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Aprataxin the causative protein for EAOH is a nuclear protein with a potential role as a DNA repair protein.

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Aprataxin, the Causative Protein for
EAOH Is a Nuclear Protein with a Potential
Role as a DNA Repair Protein
Yasuteru Sano, MD,1,5 Hidetoshi Date, MS,1 Shuichi Igarashi, MD, PhD,1,3 Osamu Onodera, MD, PhD,1,3
Mutsuo Oyake, MD, PhD,1 Toshiaki Takahashi, MD,1 Shintaro Hayashi, MD, PhD,2
Mitsunori Morimatsu, MD,5 Hitoshi Takahashi, MD, PhD,2 Takao Makifuchi, MD,3,4
Nobuyoshi Fukuhara, MD,3 and Shoji Tsuji, MD, PhD1,6
Early-onset ataxia with ocular motor apraxia and hypoalbuminemia (EAOH) is an autosomal recessive neurodegenerative disorder characterized by early-onset ataxia, ocular motor apraxia, and hypoalbuminemia. Recently, the causative gene for EAOH, APTX, has been identified. Of the two splicing variants of APTX mRNA, the short and the long
forms, long-form APTX mRNA was found to be the major isoform. Aprataxin is mainly located in the nucleus, and,
furthermore, the first nuclear localization signal located near the amino terminus of the long-form aprataxin is essential for its nuclear localization. We found, based on the yeast two-hybrid and coimmunoprecipitation experiments,
that the long-form but not the short-form aprataxin interacts with XRCC1 (x-ray repair cross-complementing group
1). Interestingly the amino terminus of the long-form aprataxin is homologous with polynucleotidekinase-3ⴕphosphatase, which has been demonstrated to be involved in base excision repair, a subtype of single-strand DNA
break repair, through interaction with XRCC1, DNA polymerase ␤, and DNA ligase III. These results strongly
support the possibility that aprataxin and XRCC1 constitute a multiprotein complex and are involved in single-strand
DNA break repair, and furthermore, that accumulation of unrepaired damaged DNA underlies the pathophysiological
mechanisms of EAOH.
Ann Neurol 2004;55:241–249
Early-onset ataxia with ocular motor apraxia and hypoalbuminemia (EAOH) is an autosomal recessive neurodegenerative disorder characterized by early-age onset, progressive ataxia, absence of tendon reflexes, distal
loss of sense of position and vibration, and pyramidal
weakness of the legs.1–5 Ocular motor apraxia is most
prominent in childhood and becomes less prominent
with advancing age, whereas hypoalbuminemia becomes prominent only in adulthood. Neuropathological studies have showed a severe loss of Purkinje cells,
degeneration of posterior columns and spinocerebellar
tracts of the spinal cord, and a marked loss of myelinated and unmyelinated fibers of peripheral nerves.6,7 Recently, we and Moreira and colleagues have identified
From the Departments of 1Neurology and 2Pathology, 3Molecular
Neuroscience, Brain Research Institute, Niigata University; 4Department of Neurology, Saigata Hospital, Niigata; 5Department of Neurology, Yamaguchi University, Yamaguchi; and 6Department of
Neurology, University of Tokyo, Tokyo, Japan.
Received Mar 19, 2003, and in revised form Sept 5. Accepted for
publication Sep 8, 2003.
the causative gene for EAOH, aprataxin gene (APTX).8,9
Two mRNA species of APTX, the “short-form” and
“long-form” APTX mRNAs,8 are produced as a result of
alternative splicing involving exon 3, leading to production of two protein isoforms. The long-form aprataxin is
a protein consisting of 342 amino acids harboring three
nuclear localizing signals (NLSs) with a calculated molecular weight of 39,100, containing a histidinetriad10,11 motif and a DNA-binding C2H2 zinc-finger
motif.12 In addition, the amino-terminal portion of the
long-form aprataxin has a homology with that of
polynucleotide kinase 3⬘-phosphatase (PNKP).13,14 Despite these motifs, the physiological functions of
aprataxin, however, remain unknown.
Address correspondence to Dr Tsuji, Department of Neurology,
University of Tokyo, 7-3-1, Hongou, Bunkyoku, Tokyo, Japan.
