are
stained with proteasome subunits
(6) and molecular chaperones
such as Hsp40 and Hsp70 (24).
Thus, we next examined whether the inclusion bodies in COS-7 cells contain the
20 S proteasome core subunit and Hsp70. We found both the 20 S proteasome
subunit and Hsp70 to be colocalized with synphilin-1 inclusion bodies
(Fig. 2, GL).
Dorfin binds specifically to UbcH7 as an E2 through the RING finger/IBR domain
(40). UbcH7 was also localized
with Dorfin in these inclusions (Fig. 2,
MO). These observations suggest that large
juxtanuclear inclusions formed by synphilin-1 in our cell culture system have
many characteristic features of LBs, that synphilin-1 can aggregate when
overexpressed, and that this process may be associated with its
ubiquitylation.
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Expression of the Central Portion of Synphilin-1 Induces Large Juxtanuclear Inclusions as Full-length Proteins, but Small Punctate Aggregates Are Also FormedTo further analyze which part of synphilin-1 is related to aggregation formation, we prepared a series of deletion mutants of synphilin-1. We divided synphilin-1 into three parts, the N terminus of synphilin-1 (synphilin-1-N) containing amino acids 1348, the central portion of synphilin-1 (synphilin-1-M) containing amino acids 349555, and the C terminus of synphilin-1 (synphilin-1-C) containing amino acids 556919, and fused them to DsRed at their C termini (Fig. 3, A and B). Inclusions were not seen with overexpression of DsRed alone, synphilin-1-N, or synphilin-1-C in COS-7 cells (Fig. 3, CE). However, expression of synphilin-1-M resulted in the production of two types of inclusions: large juxtanuclear inclusions (Fig. 3, FK) and small punctate aggregates scattered throughout the cytoplasm (LQ). The large inclusions were stained with ubiquitin (Fig. 3, FH) and Dorfin (IK), as were inclusions induced by full-length synphilin-1. However, neither ubiquitin nor Dorfin was colocalized with the small punctate aggregates scattered throughout the cytoplasm (Fig. 3, LQ).
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Expression of the Central Portion of Synphilin-1 Compromises Cell ViabilityWe examined the frequency of the inclusion formation by synphilin-1 and its deletion mutants. The number of inclusions was counted with and without the proteasome inhibitor MG132 in COS-7 cells (Fig. 4A). Both synphilin-1-N and synphilin-1-C formed almost no inclusions in either the presence or absence of MG132. Full-length synphilin-1 and synphilin-1-M produced inclusions with high frequency even in the absence of MG132, and the number of cells with inclusions induced by full-length synphilin-1 was significantly greater than that induced by synphilin-1-M (Fig. 4A). Treatment with MG132 significantly increased the number of inclusions. We next measured the ratio of cells that contained small punctate aggregates to total cells bearing all inclusions (Fig. 4B) because two types of aggregates, large juxtanuclear inclusions and small punctate scattered aggregates, were observed. In contrast to full-length synphilin-1, the inclusions induced by overexpression of synphilin-1-M were predominantly small punctate aggregates scattered through the cytoplasm (Fig. 4B). Treatment with MG132 decreased the ratio of small aggregates induced by synphilin-1-M (Fig. 4B).
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The effects of synphilin-1 expression on cell viability are poorly understood. O'Farrell et al. (36) reported that cells transfected with synphilin-1 are more viable than cells transfected with LacZ. On the other hand, Lee et al. (37) reported that synphilin-1 compromises cell viability. Thus, we examined the effects of synphilin-1 and its deletion mutants on cell viability using the MTS assay in the neuronal cell line Neuro2a (Fig. 4C). We found that synphilin-1-M had a cytotoxic effect, whereas overexpression of full-length synphilin-1 or the N- or C-terminal deletion mutant of synphilin-1 did not (Fig. 4C). We used synphilin-1-V5 fusion constructs, but synphilin-1-DsRed fusion constructs gave the same results (data not shown).
