Ataxin-3issubjecttoautolytic cleavage
Pier Luigi Mauri
1
, Matteo Riva
2
, Daniela Ambu
1
, Antonella De Palma
1
, Francesco Secundo
3
,
Louise Benazzi
1
, Marco Valtorta
2
, Paolo Tortora
2,
* and Paola Fusi
2,
*
1 Istituto di Tecnologie Biomediche, CNR, Milano, Italy
2 Dipartimento di Biotecnologie e Bioscienze, Universita
`
di Milano-Bicocca, Milano, Italy
3 Istituto di Biocatalisi e Riconoscimento molecolare del CNR, Milano, Italy
Spinocerebellar ataxia type 3, also known as Mach-
ado–Joseph disease, is one of the nine polyglutamine
(polyQ) disorders described to date [1]. They share a
number of clinical features, such as late onset and anti-
cipation, yet each affects a different area of the brain
[1]. Each polyQ disorder is associated with a protein
harboring, in its pathologic form, an uninterrupted
stretch of polyQ, with a length in excess of a typical
threshold [2]. Misfolding of these polyQ-carrying pro-
teins has long been known to be associated with the
formation of insoluble inclusions [3]. Although such
aggregates are a hallmark of neurodegenerative dis-
orders, their precise role in the pathologic process
remains elusive [4–7]. The polyQ stretch is responsible
for protein misfolding, which in turn leads to the gain
of a new toxic function, resulting in cell death. This
gain of function may account for all the similarities
among polyQ diseases [3]. However, it was recently
suggested that a loss of function, due to the impair-
ment of wild-type protein function caused by the mis-
folding and aggregation, could also play a role in the
pathogenesis of Huntington disease [8,9]. This led to
many studies to define the physiologic and biochemical
properties of the nonexpanded proteins.
Ataxin-3 (AT-3) is a 42 kDa protein that is respon-
sible for spinocerebellar ataxia type 3. In its normal
Keywords
ataxin-3; mass spectrometry; polyglutamine
diseases; proteolysis
Correspondence
P. Tortora, Dipartimento di Biotecnologie e
Bioscienze, Universita
`
di Milano-Bicocca,
Piazza della Scienza 2, I-20126 Milano, Italy
Fax: +39 02 6448 3565
Tel: +39 02 6448 3401
E-mail: paolo.tortora@unimib.it
*These authors contributed equally to this
work
(Received 21 February 2006, revised 15 July
2006, accepted 20 July 2006)
doi:10.1111/j.1742-4658.2006.05419.x
The protein ataxin-3is responsible for spinocerebellar ataxia type 3, a neu-
rodegenerative disease triggered when the length of a stretch of consecutive
glutamines exceeds a critical threshold. Different physiologic roles have
been suggested for this protein. More specifically, recent papers have
shown that the highly conserved N-terminal Josephin domain of ataxin-3
binds ubiquitin and has ubiquitin hydrolase activity, thanks to a catalytic
device specific to cysteine proteases. This article shows that the protein also
has autoproteolytic activity, sustained by the same residues responsible for
the ubiquitin hydrolase activity. The autolytic activity was abolished when
these residues, i.e. Cys14 and His119, were replaced by noncatalytic ones.
Furthermore, we found that pretreatment of the protein with tosyl l-phe-
nylalanine chloromethyl ketone also abolished this activity, and that this
site-specific reagent covalently bound His119, findings supported by MS
experiments. MS also allowed us to establish that the attack was aspecific,
as cleavage sites were observed at the carboxyl side of apolar, acidic and
polar uncharged residues, clustered in the C-terminal, unstructured domain
of the protein. In contrast, the Josephin domain was preserved from attack.
We propose that the autolytic activity reported here may play a role in
pathogenesis, as fragments carrying expanded polyglutamines are thought
to be significantly more toxic than the whole protein.
Abbreviations
AT-3, ataxin-3; AT-3-Q6, murine ataxin-3 carrying six consecutive glutamines; AT-3-Q26, human ataxin-3 carrying 26 consecutive glutamines;
EIC, extracted ion chromatogram; polyQ, polyglutamine; GST, glutathione-S-transferase; LC-MS, liquid chromatography coupled to mass
spectrometry; LC-MS ⁄ MS, liquid chromatography coupled to tandem mass spectrometry; pCMBS, p-chloro-mercuribenzoate; TLCK, tosyl-
L-lysine chloromethyl ketone; TPCK, tosyl-L-phenylalanine chloromethyl ketone.
FEBS Journal 273 (2006) 4277–4286 ª 2006 The Authors Journal compilation ª 2006 FEBS 4277
form, it contains 12–36 glutamines; however, the
length of the polyQ in its pathologic variant is in the
range 55–84. Its primary structure shows the presence
of a conserved N-terminal region, the so-called Jose-
phin domain, and of a less conserved C-terminus con-
taining the polyQ stretch [10–13].
The increasing interest in the ‘loss of function’
mechanism of pathogenesis has led to many hypothe-
ses about the physiologic role of AT-3. Many data
suggest that transcriptional repression might be the
mechanism for polyQ disorder pathology [14]. On the
other hand, a growing number of recent reports sug-
gest a role of AT-3 in protein degradation [15,16].
These data are supported by the fact that AT-3 has
either two or three potential ubiquitin-interacting
motifs, depending on the splice variant [17]. Donald-
son et al. [18] also reported that AT-3 is a ubiquitin-
binding protein, its functional ubiquitin-binding motifs
being required for protein localization into aggregates.
In keeping with these observations, through surface
plasmon resonance binding analysis, Chai et al. [19]
showed that AT-3 is a polyubiquitin-binding protein.