© 2004 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
To elucidate the functions of aprataxin and the
molecular mechanisms of EAOH caused by mutations
in APTX gene, we conducted detailed analyses on the
aprataxin mRNA and protein species in tissues including those of patients with EAOH and furthermore explored proteins that interact with aprataxin.
We herein report that the long-form aprataxin, the
major form of the APTX gene products, binds to
XRCC1 (x-ray repair cross-complementing group 1),
strongly suggesting that the long form of aprataxin is
involved in the repair of single-strand (ss) DNA break
and gradual accumulation of DNA damages as the
result of dysfunction of the long-form aprataxin underlying the pathophysiological mechanisms of
Materials and Methods
Quantitative Reverse Transcription Polymerase Chain
Reaction Analysis
First-strand cDNA was synthesized with 5␮g of RNA (Human Total RNA panel I; Clontech, Palo Alto, CA) using
Superscript II RNase H reverse transcriptase (RT; Invitrogen, La Jolla, CA) at 42°C for 60 minutes. Both the longand short-form APTX cDNAs were simultaneously amplified using primers located in exon 2 (fluorescein isothiocyanate [FITC] 5⬘- CAGTTGAAAGCAGAGTGTAA-3⬘)
and exon 3 (5⬘-CCTGATCTCTTTCTCTTCCT-3⬘).
Polymerase chain reaction (PCR) amplification was conducted with the following temperature cycle conditions; 30
seconds at 95°C, 30 seconds at 50°C, and 60 seconds at
72°C. PCR consisting of 26 cycles was selected based on
preliminary experiments for the quantitative analysis.
Quantification of the fluorescence-labeled PCR products
was accomplished using an ABI PRISM 3100 Genetic Analyzer and the Genescan software (version 3.7; Applied Biosystems, Foster City, CA). Using the long- and short-form
cDNAs mixed at varying ratios of 0 to 10, 1 to 9, 2 to 8,
3 to 7, 4 to 6, 5 to 5, and 6 to 4 in molar basis, a standard
curve was generated by plotting the molar percentage of the
cloned cDNAs as a function of the percentage of the area
of the peaks of the PCR products. Using the standard
curve, we determined the relative abundance of the longform APTX cDNA (and hence long-form APTX mRNA)
for each human tissue.
Production of Antiaprataxin Antibodies
Four peptides corresponding to long-form aprataxin (ME41,
ME43, CLKNKKHWNSFNTEY; ME44, CIPQLKEHLRKHWTQ) were synthesized and conjugated with bovine
thyroglobulin as a carrier protein. Rabbits were immunized
with the bovine thyroglobulin-conjugated peptides. The rabbit sera were affinity purified.
Immunoblot Analysis
Proteins were extracted from cells or tissues in the lysis
buffer containing 50mM Tris-HCl, pH 7.4, 5mM EDTA,
0.5% NP40, 0.5% sodium deoxycholate, 150mM NaCl
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and a protease inhibitor cocktail tablet (Roche). Tissues
were homogenized using a Potter-Elvehjem type homogenizer at 1,200rpm for 5 minutes at 4°C. The supernatant
after centrifugation (8,000g, 5 minutes) was subjected to
electrophoresis through a 10% polyacrylamide gel containing 0.1% sodium dodecyl sulfate (SDS) under a reducing
condition (50mM Tris-HCl, pH 6.8, and 6.0%
2-mercaptoethanol) followed by blotting onto a polyvinylidene difluoride membrane. The membrane was incubated
with a primary antibody (1:2,000) at 4°C for 18 hours followed by incubation with a secondary antibody (1:2,000)
at room temperature for 1 hour. The antibodies used in
this study were a goat polyclonal anti–actin antibody, goat
polyclonal anti–cytochrome b (mitochondrial marker), goat
polyclonal anti–hnRNP (A1; nuclear marker; all from Santa
Cruz Biotechnology).
Subcellular Fractionation
A control human cerebral cortex was homogenized as described above and subfractionated using NE-PER Nuclear
and Cytoplasmic Extraction Reagents (Pierce, Rockford, IL)
according to the manufacturer’s instructions (differential centrifugation method).