Dorfin Interacts with Synphilin-1We examined whether Dorfin interacts with synphilin-1 because Dorfin localizes in LBs and cytoplasmic juxtanuclear inclusions formed by synphilin-1. To identify which portion of synphilin-1 binds to Dorfin, we expressed a series of deletion mutants of V5-tagged synphilin-1 and Xpress-tagged Dorfin in COS-7 cells (Fig. 5B). Co-immunoprecipitation confirmed that Dorfin bound to full-length synphilin-1 (Fig. 5A) and interacted strongly with synphilin-1-M and weakly with synphilin-1-N, but Dorfin failed to bind to synphilin-1-C (Fig. 5C). Thus, Dorfin interacts with synphilin-1 mainly through its central portion, which contains the ankyrin-like repeat, the coiled-coil domain, and the ATP/GTP-binding domain. Dorfin has a unique primary structure containing a RING finger/IBR motif at its N terminus and can be structurally divided into two parts, the N-terminal region containing a RING finger/IBR motif (Dorfin-N) that interacts with E2 and the C-terminal region with no similarity to any other known proteins (Dorfin-C) (Fig. 5B) (40). We found that Dorfin-C, but not Dorfin-N, specifically bound synphilin-1, indicating that Dorfin binds to synphilin-1 via its C-terminal region (Fig. 5D).
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Dorfin Ubiquitylates Synphilin-1 through Its Central Domain In
VitroThe physical interaction between Dorfin and synphilin-1
prompted us to investigate whether synphilin-1 itself is ubiquitylated by
Dorfin. We first examined whether synphilin-1 is ubiquitylated in a culture
cell model. V5-tagged full-length synphilin-1 or its deletion mutants were
cotransfected with FLAG-tagged ubiquitin in HEK293 cells. When full-length
synphilin-1 or its deletion mutants were immunoprecipitated after treatment
with the proteasome inhibitor MG132, full-length synphilin-1 and synphilin-1-M
were found to be polyubiquitylated, but synphilin-1-N and synphilin-1-C were
not (Fig. 6A).
Wild-type and mutant -synuclein were found not to be polyubiquitylated,
whereas, as previously reported
(44), mutant SOD1 was
polyubiquitylated (Fig.
6A).
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We next examined whether Dorfin is involved in the ubiquitylation of
synphilin-1 in vitro. For this purpose, we immunopurified
Xpress-Dorfin and synphilin-1-V5 independently after transfection into HEK293
cells. When these immunopurified proteins were incubated with recombinant E1,
E2 (UbcH7), His-tagged ubiquitin, and ATP, high molecular mass ubiquitylated
bands were observed in the presence of Xpress-Dorfin with synphilin-1, whereas
no signal was noted with synphilin-1 in the absence of either E1 or E2
(Fig. 6B). Dorfin
ubiquitylated mutant SOD1 in vitro, as previously reported
(44). Dorfin did not
ubiquitylate either wild-type or mutant -synuclein
(Fig. 6B). In
vitro ubiquitylation assay of a series of synphilin-1 deletion mutants
with Dorfin revealed that synphilin-1-M was ubiquitylated, whereas
synphilin-1-N and synphilin-1-C were not ubiquitylated at all
(Fig. 6C).
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DISCUSSION |
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For the analysis of LB formation by synphilin-1, various cell culture
models have been reported (31,
3537).
In our cell culture model, overexpression of synphilin-1 alone induced large
juxtanuclear cytoplasmic inclusions. In these large inclusions, Dorfin was
colocalized with ubiquitin/proteasome pathway-related proteins such as
ubiquitin, the 20 S proteasome core subunit, and Hsp70, just as Dorfin in LBs.
The central portion of synphilin-1 contains the ankyrin-like repeat, the
coiled-coil domain, and the ATP/GTP-binding domain
(31). This region is reported
to be necessary for interaction with -synuclein
(32). We found that the
central portion of synphilin-1 also bound with Dorfin and that overexpression
of this region alone led to inclusion body formation, whereas neither the N-
nor C-terminal regions induced aggregates. Overexpression of this central
portion of synphilin-1 produced small punctate aggregates scattered throughout
the cytoplasm as well as large juxtanuclear inclusions, but the former
predominated. The small punctate aggregates did not colocalize with either
ubiquitin or other proteasome pathway-associated proteins and had cytotoxic
effects as revealed by MTS assays. Recently, Lee and Lee
(50) reported that
overexpression of
-synuclein in culture cells produces two distinct
types of aggregates: large juxtanuclear inclusions and small punctate
aggregates scattered throughout the cytoplasm. The juxtanuclear inclusion
bodies are filled with amyloid-like
-synuclein fibrils, whereas the
small aggregates contain non-fibrillar spherical aggregates
(50). They suggested that
these aggregates appear sequentially, with the smallest population appearing
first and the fibrillar inclusions last, and that the small spherical
aggregates are the cellular equivalents of protofibrils
(50). Protofibrils are
recognized to be more important in terms of cytotoxicity than mature fibrils
in A
(51,
52) and
-synuclein
(53,
54). In our cell culture
model, overexpression of synphilin-1 produced two distinct types of
aggregates, very closely resembling two types of
-synuclein aggregates
(50). Thus, the small punctate
aggregates scattered throughout the cytoplasm induced by the central portion
of synphilin-1 might have characteristics similar to those of protofibrils.