Based on a bioinformatics approach, Scheel et al. [20]
also recently discovered that the highly conserved Jose-
phin domain of AT-3 possesses the predicted catalytic
triad of amino acids found in cysteine proteases, puta-
tively consisting of Cys14, His119 and Asn134. This led
them to predict that AT-3 can remove ubiquitin from
polyubiquitinated proteins. Actually, its putative cata-
lytic site is similar to that found in ubiquitin C-terminal
hydrolase (family 1)-type and ubiquitin C-terminal
esterase (ubiquitin thiolesterase)-type ubiquitin proteas-
es. This hypothesis was confirmed by replacing the pre-
dicted catalytic cysteine (Cys14) by alanine [21], which
abolished ubiquitin hydrolase activity towards both the
polyubiquitylated
131
I-labeled lysozyme and the fluoro-
genic substrate ubiquitin-7-amido-4-methylcoumarin.
Although the above-mentioned investigations
assigned a ubiquitin hydrolase activity to AT-3, we
consistently observed that normal variants of AT-3
underwent slow proteolytic fragmentation when incu-
bated at room temperature or even at 4 °C. This led
us to check whether AT-3 degradation isautolytic and
sustained by the same residues responsible for ubiqu-
itin hydrolase activity. For this reason, we replaced the
putative catalytic residues by noncatalytic ones, and
this abolished the autolytic activity. Although the
physiologic significance of this finding has still to be
defined, this property may be involved in events lead-
ing to pathogenesis, because fragments carrying polyQ
stretches are suspected to be more toxic than the whole
protein [22].
Results
AT-3 carrying six consecutive glutamines
(AT-3-Q6) undergoes fragmentation upon
incubation at room temperature
In previous experiments, we consistently observed that
both murine AT-3-Q6 and human AT-3-Q26 under-
went slow proteolytic fragmentation when incubated at
room temperature for several hours (data not shown).
A typical degradation pattern of wild-type murine
AT-3-Q6 at 24 °C is shown in Fig. 1 (lanes referred to
as wt).
HL
9
11DMHL911
D
MH L9
1
1WT
M
DT
W
CA41T
W
CA
4
1CA41
h
0h
4
2h
8
4h
4
2
h8
4h0
M
5
4
-
66 -
03 -
4
.
7
9
-
5.12 -
3.
41 -
022 -
a
b
c
Fig. 1. SDS ⁄ PAGE (12% gel) of murine ataxin-3 carrying six consecutive glutamines (AT-3-Q6) and its mutants after incubation at 24 °C.
After incubation for the indicated times, 6 lg samples of wild-type and mutated proteins were subjected to electrophoresis and revealed by
Gel Code staining. DM, double mutant. Lane M shows marker proteins with their relative molecular masses (· 10
)3
). Bands referred to as
‘a’, ‘b’ and ‘c’ were subjected to in-gel tryptic digestion, and the digestion mixtures analyzed by LC-MS ⁄ MS, which led to their identification
as N-terminal fragments covering the regions 1–346, 1–250 and 1–241, respectively. For other details, see Experimental procedures.
Autolytic cleavage of ataxin-3 P. L. Mauri et al.
4278 FEBS Journal 273 (2006) 4277–4286 ª 2006 The Authors Journal compilation ª 2006 FEBS
C14A, H119L and C14A/H119L mutants of
AT-3-Q6 do not undergo proteolytic fragmentation
on incubation at room temperature
To check whether AT-3-Q6 degradation is autolytic
and sustained by the same residues responsible for
ubiquitin hydrolase activity, we replaced two putatively
catalytic residues, i.e. Cys14 and His119, by noncata-
lytic ones, producing the single mutants C14A and
H119L and the double mutant C14A ⁄ H119L. The
mutants were incubated at 24 °C and subjected to
SDS ⁄ PAGE. Figure 1 shows that only faint lower
molecular weight bands were detected after incubating
C14A for 24 and 48 h, whereas no proteolytic frag-
mentation occurred during incubation of both H119L
and C14A ⁄ H119L. Far UV-CD spectra of AT-3-Q6
and the mutants collected at 24 °C showed that the
two mutations did not significantly affect the overall
secondary structure of the protein (Fig. 2). Thus, our
results demonstrate a direct involvement of Cys14 and
His119 in the autolytic activity.
Tosyl-
L-phenylalanine chloromethyl ketone
(TPCK) inhibits AT-3-Q6 fragmentation
Several well-known protease inhibitors, such as
phenylmethanesulfonyl fluoride, TPCK, tosyl-l-lysine
chloromethyl ketone (TLCK), EDTA, HgCl
2
and
p-chloro-mecuribenzoate (pCMBS), were assayed for
their ability to prevent the autolytic activity of AT-3-
Q6. Except for TPCK, none of them was effective
(data not shown). In contrast, TPCK prevented the
appearance of proteolytic fragments, as shown by
SDS ⁄ PAGE (Fig. 3).
To check whether the pattern of covalent modifica-
tions of AT-3-Q6 effected by TPCK was consistent
with the catalytic role assigned to His119, bands of
AT-3-Q6 pretreated with TPCK were excised from the
gel and digested with trypsin. Then, the resulting
mixtures were analyzed by LC-MS ⁄ MS. This made it
possible to identify 81% of the AT-3-Q6 sequence.
Moreover, it was shown by LC-MS ⁄ MS analysis that
His17, His119 and His198, located in the tryptic
peptides T
17)45
,T
111)124
and T
196)206
, respectively,
were modified by TPCK. In contrast, the other histi-
dines (His6, His38, His187) were not (Table 1). As a
control, the same analysis performed on AT-3-Q6 not
subjected to TPCK preincubation did not show any
modification of the residue His119 (data not shown).
As a representative profile, the MS ⁄ MS spectrum of
the TPCK-modified peptide T
111)124
is shown in
Fig. 4.