Plasmid Construction
To investigate subcellular localizations of the long- and
short-form aprataxins and the truncated proteins, we constructed plasmid DNAs using various forms of myc-tagged
aprataxin cDNAs prepared by PCR with the Human Brain
Marathon Ready cDNA library (Clontech) as templates.
The myc-tagged long-form aprataxin was amplified using
the primer pair 5⬘-GGG GAT CCG CCG CCA CCA
myc-tagged short-form aprataxin was amplified using the
primer pair 5⬘-GGG GAT CCG CCG CCA CCA TGC
TCC TGT GTC CAG TGC TTC CT-3⬘. To investigate
functional roles of nuclear localization signals of aprataxin,
we generated cDNAs with various lengths of deletions
(delta 1, delta 2, and delta 3) using 5⬘-GGG GAT CCG
CAC CTA TCA AAA AGG AAT-3⬘(delta 2), and 5⬘-GGG
TCA ATA C-3⬘ (delta 3) as forward primers, and 5⬘-GGC
common reverse primer. To achieve high levels of expression of cDNAs, we included Kosak’s consensus sequence in
the forward primers.
The PCR products were digested with BamHI and XhoI
and subcloned into the corresponding sites of the
pcDNA3.1(⫹) vector (Invitrogen). The nucleotide sequences
of all the cloned aprataxin cDNAs were confirmed by nucleotide sequence analysis.
Fig 1. Immunoblot analysis of aprataxin. Tissue
homogenates were subjected to SDS-PAGE (10%
polyacrylamide gel electrophresis) and analyzed
using the ME44 antibody. The molecular weight
markers are shown to the left. (A) Total protein
extracts (30␮g each) from the cerebral cortex of
normal individuals (lanes 1 and 2) and an
early-onset ataxia with ocular motor apraxia and
hypoalbuminemia (EAOH) patient (lane 3:
compound-heterozygous carrying 689insT/
840delT) was immunoblotted with the ME44
antibody (a). The same filter was reprobed with
the anti–actin antibody after stripping the anti–
aprataxin antibody (b). The ME44 antibody
detected an intense band with an estimated molecular mass of 39kDa (aprataxin[L]), 34kDa,
32kDa, 29kDa, and a faint band with approximately 20kDa (aprataxin[S]) in extracts from
the normal human cerebral cortex, whereas these
bands were barely detectable in the protein extract from the EAOH patient. Although the
short-form aprataxin of control 2 (lane 2) is not
detectable in this figure, faint bands corresponding to the short-form aprataxin were detectable
at longer exposure (data not shown). The ME44
antibody also detected a 60kDa cross-reactive
band in all the lanes. (B) Immunoblot of lymphoblast cell line extracts from a control individual (lane 1) and EAOH patients (lanes 2–4). In
the case of the compound heterozygote carrying
689insT and 840delT (Pt1), the 39kDa band
was absent, whereas faint 39kDa bands were
detected in extracts from the patient carrying
homozygous P206L (Pt2) and the patient carrying P206L and V263G (Pt3). The 20kDa band
was detected only in lane 1 (a). The same filter
was reprobed with the anti–actin antibody after
stripping the anti–aprataxin antibody (b). The
age at onset and the clinical severity of the
EAOH patients are shown along with the genotypes under the Western blot results (c).
Yeast Two-Hybrid Assay
COS7 cells were plated in two-well chamber slides at a density of 2.5 ⫻ 104 cells/cm2 in each well. The cells were transfected with 1␮g of each expression plasmid described above
using the PolyFect transfection reagent (Qiagen, Chatsworth,
CA) according to the manufacturer’s instructions. Twentyfour hours after transfection, the cells were washed with
phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde/PBS for 30 minutes, and permeabilized in 0.5% Triton X-100/PBS for 15 minutes at room temperature. The
cells were incubated in a blocking solution (PBS containing
5% goat serum and 0.02% Triton X-100) for 1 hour at
room temperature and then with the anti–c-myc monoclonal
antibody (Roche) diluted 1 to 500 in the blocking solution
for 1 hour at 37°C. The secondary antibody conjugated to
FITC (Molecular Probes, Eugene, OR) was used at a 1 to
2,000 dilution.