Our cell culture system will allow detailed characterization of LB formation
and cytotoxic processes in further studies.
We reported previously that Dorfin localizes in the inclusion bodies of
familial ALS with SOD1 mutations as well as in those of sporadic ALS and
ubiquitylates various SOD1 mutants derived from familial ALS patients
(44). Based on these findings,
it is conceivable that familial and sporadic forms of ALS share a common
mechanism involving the dysfunction of the ubiquitin/proteasome pathway,
despite having distinct etiological mechanisms. In sporadic ALS, unknown
substrate(s) of Dorfin might play a role in the pathogenesis of the disease
and accumulate in ubiquitylated inclusion bodies. The following results
support the view that Dorfin plays an important role in the formation of LBs
of PD: (i) the presence of Dorfin in LBs and large juxtanuclear inclusions of
synphilin-1 in our cell culture model, (ii) the parallel distribution patterns
of ubiquitin and Dorfin in LBs and inclusion bodies induced by synphilin-1 in
cultured cells, and (iii) the E3 function of Dorfin to ubiquitylate
synphilin-1. Dorfin did not ubiquitylate either wild-type or mutant
-synuclein; however, our results cannot exclude the possibility that
post-translational modification, such as glycosylation
(55) or phosphorylation
(56,
57), of
-synuclein may
be necessary for it to become a substrate for Dorfin because overexpressed
-synuclein was not phosphorylated in our cell culture system (data not
shown). The relation between Dorfin and PD shows striking similarities to the
relation between Dorfin and ALS. Our findings raise the possibility that PD
and ALS are etiologically distinct, but share a biochemically common metabolic
pathway through Dorfin, leading to the formation of ubiquitylated inclusion
bodies and to neuronal cell degeneration.
Parkin has been shown to have E3 ubiquityl ligase activity
(1012).
It was recently demonstrated that an O-glycosylated -synuclein
(55) and synphilin-1
(35) are the substrates of
parkin and that parkin localizes to LBs of sporadic PD
(49). The link between
sporadic and familial forms of PD through
-synuclein, synphilin-1, and
parkin sheds new light on underlying common molecular pathogenic mechanisms in
PD. What roles, then, do Dorfin and parkin play with respect to each other in
the pathogenesis of PD and/or LB formation? Both proteins have a RING
finger/IBR domain and E3 ubiquitin ligase activities. Parkin interacts with
both
-synuclein (55)
and synphilin-1 (35), whereas
Dorfin binds and ubiquitylates only synphilin-1. Parkin resides in the core of
LBs (49), whereas Dorfin
localizes predominantly to the rim. Impaired function of parkin as an E3
ubiquityl ligase is responsible for one of the most common forms of familial
PD, autosomal recessive juvenile parkinsonism
(9,
10). However, there has been
no analysis of whether Dorfin gene mutation causes familial PD.
Recently, Valente et al.
(58,
59) identified a locus,
PARK6, on chromosome 1p35
[PDB]
1p36 that is involved in the
autosomal recessive form of parkinsonism. Interestingly, a human paralog of
Dorfin (Dj1174N9.1) has been mapped at 1p34.11p35.3
(60). Furthermore, Dorfin was
identified by a phage display system to be one of the binding proteins with
2-methylnorharman, an analog of the parkinsonism-inducing toxin,
1-methyl-4-phenylpyridinium cation
(61). These findings suggest
the utility of analyzing Dj1174N9.1 or Dorfin mutation for potential
involvement in familial PD. Production of Dorfin knockout mice will also
answer the question of whether Dorfin is essential for pathogenesis and/or
ubiquitylated inclusion body formation in PD.
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FOOTNOTES |
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Research Fellow of the Japan Society for the Promotion of Science for Young
Scientists.
To whom correspondence should be addressed: Dept. of Neurology, Nagoya
University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya
466-8550, Japan. Tel.: 81-52-744-2385; Fax: 81-52-744-2384; E-mail:
sobueg{at}med.nagoya-u.ac.jp.
1 The abbreviations used are: PD, Parkinson's disease; LBs, Lewy bodies; E1,
ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3,
ubiquitin-protein isopeptide ligase; HEK293, human embryonic kidney 293; ALS,
amyotrophic lateral sclerosis; SOD1, superoxide dismutase-1; IP,
immunopurified; MTS,
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,
inner salt.
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ACKNOWLEDGMENTS |
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REFERENCES |
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