Characterization of the large-sized autolytic
fragments of AT-3-Q6
To characterize the fragments resulting from autolytic
cleavage of AT-3-Q6, the protein was incubated for
48 h at 24 °C and subjected to SDS ⁄ PAGE. Three
Fig. 2. Far UV-CD spectra of murine ataxin-3 carrying six consecu-
tive glutamines (AT-3-Q6) and the mutants C14A, H119L and
C14A ⁄ H119L. Protein samples (0.1 mgÆmL
)1
) were dissolved in
50 m
M Tris ⁄ HCl, pH 7.0. The optical pathlength was 0.1 cm.
- TPCK
M - TPCK + TPCK + TPCK
0 h 48 h
220 -
97.4 -
66 -
45 -
30 -
21.5 -
14.3 -
Fig. 3. SDS ⁄ PAGE (12% gel) of murine ataxin-3 carrying six con-
secutive glutamines (AT-3-Q6) incubated at 24 °C in the presence or
the absence of 1 m
M tosyl-L-phenylalanine chloromethyl ketone
(TPCK). AT-3 was incubated for the indicated times. Six-microgram
samples were then subjected to electrophoresis and revealed by Gel
Code staining. Lane M shows marker proteins with relative molecu-
lar masses (· 10
)3
). For other details, see Experimental procedures.
P. L. Mauri et al. Autolyticcleavage of ataxin-3
FEBS Journal 273 (2006) 4277–4286 ª 2006 The Authors Journal compilation ª 2006 FEBS 4279
major bands, referred to as ‘a’, ‘b’ and ‘c’, were detec-
ted (Fig. 1). They were subsequently subjected to in-gel
tryptic digestion, and the digestion mixtures analyzed
by LC-MS ⁄ MS. The identified tracts of the sequences
represent 68%, 51% and 34.2% of the total, respect-
ively. Also, the bands were identified as N-terminal
fragments covering the regions 1–346, 1–250 and 1–
241, respectively. Edman degradation of the fragments
also confirmed that they carried the expected N-ter-
minal sequence resulting from the cleavage of the
glutathione-S-transferase (GST)–AT-3 fusion protein
(data not shown).
On the assumption that cleavage sites autolytically
attacked by AT-3-Q6 were different from those
attacked by tryptic digestion, we searched for nontryp-
tic peptides in the in-gel tryptic digestion mixtures of
bands ‘a’, ‘b’ and ‘c’. For this reason, the ‘no-enzyme
mode’ of the bioworks software was used, and con-
firmed the identification of the C-terminal site D
241
.
Surprisingly, we also identified a small number of pep-
tides resulting from nontryptic digestion, distant from
the putative C-termini of the three fragments under
investigation. Non-canonical cleavage sites on protein
incubation with trypsin have been already reported,
although it is unclear whether this phenomenon is
accounted for by a side activity of the protease or by
Fig. 4. Fragmentation spectrum (MS ⁄ MS) of
the peptide T
111)124
. Peptide identification
was achieved by means of
SEQUEST data
handling. The peptide contains tosyl-
L-phe-
nylalanine chloromethyl ketone (TPCK)-modi-
fied His119. In the inserted table, the
calculated masses of B and Y ion fragments
are shown, and the matched fragments
from the raw spectrum are shown in bold in
the table. H* indicates modified His119.
Table 1. List of the histidine-containing tryptic fragments from
murine ataxin-3 carrying six consecutive glutamines (AT-3-Q6). His-
tidines are indicated in bold. Histidines modified by tosyl-
L-phenylal-
anine chloromethyl ketone (TPCK) are marked with an asterisk.
Amino acid numbering starts from the N-terminal methionine of
authentic AT-3-Q6. PreScission protease cleavage of the GST–AT-3
fusion protein releases an AT-3 form carrying five residues (GPLGS)
upstream of the authentic N-terminus.
Peptide fragments Sequence
T
1)8
(GPLGS)MESIFHEK
T
17)45
H*CLNNLLQGEYFSPVELSSIAHQLDEEER
T
35)45
SIAHQLDEEER
T
111)124
SFICNYKEH*WFTVR
T
196)206
LAH*LKEQSALK
T
183)200
VQQMHRPKLIGEELAHLK
Autolytic cleavage of ataxin-3 P. L. Mauri et al.
4280 FEBS Journal 273 (2006) 4277–4286 ª 2006 The Authors Journal compilation ª 2006 FEBS
additional, contaminating proteolytic activities [23].
Whatever the reason for this observation, it cast no
doubt on the identification of the three large-sized
fragments described here.
Characterization of small-sized autolytic
fragments of AT-3-Q6
As shown above, the largest fragments detected in
SDS ⁄ PAGE cover the N-terminal region of AT-3-Q6.
In order to characterize small-sized polypeptides,
which are not retained by the electrophoretic gel, we
preincubated preparations of AT-3-Q6 at 24 °C for
48 h and then subjected them to LC-MS analysis with
no preliminary tryptic digestion. Polypeptides identi-
fied on the basis of their molecular masses cover the
C-terminal region only, with sizes in the range 1.7–
9.6 kDa (Table 2). In contrast, no peptide was detected
when the mutant H119L was subjected to the same
analysis after a 48 h preincubation at 24 °C (data not
shown). As a representative profile, Fig. 5 shows the
multicharge mass spectrum of peptide 306–355 and the
extracted ion chromatograms (EICs) of ion m ⁄ z 784.5
([M + H
+
]
7+
) in wild-type and C14A mutant sam-
ples. Similar results were obtained for the other pep-
tides listed in Table 2 (data not shown). These findings
also confirm that the observed polypeptides actually
result from autolytic activity, and are released by the
wild-type protein only, and, on the whole, point to a
cleavage pattern involving several sites of autolytic
attack.