The MATCHMAKER GAL4 Two-Hybrid System 3
(Clontech) was used in this experiment. All the experiments
were performed using yeast strain AH109. We subcloned
DNA fragments encoding the full-length long- and shortform aprataxins (LAPTX and SAPTX, respectively) into the
pGBKT7 bait vector. The plasmid, pGBKT7-LAPTX, was
transformed into AH109 cells using the lithium acetate
method according to the supplier’s protocol. A single colony of AH109 cells harboring the pGBKT7-LAPTX was
grown overnight in SD-Trp medium and then transformed
with the MATCHMAKER GAL4 human fetal brain cDNA
library constructed in the pACT2 vector (Clontech). The
transformants were grown on selection plates without Trp,
Leu, and His. HIS⫹ clones were further selected on SDTry-Leu/X-a-Gal plates three times. The positive blue colonies on the selection plates were used in subsequent ex-
Sano et al: Aprataxin Is a DNA Nuclear Repair Protein
periments. Plasmid DNAs from positive clones were
isolated using the YEASTMAKER Yeast Plasmid Isolation
kit (Clontech). The nucleotide sequences of the plasmids
were determined using a 3100 DNA sequencer (ABI). The
pair of pGBKT7-p53 and pGADT7-T p53 and large T antigen was used as a positive control, and the pair of
pGBKT7-Lam and pGADT7-T human Lamin C and large
T antigen was used as a negative control (Clontech).
Coprecipitation Assay
HEK293 cells (1 ⫻ 107 cells) were transfected with either
c-myc–tagged long-form aprataxin or c-myc–tagged shortform aprataxin in pcDNA3.1(⫹) vector (Invitrogen) described in the method of plasmid construction. Whole-cell
extracts were prepared using 500␮l of IP buffer containing
25mM Hepes, pH 7.5, 50mM NaCl, 1% NP40, 10% glycerol 48 hours after transfection. After centrifugation
(10,000g, 10 minutes), 500␮l of the supernatants of the
cell extracts was incubated with 5␮l of anti–c-myc mouse
monoclonal antibody (1␮g/␮l; Roche) for 1 hour at 4°C
and then incubated with 1.5 ⫻ 108 beads of Dynabeads
M-450 (anti–mouse sheep IgG; Dynal, Fort Lee, NJ) for 2
hours at 4°C. The beads were pulled down with a magnet
and washed with the washing buffer (25mM Hepes, pH
7.5, 50mM NaCl, 0.1% NP40, and 10% glycerol). The
beads were dissolved in the Laemmeli sample buffer (BioRad, Richmond, CA) and subjected 0.1% SDS–polyacrylamide electorphoresis. Immunoblots were probed with either anti–XRCC1 (C-15) goat polyclonal antibody (Santa
Cruz Biotechnology; 1:100 dilution) or anti–c-myc mouse
monoclonal antibody (9E10; Roche) and developed using
enhanced chemiluminescence (Amersham, Arlington
Heights, IL).
The Long-Form Aprataxin Is the Major Form
The relative abundance of the long- and short-form
APTX mRNA species in various tissues were determined by quantitative RT-PCR. The relative abundance of long-form APTX mRNA were 58.3%, 75.0%,
61.6%, 79.7%, 73.1%, and 61.8% in the brain, heart,
lung, kidney, liver, and trachea, respectively. These results indicate that the long-form APTX is invariably the
dominant isoform among these tissues.
Of the antibodies raised against four synthetic peptides of aprataxin, the antibody against the C-terminal
fragment of aprataxin (ME44) strongly detected a protein with an estimated molecular mass of 39kDa in extracts from the normal human brain. Moreover, a faint
20kDa band presumably corresponding to the shortform aprataxin also was observed in the control extracts. In addition, faint 34, 32, and 29kDa bands also
were observed in the control extracts (Fig 1A, lanes 1
and 2). In homogenates of autopsied brain tissues of an
EAOH patient (compound heterozygote carrying
689insT and 840delT), these 39 , 34, 32, 29, and
20kDa proteins were absent (see Fig 1A, lane 3). In
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addition to these bands, ME44 detected a 60kDa band
which is presumably a cross-reactive protein.
Western blot analysis of lymphoblastoid cell lines
established from a normal control using ME44 also
showed the 39kDa band corresponding to the longform aprataxin (see Fig 1B, a, lane 1), which was absent in EAOH patients carrying compound heterozygous mutations of 689insT and 840delT (Patient 1;
see Fig 1B, a, lane 2). In contrast, the 39kDa band
was observed in the cases with missense mutations
(the homozygous mutation of P206L (Patient 2; see
Fig 1B, a, lane 3), and the compound heterozygous
mutations of P206L and V263G (Patient 3; Fig 1B,
a, lane 4)). Interestingly, Patients 2 and 3 showed
milder clinical presentations than that of Patient 1
(see Fig 1B, c).
Western blot analysis using ME44 demonstrated
that the 39kDa band was widely expressed among tissues (Fig 2A). The long-form aprataxin was more
strongly detected in the cerebral cortex and cerebellum
than the short-form one, confirming the results of RTPCR analyses of the long- and short-form APTX
mRNA species. The expression levels of aprataxin in
the posterior cortex, cerebellum, hippocampus, and olfactory bulb appeared to be higher than those in other
somatic tissues when analyzed by Western blot using
ME44 (see Fig 2B.).
Fig 2. Regional expressions of aprataxin. (A) The total protein
extracts (30␮g each) from various control tissues were subjected to SDS-PAGE (10% polyacrylamide gel electrophresis)
and immunoblotted with the ME44 antibody. Although the
short-form aprataxin is not detectable in this blot, longer exposure led to its detection in the cerebral cortex, cerebellum, and
heart (data not shown). (B) Regional distributions of
aprataxin in central nervous system of newborn mouse. In the
central nervous system, aprataxin is similarly widely distributed.
Fig 3. Immunocytochemical analysis of aprataxin-myc fusion protein expressed in COS-7 cells. (A) Structures of plasmid constructs.
(a) Full-length long-form aprataxin fused with c-myc tag (pcDNA3.1[⫹]-L-myc). (b) Full-length short-form aprataxin fused with
c-myc (pcDNA3.1[⫹]-S-myc). (c) Truncated aprataxin (amino acid residues 117–342) (aprataxin-delta 1) lacking the first NLS
(pcDNA3.1[⫹]-d1-myc). (d) Truncated aprataxin (amino acid residues 156 –342) (aprataxin-delta 2) lacking the first two NLSs
(pcDNA3.1[⫹]-d2-myc). (e) Truncated aprataxin (amino acid residues 277–342) (aprataxin-delta 3) lacking all the NLSs
(pcDNA3.1[⫹]-d3-myc). Aprataxin-delta 1– 3 also were fused with c-myc tag at the 3⬘ ends. (B) COS-7 cells expressing each
aprataxin-myc protein were stained with the FITC-conjugated anti–c-myc antibody (top). The cells were co-stained with DAPI to
visualize the nuclei (middle). The overlayed images are shown at the bottom. Cytoplasmic localization of all of these truncated
aprataxin molecules (aprataxin delta 1–3) suggests that the first NLS located near the amino terminus is essential in the localization of aprataxin in the nucleus.
Sano et al: Aprataxin Is a DNA Nuclear Repair Protein
Aprataxin Is a Nuclear Protein
Expression of myc-tagged long-form aprataxin was
observed exclusively in the nuclei, whereas cytoplasmic staining patterns were obtained in the cells expressing the myc-tagged short-form aprataxin (Fig
3B). To evaluate the effect of NLS in aprataxin, we
constructed three plasmids (delta 1, delta 2, and delta
3) coding for myc-tagged long-form aprataxin with
various lengths of deletions at the amino-terminal
portion (see Fig 3A). The COS7 cells expressing any
of these deletion mutants invariably exhibited cytoplasmic patterns (see Fig 3B), suggesting that the first
NLS of the long-form aprataxin is essential for its nuclear localization.
Nuclear localization of the long-form aprataxin
also was confirmed by Western blot analysis of
subcellular fractionations of the cerebral cortex
(Fig 4).