Discussion
In recent years, the idea has become widely accepted
that many neurodegenerative diseases result not only
from the gain of a toxic function of the proteins
involved, but also from the loss of their physiologic
function [8,9]. This, in turn, led to several investiga-
tions aimed at clarifying their physiologic role(s).
Different hypotheses have recently been put forward
regarding the function of AT-3. Initially, it was sugges-
ted that it might play a role in transcriptional regula-
tion [14]. However, a growing body of data also points
Table 2. Peptides identified by LC-MS of murine ataxin-3 carrying
six consecutive glutamines (AT-3-Q6) preparations preincubated for
48 h at 24 °C. For the identification of the peptides, a tool of
BIO-
WORK
software was used.
Detected molecular mass Putative AT-3-Q6 region
1731.4 341–355
5484.6 306–355
7414.4 192–257
7565.9 239–302
9553.7 257–341
9555.9 272–355
Fig. 5. Multicharge mass spectrum of the fragmentation peptide T
306)355
obtained by preincubation of wild-type ataxin-3 (AT-3) at 24 °C for
48 h (lower panel). The corresponding extracted ion chromatograms (EICs) of ion m ⁄ z 784.5 ([M + H
+
]
7+
) are shown in the upper panel,
along with the C14A mutant sample obtained under the same conditions.
P. L. Mauri et al. Autolyticcleavage of ataxin-3
FEBS Journal 273 (2006) 4277–4286 ª 2006 The Authors Journal compilation ª 2006 FEBS 4281
to its possible involvement in proteasome-mediated
protein degradation. Actually, AT-3 was shown to
bind both ubiquitin, through the two ubiquitin-inter-
acting motifs located in its sequence [18], and a num-
ber of proteins involved in protein degradation, such
as VCP, Rad23 and E4 [24–26]. Furthermore, a recent
bioinformatic study suggested that the ubiquitin hy-
drolase activity of AT-3 might be sustained by a cata-
lytic triad consisting of Cys14, His119 and Asn134
[20]. An ensuing paper provided experimental evidence
in support of this hypothesis, in that the mutagenesis
of Cys14 abolished ubiquitin hydrolase activity [21].
We consistently observed that pure AT-3 undergoes
proteolysis when incubated at room temperature for
several hours, and this prompted us to check whether
this phenomenon is supported by autolytic cleavage.
Based on the hypothesis that the autolytic and ubiqu-
itin hydrolase activities are effected by the same resi-
dues, we replaced Cys14 and His119 by alanine and
leucine, respectively, and this prevented the observed
fragmentation. We did not mutagenize residue Asn134,
as one cannot expect clearcut results from such
replacement. This is because the replacement of the
putatively catalytic asparagine by alanine may reduce
but does not necessarily abolish enzyme activity, as
observed in the case of papain and other cysteine pep-
tidases [27].
The occurrence of faint, lower molecular weight
bands, even in the electropherograms of the mutated
AT-3 forms (Fig. 1), might suggest that the observed
fragmentation is at least in part accounted for by con-
taminating proteolytic activities. However, this can be
definitely ruled out on the basis of the following consid-
erations: (a) no time-dependent disappearance of the
full-length protein and concomitant accumulation of
degradation products was detectable during the incuba-
tion of the mutants, as observed in the case of the wild
type; (b) although a faint band with size comparable to
that of fragment ‘c’ was apparent even in the electro-
pherograms of the mutated proteins, additional smaller
fragments (in the molecular mass range of about 14–
25 kDa) were consistently detected only in the case of
the wild-type protein (Fig. 1); (c) whereas bands ‘a’, ‘b’
and ‘c’ could be identified as fragments of wild-type AT-
3 by in-gel tryptic digestion and LC-MS ⁄ MS analysis,
similar analyses run in parallel on the mutated forms
did not reveal any such fragment.
To further substantiate our conclusions, we used the
Escherichia coli protein database (downloaded from
the NCBI website) for data handling of mass spectra
obtained from wild-type and C14A LC-MS ⁄ MS analy-
ses: this allowed us to definitely rule out the presence
of any contaminating protease from the microorganism
in our samples. Tiny amounts of other contaminants
were found instead, including the Hsp70 chaperone
protein (data not shown).
Our experiments also showed that the autolytic frag-
mentation is inhibited by TPCK, and that this site-
specific reagent covalently modifies His17, His119 and
His198, as supported by MS data. Although we have
no obvious explanation for the reactivity of TPCK
toward the residues His17 and His198, its ability to
covalently bind His119 and at the same time to abolish
the self-degradation of the protein is consistent with
the above-mentioned hypothesis.
The location of the sites of autolyticcleavage (sum-
marized in Fig. 6) makes it immediately apparent that
no defined specificity can be assigned to this activity,
and that these sites are clustered in the C-terminal
domain of AT-3, the closest to the N-terminus being
next to Leu191. Thus, the Josephin domain, which
approximately spans residues 1–182 and is reported as
the only one structured in AT-3 [11,12], is completely
preserved from proteolytic attack. Our analysis may
well have failed to identify all of the cleavage sites: in
particular, we observed slightly different cleavage pat-
terns in different experiments. However, this does not
contradict the above-mentioned conclusions, as proteo-
lytic fragmentation was never observed outside the
C-terminal, unstructured domain. As the identified
Fig. 6. Location of the autolytic cleavage
sites of murine ataxin-3 carrying six con-
secutive glutamines (AT-3-Q6). Arrows indi-
cate the identified cleavage sites. The
Josephin domain is shaded.
Autolytic cleavage of ataxin-3 P. L. Mauri et al.
4282 FEBS Journal 273 (2006) 4277–4286 ª 2006 The Authors Journal compilation ª 2006 FEBS
sites of cleavage are at the carboxyl side of apolar, aci-
dic and polar uncharged residues, it seems unlikely
that cleavage also occurs next to basic residues, in
keeping with the lack of any inhibitory effect by
TLCK. Furthermore, as only one site close to an aci-
dic residue was identified (i.e. Asp241), we conclude
that AT-3 activity is not caspase-like.
The ability of AT-3 to undergo autolytic degradation
might be related to the mechanisms of pathogenesis, as
proteolysis is widely thought to play a role in the cas-
cade of events that eventually lead to the onset of many
neurodegenerative diseases [28]. In particular, fragments
containing polyQ stretches have been reported to be
more toxic than the whole proteins [29–34], although
their proteolytic fragmentation is generally ascribed to
caspases. In vivo studies also showed that expanded
AT-3 undergoes proteolysis, with the resulting formation
of a cytotoxic, polyQ-containing fragment [35]. Possibly,
autolysis might play an additional role in releasing
polyQ-containing, pathogenic fragments in vivo.
At this stage, it cannot be established whether the
observed fragmentation is involved in physiologic pro-
cesses such as protein turnover or nuclear localization.
It should be pointed out, however, that a recent paper
presenting the Josephin architecture of AT-3 solved by
NMR spectroscopy showed that it bears a clear simi-
larity to members of the papain-like cysteine proteases
[36]. This finding nicely confirms our results, and fur-
ther supports the idea that the proteolytic activity that
we assigned to AT-3 may play a still unidentified
physiologic role.
Finally, the fact that AT-3 issubjectto autolysis
also has methodologic relevance. Actually, this prop-
erty, along with the occurrence of the unstructured
C-terminal domain, explains why attempts to crystal-
lize the protein have been unsuccessful so far. We
expect that this goal may be achieved when the isola-
ted Josephin domain is subjected to crystallization tri-
als, where Cys14 and ⁄ or His119 have been replaced by
noncatalytic residues.
Experimental procedures
Gene cloning and mutagenesis
Murine AT-3-Q6 was cloned into plasmid pGEX6P-1, from
its cDNA identified in the EST database (dbEST), as previ-
ously reported [37]. The recombinant protein was then
expressed as a GST fusion protein in the E. coli Codon
Plus-RIL strain, and retrieved by means of a PreScission
Protease (Amersham Biosciences UK Ltd, Little Chalfont,
UK) cleavage site located in-between the two coding
sequences. Murine AT-3-Q6 mutagenesis to produce C14A,
H119L and C14AH119L was carried out with the Quick
Change Mutagenesis Kit (Stratagene, La Jolla, CA).
According to the manufacturer’s instructions, specific prim-
ers carrying the mutated codon were used to produce and
amplify the mutated plasmid by means of PCR. Parental
DNA was then digested with DpnI endonuclease for 1 h at
37 °C, and E. coli XL-Blue cells were transfected with
recombinant plasmids (1 lL of the reaction mixture was
used to transfect 50 lL of supercompetent XL-Blue cells).
Protein purification
All proteins were expressed in E. coli strain BL21 Codon
Plus RIL as GST fusion proteins containing a PreScission
Protease recognition site. Cells were grown at 37 °Cin
LB ⁄ ampicillin medium and induced with 50 lm isopropyl
thio-b-d-galactoside (IPTG) at A
600
¼ 1.0 for 2 h. To
obtain crude extracts, cells were frozen and thawed, incuba-
ted for 1 h at 4 ° C in 100 mL of lysis buffer (10 mm
sodium phosphate, pH 7.2, 150 mm NaCl, 1 mm phenyl-
methanesulfonyl fluoride, 10 mm dithiothreitol, 100 mm
MgCl
2
, 0.5 mg mL
)1
lysozyme), and again frozen and
thawed. Triton X-100 (1%) and DNase [0.2 mgÆ(g cells)
)1
]
were then added, and the samples were further incubated
for 30 min at room temperature. Finally, they were centri-
fuged for 30 min at 47 000 g in an Avanti J-20 centrifuge
with a JA20 rotor (Beckman Coulter, Fullerton, CA). The
supernatants were incubated in batches with Glutathione
Sepharose 4B affinity resin [0.1 mLÆ(mL crude extract)
)1
]
for 45 min at room temperature. The resin was subse-
quently packed into a column and washed with three vol-
umes of 10 mm sodium phosphate (pH 7.2) and 150 mm
NaCl, and then three volumes of cleavage buffer (50 mm
Tris ⁄ HCl, pH 7.0, 150 mm NaCl, 1mm EDTA, 1 mm di-
thiothreitol). Removal of the GST affinity tail was achieved
by incubating the resin-bound proteins overnight at 4 °Cin
the presence of PreScission Protease [400 UÆ(mL resin)
)1
].
Mature AT-3-Q6 proteins were then eluted with cleavage
buffer, while PreScission Protease, a GST fusion protein,
remained bound to the resin. PreScission protease cleavage
of GST–AT-3 fusion protein released an AT-3 form carry-
ing five residues (GPLGS) upstream from the authentic
N-terminus. Amino acid numbering, where presented, starts
from the N-terminal methionine of authentic AT-3-Q6.
Incubation of AT-3-Q6 and its mutants
To monitor the appearance of proteolytic fragments, wild-
type AT-3-Q6, as well as C14A, H119L and C14AH119L
mutants, was incubated at 24 °C for different times in the
presence of 50 mm Tris ⁄ HCl, pH 7.0, 150 mm NaCl, 1 mm
EDTA, and 1 mm dithiothreitol (cleavage buffer). Each
protein had a final concentration in the mixture of about
0.4 mgÆmL
)1
. Fifteen-microliter samples were taken at dif-
ferent times and subjected to SDS ⁄ PAGE.