Aprataxin Interacts with XRCC1
To investigate the physiological functions of
aprataxin, we conducted a yeast two-hybrid screening
to search for aprataxin-interacting proteins. Using the
long-form aprataxin as the bait, we screened a human
fetal brain cDNA library. By screening approximately
7.0 ⫻ 104 Trp/Leu auxotrophic transformants, we
isolated five HIS⫹ colonies. These plasmids contained
Fig 4. Subcellular distribution of aprataxin. Proteins from
the normal cerebral cortex were separated into nuclear and
cytoplasmic fractions and subjected to Western blot using the
ME44 antibody. After deprobing, this membrane was reprobed with antibodies against the nuclear and mitochondrial marker proteins, hnRNP, and cytochrome b, respectively. In this procedure, mitochondrial proteins were
predominantly fractionated into the cytoplasmic fraction as
demonstrated using the anti–cytochrome b antibody.
Aprataxin is codistributed with the nuclear marker protein.
A faint band with an estimated molecular mass of approximately 20kDa corresponding to the short-form aprataxin also
was detected in the nuclear fraction.
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cDNAs for x-ray repair cross-complementing group 1
(XRCC1), sodium/potassium-transporting ATPase
beta-1, Jarkat T-cells VI, and the other two plasmids
for unknown cDNAs. Among these plasmids,
pACT2-fb4 coding for the C-terminal region (nucleotides 1473–1902 of the full-length cDNA [1–1902])
of XRCC1, is of particular interest, because XRCC1
has been shown to be involved in base excision repair,
a subtype of ssDNA break repair, associating with
PNKP,13,14 DNA polymerase ␤, and DNA ligase
To confirm the interaction between aprataxin and
XRCC1, five types of simultaneous transformation experiments were performed. In the case of the positive
control (p53 and large T antigen) and in the case of
aprataxin and XRCC1 (pACT2-fb4), the transformants
grew on SD-Trp-Leu-His and SD-Trp-Leu-His-Ade
selection plates. On the other hand, in the case of the
short-form aprataxin and fb4 and that of p53 and fb4,
the transformants did not grow on these selection
plates similarly to the case of the negative control (human Lamin C and large T antigen). These results
strongly indicate that the long-form aprataxin interacts
with the C-terminal domain of XRCC1 in the yeast
two-hybrid system (Table).
To further confirm the interaction of aprataxin with
XRCC1, we expressed myc-tagged long- and shortform aprataxin isoforms in HEK293 cells. The myctagged long- and short-form aprataxin isoforms in the
whole-cell extract were immunoprecipitated with anti–
myc antibody and then subjected to Western blot analysis using the anti–XRCC1 antibody. The interaction
between long-form aprataxin and XRCC1 was clearly
demonstrated, whereas short-form aprataxin did not
interact with XRCC1 (Fig 5). Taken together, these
results indicated that long-form aprataxin interacts
with XRCC1.
We demonstrated that the long-form aprataxin is localized in the nucleus, and, furthermore, that the first
NLS of the long-form aprataxin is essential for the
nuclear transport. Interestingly, we found that the
long-form aprataxin but not the short-form interacts
with the C-terminal domain of XRCC1, suggesting
that the amino-terminal region of the long-form
aprataxin that is absent in the short-form aprataxin is
essential in the interaction with XRCC1. Because the
amino terminus of the long-form aprataxin is homologous with that of PNKP,13,14 the results raise the
possibility that the PNKP-like domain of the longform aprataxin is involved in the interaction with
XRCC1. Our results also confirm the very recent report demonstrating the interaction between APTX
and XRCC1 by yeast two-hybrid system.15 Interestingly, human PNKP has been shown to interact with
Table. Interaction of XRCC1 and the Long-Form Aprataxin Demonstrated by Yeast Two-Hybrid System
Leu⫺, Trp⫺
Leu⫺, Trp⫺, His⫺
Leu⫺, Trp⫺, His⫺, Ade⫺
Positive Control
Negative Control
Short-form APTX
(C-terminal of
Long-form APTX
(C-terminal of
(C-terminal of
Simultaneous transformation experiences were performed and each the transformats were plated on Trp, Leu depleted plate (top), Trp, Leu, His
depleted plate (middle), or Trp, Leu, His, Ade depleted plate (bottom). The transformants cotransfected with pACT2-fb4 and pGBK7-LAPTX
grew on the Trp, Leu, His, Ade-depleted plate, indicating that the long-form aprataxin interacts with XRCC1 (⫹⫹, more than 50 colonies;
⫹, a few colonies; and ⫺; no colony on the selection plates).