P. L. Mauri et al. Autolyticcleavage of ataxin-3
FEBS Journal 273 (2006) 4277–4286 ª 2006 The Authors Journal compilation ª 2006 FEBS 4283
SDS
⁄
PAGE and western blotting
SDS ⁄ PAGE was carried out according to Laemmli [38], in
a Mighty Small apparatus (Hoefer Scientific Instruments,
San Francisco, CA) using a 12% running gel and a 4%
stacking gel. Proteins were revealed by Gel Code staining
(Pierce Biotechnology, Rockford, IL).
CD spectroscopy
All far-UV CD spectra were collected at 25 °C using a
Jasco-600 spectrophotometer (Jasco, Tokyo, Japan), and a
cuvette with 0.1-cm pathlength. All experiments were per-
formed in 50 mm Tris ⁄ HCl, pH 7.0, at a protein concen-
tration of 0.1 mgÆmL
)1
. The spectra were registered from
198 to 250 nm, and ran at a scan speed of 10 nmÆmin
)1
,
with a time response of 4 s and a data pitch of 0.2 nm.
All the spectra were baseline-corrected and smoothed. The
molar mean residue ellipticity [h] was expressed in degree-
sÆcm
2
Ædmol
)1
, and calculated as
½h¼h
obs
MWR=ð10lcÞ
where h
obs
is the observed ellipticity in degrees, MWR the
mean residue molar weight of the protein (121.1 mgÆ
mmol
)1
), l the optical path length in centimeters, and c the
protein concentration in grams per milliliter.
Tryptic fragmentation of AT-3-Q6 in solution
Sequence-grade modified trypsin (Promega, Madison, WI)
was added to 50 lL of conditioned medium containing
4 lg of protein at a 1 : 50 enzyme ⁄ protein ratio (wt ⁄ wt) in
100 mm ammonium bicarbonate, pH 8.9, and incubated at
37 °C. Following an overnight incubation, the pH was
adjusted to 2.0 by adding trifluoroacetic acid to stop the
reaction. Ten microliters of the peptide mixture diluted
1 : 10 were subjected to LC ⁄ MS ⁄ MS.
Trypsin digestion of AT-3-Q6 in the
electrophoretic gel
Protein bands were excised from the gel, cut into small
fragments ( 1 · 1 mm) using a scalpel, and placed in a
1.5 mL siliconized tube. After dehydration in 100% meth-
anol for 5 min at room temperature, fragments were
rehydrated in 30% methanol for 5 min, and washed twice
in ultrapure water for 10 min. The gel bands were
washed three times for 10 min with 100 mm ammonium
bicarbonate, pH 7.9, containing 30% acetonitrile, and fur-
ther washed in ultrapure water. Gel fragments were thor-
oughly dried and resuspended in 50 mm ammonium
bicarbonate. Then, trypsin digestion was performed as
previously described [39]. Trypsin was added at an
enzyme ⁄ substrate ratio of 1 : 30 (wt ⁄ wt) in 100 mm
ammonium bicarbonate, pH 7.9, and incubated over-
night at 37 °C. The peptides were extracted from the gel
at room temperature with 50% acetonitrile containing
0.1% trifluoroacetic acid. Aliquots containing 10 lLof
sample were injected into the LC-MS ⁄ MS mass spectro-
meter.
LC conditions for LC-MS analysis
For the analysis of enzymatic digests, a Phoenix 40
HPLC (Thermo Electron Corp., Milan, Italy) equipped
with 7725i Rheodyne injector was coupled to an LCQ-
Deca ion trap mass spectrometer by an electrospray inter-
face. A Nucleosil C
18
column (0.5 · 150 mm, 5 lm;
Waters, Milford, MA) with an acetonitrile gradient was
used (eluent A, 0.1% formic acid in water; eluent B,
0.1% formic acid in acetonitrile). The flow rate was
25 lLÆmin
)1
. The gradient profile was 10% B for 3 min,
followed by 10–80% B for 60 min. For the analysis of
intact AT-3-Q6 and polypeptides resulting from autolysis,
aC
8
column (150 · 1 mm, 5 lm; Luna, Phenomenex,
Torrance, CA) was used with a flow rate of 20 lLÆmin
)1
.
The gradient profile was 10% B for 3 min, followed by
10–80% B for 40 min.
MS conditions
The heated capillary was held at 260 °C at a voltage of
30 V. The spray voltage was 4.5 kV. For peptide analysis,
spectra were acquired in automated MS ⁄ MS mode: each
MS full scan (in the range 300–1600 m ⁄ z) was followed by
three MS ⁄ MS scans of the most abundant ions, using a rel-
ative collision energy of 35%. For protein and polypeptide
analysis, MS full scan (in the range of 400–1800 m ⁄ z)in
positive mode was used.
Data handling of MS results
Computer analysis of peptide MS ⁄ MS spectra was per-
formed using Bioworks 3.1 SR1, based on the sequest
algorithm (University of Washington, USA, licensed to
ThermoFinnigan Corp., Austin, TX). The ‘no enzyme’
option was used. The experimental mass spectra produced
were correlated to peptide sequences obtained by compar-
ison with theoretical mass spectra of the Machado–Joseph
disease protein 1 (AT-3) database downloaded from the
Swiss-Prot website (http://www.expasy.org) (Q9CVD2). For
peptide matching, the following limits were used: Xcorr
scores greater than 1.5 for singly charged peptide ions, and
2.0 and 2.5 for doubly and triply charged ions, respectively.
Data handling of MS spectra from LC-MS analysis of
intact AT-3-Q6 and related polypeptides was performed
with suitable tools for peptide fragment characterization in
bioworks 3.1 SR1.
Autolytic cleavage of ataxin-3 P. L. Mauri et al.
4284 FEBS Journal 273 (2006) 4277–4286 ª 2006 The Authors Journal compilation ª 2006 FEBS
Acknowledgements
We are indebted to Gabriella Tedeschi for the N-ter-
minal sequencing of AT-3 fragments. The excellent
technical assistance of Enrico Rosti is gratefully
acknowledged. This research was supported by grants
from the Ministero dell’Universita
`
e della Ricerca Sci-
entifica e Tecnologica (MIUR, Rome, Italy) and from
the Fondazione Cariplo (NOBEL project).
References
1 Zoghbi HY & Orr HT (2000) Glutamine repeats and
neurodegeneration. Annu Rev Neurosci 23, 217–247.
2 Cummings CJ & Zoghbi HY (2000) Trinucleotide
repeats: mechanisms and pathophysiology. Annu Rev
Genomics Hum Genet 1, 281–328.
3 Paulson HL, Das SS, Crino PB, Perez MK, Patel SC,
Gotsdiner D, Fischbeck KH & Pittman RN (1997)
Machado–Joseph disease gene product is a cytoplasmic
protein widely expressed in brain. Ann Neurol 41, 453–
462.
4 Ross CA, Poirier MA, Wanker EE & Amzel M (2003)
Polyglutamine fibrillogenesis: the pathway unfolds. Proc
Natl Acad Sci USA 100, 1–3.
5 Clarke G, Collins RA, Leavitt BR, Andrews DF, Hay-
den MR, Lumsden CJ & McInnes RR (2000) A one-hit
model of cell death in inherited neuronal degenerations.
Nature 406, 195–199.
6 Perutz MF & Windle AH (2001) Cause of neural death
in neurodegenerative diseases attributable to expansion
of glutamine repeats. Nature 412, 143–144.
7 Bucciantini M, Giannoni E, Chiti F, Baroni F, Formigli
L, Zurdo J, Taddei N, Ramponi G, Dobson CM & Ste-
fani M (2002) Inherent toxicity of aggregates implies a
common mechanism for protein misfolding diseases.
Nature 416, 507–511.
8 Zuccato C, Tartari M, Crotti A, Goffredo D, Valenza
M, Conti L, Cataudella T, Leavitt BR, Hayden MR,
Timmusk T et al. (2003) Huntingtin interacts with
REST ⁄ NRSF to modulate the transcription of
NRSE-controlled neuronal genes. Nat Genet 35,
76–83.
9 Zuccato C, Ciammola A, Rigamonti D, Leavitt BR,
Goffredo D, Conti L, MacDonald ME, Friedlander
RM, Silani V, Hayden MR et al. (2001) Loss of hunt-
ingtin-mediated BDNF gene transcription in Hunting-
ton’s disease. Science 293 , 493–498.
10 Albrecht M, Hoffmann D, Evert BO, Schmitt I, Wull-
ner U & Lengauer T (2003) Structural modeling of
ataxin-3 reveals distant homology to adaptins. Proteins
50, 355–370.
11 Masino L, Musi V, Menon RP, Fusi P, Kelly G, Frenkiel
TA, Trottier Y & Pastore A (2003) Domain architecture
of the polyglutamine protein ataxin-3: a globular domain
followed by a flexible tail. FEBS Lett 549, 21–25.
12 Albrecht M, Golatta M, Wullner U & Lengauer T
(2004) Structural and functional analysis of ataxin-2
and ataxin-3. Eur J Biochem 271, 3155–3170.
13 Chow MK, Mackay JP, Whisstock JC, Scanlon MJ &
Bottomley SP (2004) Structural and functional analysis
of the Josephin domain of the polyglutamine protein
ataxin-3. Biochem Biophys Res Commun 322, 387–394.
14 Li F, MacFarlan T, Pittman RN & Chakravarti D
(2002) Ataxin-3is a histone-binding protein with two
independent transcriptional corepressor activities. J Biol
Chem 277, 45004–45012.
15 Wang G, Sawai N, Kotliarova S, Kanazawa I &
Nukina N (2000) Ataxin-3, the MJD1 gene product,
interacts with the two human homologs of yeast DNA
repair protein RAD23, HHR23A and HHR23B. Hum
Mol Genet 9, 1795–1803.
16 Madura K (2002) The ubiquitin-associated (UBA)
domain: on the path from prudence to prurience. Cell
Cycle 1, 235–244.
17 Hofmann K & Falquet L (2001) A ubiquitin-interacting
motif conserved in components of the proteasomal and
lysosomal protein degradation systems. Trends Biochem
Sci 26, 347–350.
18 Donaldson KM, Li W, Ching KA, Batalov S, Tsai CC
& Joazeiro CA (2003) Ubiquitin-mediated sequestration
of normal cellular proteins into polyglutamine aggre-
gates. Proc Natl Acad Sci USA 100, 8892–8897.
19 Chai Y, Berke SS, Cohen RE & Paulson HL (2004)
Poly-ubiquitin binding by the polyglutamine disease
protein ataxin-3 links its normal function to protein sur-
veillance pathways. J Biol Chem 279, 3605–3611.
20 Scheel H, Tomiuk S & Hofmann K (2003) Elucidation
of ataxin-3 and ataxin-7 function by integrative bio-
informatics. Hum Mol Genet 12, 2845–2852.
21 Burnett B, Li F & Pittman RN (2003) The polygluta-
mine neurodegenerative protein ataxin-3 binds polyubi-
quitylated proteins and has ubiquitin protease activity.
Hum Mol Genet 12, 3195–3205.
22 Berke SJ, Schmied FA, Brunt ER, Ellerby LM & Paul-
son HL (2004) Caspase-mediated proteolysis of the
polyglutamine disease protein ataxin-3. J Neurochem 89,
908–918.