the XRCC1 protein that also interacts with DNA
polymerase ␤ and DNA ligase III, and these four proteins constitute a multiprotein complex which is involved in base excision repair, a subtype of ssDNA
break repair.16 It also was described that XRCC1
stimulates both the DNA kinase and DNA phosphatase activities of PNKP at damaged termini and
thereby accelerates the overall repair reaction.16 These
results strongly support the possibility that aprataxin
and XRCC1 constitute a multiprotein complex and
are involved in ssDNA break repair.
On the other hand, aprataxin belongs to the
histidine-triad superfamily.8 –11 Recently, it was reported that the loss of the enzyme activity of yeast
HINT, Hnt1, leads to hypersensitivity to mutations in
Ccl, Tfb3, and Kin28, which constitute the TFII IIIK
Fig 5. Interaction of XRCC1 and the long-form aprataxin demonstrated by coprecipitation experiment. The myc-tagged long- and
short-form aprataxin in the whole-cell extract was immunoprecipitated with anti–myc antibody and then subjected to Western blot
analysis using the anti–XRCC1 antibody. IP ⫽ immunoprecipitation; IB ⫽ immunoblot.
Sano et al: Aprataxin Is a DNA Nuclear Repair Protein
kinase subcomplex of the general transcription factor
TFIIH.17 Because TFIIH is not only a general transcription factor but also a nucleotide excision repair
factor,18,19 it is suggested that Hnt1 is indirectly involved in nucleotide excision repair, a subtype of
ssDNA break repair. Taken together, aprataxin has two
motifs, PNKP and histidine-triad motifs, which potentially are involved in ssDNA break repair. The roles of
these two motifs in the ssDNA break repair should be
thoroughly investigated.
Notably, progressive neurological dysfunction including cerebellar ataxia is one of the hallmarks of
subtypes of xeroderma pigmentosum (XP), Cockayne
syndrome (CS), and tricothiodystrophy (TTD) associated with defects in nucleotide excision repair, another type of ssDNA repair,20 –24 and loss of Purkinje
cells also has been reported in some subtypes of XPA
and CS cases.25 Moreover, mice lacking both the XPA
and CSB genes exhibit ataxia and their cerebella show
morphological abnormalities.26 Thus, in these diseases
including EAOH, cerebellum may be highly vulnerable to abnormalities in the repair against ssDNA
The unique extraneurological manifestation of
EAOH is decreased serum albumin levels, which becomes evident in adulthood. If aprataxin is indeed involved in ssDNA repair, genes with unrepaired damage
would accumulate and the expression levels would
gradually decrease. As a result, albumin, which is one
of the most actively transcribed and translated proteins,
might be vulnerable to such damage. The results further raise the possibility that functional abnormalities
of aprataxin–XRCC1 complex also are involved in the
transcription-coupled repair of actively transcribed
genes in EOAH.
Although this study does not provide a direct evidence that aprataxin is involved in the process of
ssDNA break repair, these observations suggest that
common pathophysiological mechanisms underlie neurodegeneration, particularly cerebellar degeneration, as
a result of impairment in DNA repair systems. The
elucidation of the physiological function of aprataxin
should lead to a better understanding of the pathophysiological mechanisms of these neurodegenerative
This study was supported by a Grant-in-Aid for Scientific Research
on Priority Areas (C)–Advanced Brain Science Project and a grantin-aid from the Ministry of Education, Culture, Sports, and Science
and Technology, Japan; grants from the Research for the Future
Program from the Japan Society for the Promotion of Science; for
the Surveys and Research on Specific Diseases; the Ministry of
Health, Labor and Welfare, Japan; Nippon Boeringer Ingelheim
Annals of Neurology
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February 2004
(S.T.); the Takeda Science Foundation; and the Suzuken Memorial
Foundation (S.T.).
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Sano et al: Aprataxin Is a DNA Nuclear Repair Protein
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potential, eaoh, nuclear, aprataxin, causative, protein, dna, role, repair
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