23 McDonald WH, Ohi R, Miyamoto DT, Mitchison TJ
& Yates JR3rd (2002) Comparison of three directly
coupled HPLC MS ⁄ MS strategies for identification of
proteins from complex mixtures: single-dimension
LC-MS ⁄ MS, 2-phase MudPIT, and 3-phase MudPIT.
Int J Mass Spectrom 219, 245–251.
24 Ghislain M, Dohmen RJ, Levy F & Varshavsky A
(1996) Cdc48p interacts with Ufd3p, a WD repeat pro-
tein required for ubiquitin-mediated proteolysis in
Saccharomyces cerevisiae. EMBO J 15, 4884–4899.
P. L. Mauri et al. Autolyticcleavage of ataxin-3
FEBS Journal 273 (2006) 4277–4286 ª 2006 The Authors Journal compilation ª 2006 FEBS 4285
25 Doss-Pepe EW, Stenroos ES, Johnson WG & Madura
K (2003) Ataxin-3 interactions with rad23 and valosin-
containing protein and its associations with ubiquitin
chains and the proteasome are consistent with a role in
ubiquitin-mediated proteolysis. Mol Cell Biol 23, 6469–
6483.
26 Matsumoto M, Yada M, Hatakeyama S, Ishimoto H,
Tanimura T, Tsuji S, Kakizuka A, Kitagawa M &
Nakayama KI (2004) Molecular clearance of ataxin-3 is
regulated by a mammalian E4. EMBO J 23, 659–669.
27 Polga
´
r L (2004) Catalytic mechanisms of cysteine pep-
tidases. In Handbook of Proteolytic Enzymes, 2nd edn
(Barrett AJ, Rawlings ND & Woessner F, eds), pp.
1072–1079. Elsevier, Academic Press, London.
28 Tarlac V & Storey E (2003) Role of proteolysis in poly-
glutamine disorders. J Neurosci Res 74, 406–416.
29 Paulson HL (2000) Toward an understanding of poly-
glutamine neurodegeneration. Brain Pathol 10, 293–
299.
30 Wellington CL, Ellerby LM, Hackam AS, Margolis RL,
Trifiro MA, Singaraja R, McCutcheon K, Salvesen GS,
Propp SS, Bromm M et al. (1998) Caspase cleavage of
gene products associated with triplet expansion disor-
ders generates truncated fragments containing the poly-
glutamine tract. J Biol Chem 273, 9158–9167.
31 Wellington CL, Singaraja R, Ellerby L, Savill J, Roy S,
Leavitt B, Cattaneo E, Hackam A, Sharp A, Thornber-
ry N et al. (2000) Inhibiting caspase cleavage of hunt-
ingtin reduces toxicity and aggregate formation in
neuronal and nonneuronal cells. J Biol Chem 275,
19831–19838.
32 Sanchez I, Mahlke C & Yuan J (2003) Pivotal role of
oligomerization in expanded polyglutamine neurodegen-
erative disorders. Nature 421, 373–379.
33 Morley JF, Brignull HR, Weyers JJ & Morimoto RI
(2002) The threshold for polyglutamine-expansion pro-
tein aggregation and cellular toxicity is dynamic and
influenced by aging in Caenorhabditis elegans. Proc Natl
Acad Sci USA 99, 10417–10422.
34 Haacke A, Broadley SA, Boteva R, Tzvetkov N, Hartl
FU & Breuer P (2006) Proteolytic cleavage of polygluta-
mine-expanded ataxin-3is critical for aggregation and
sequestration of non-expanded ataxin-3. Hum Mol
Genet 15, 555–568.
35 Goti D, Katzen SM, Mez J, Kurtis N, Kiluk J, Ben
Haiem L, Jenkins NA, Copeland NG, Kakizuka A,
Sharp AH et al. (2004) A mutant ataxin-3 putative-clea-
vage fragment in brains of Machado–Joseph disease
patients and transgenic mice is cytotoxic above a critical
concentration. J Neurosci 24, 10266–10279.
36 Nicastro G, Menon RP, Masino L, Knowles PP,
McDonald MO & Pastore A (2005) The solution struc-
ture of the Josephin domain of ataxin-3: structural
determinants for molecular recognition. Proc Natl Acad
Sci USA 102, 10493–10498.
37 Shehi E, Fusi P, Secundo F, Pozzuolo S, Bairati A &
Tortora P (2003) Temperature-dependent, irreversible
formation of amyloid fibrils by a soluble human ataxin-
3 carrying a moderately expanded polyglutamine stretch
(Q36). Biochemistry 42, 14626–14632.
38 Laemmli UK (1970) Cleavage of structural proteins
during the assembly of the head of bacteriophage T4.
Nature 227, 680–685.
39 Forler D, Kocher T, Rode M, Gentzel M, Izaurralde E
& Wilm M (2003) An efficient protein complex purifica-
tion method for functional proteomics in higher eukar-
yotes. Nat Biotechnol 21, 89–92.
Autolytic cleavage of ataxin-3 P. L. Mauri et al.
4286 FEBS Journal 273 (2006) 4277–4286 ª 2006 The Authors Journal compilation ª 2006 FEBS
. Ataxin-3 is subject to autolytic cleavage Pier Luigi Mauri 1 , Matteo Riva 2 , Daniela Ambu 1 , Antonella De Palma 1 , Francesco Secundo 3 , Louise Benazzi 1 , Marco Valtorta 2 , Paolo Tortora 2, *. conclude that AT-3 activity is not caspase-like. The ability of AT-3 to undergo autolytic degradation might be related to the mechanisms of pathogenesis, as proteolysis is widely thought to play a role in. consistently observed that pure AT-3 undergoes proteolysis when incubated at room temperature for several hours, and this prompted us to check whether this phenomenon is supported by autolytic cleavage. Based