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 de
Trang 1Pier Luigi Mauri1, Matteo Riva2, Daniela Ambu1, Antonella De Palma1, Francesco Secundo3,
Louise Benazzi1, Marco Valtorta2, Paolo Tortora2,* and Paola Fusi2,*
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-3 is 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.
Trang 2form, 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-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 131I-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 is autolytic 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 carrylead-ing 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 24C is shown in Fig 1 (lanes referred to
as wt)
H 1 9 L DM H 1 9 L DM H 1 9 L D M WT C 4 A W T C 4 A W T C 4 A
M
5
-6
-0
-4
7
-5
1
-3
4
-0
2
-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.
Trang 3C14A, 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 24C 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 24C 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, HgCl2 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 T17)45, T111)124 and T196)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 T111)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 24C 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.
Trang 4major bands, referred to as ‘a’, ‘b’ and ‘c’, were
detec-ted (Fig 1) They were subsequently subjecdetec-ted 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 trypnontryp-tic 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 D241 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 T111)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 HisHis-tidines 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
Trang 5additional, 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 24C 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 24C (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
Fig 5 Multicharge mass spectrum of the fragmentation peptide T306)355obtained 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.
Trang 6to 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 autolytic cleavage (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.
Trang 7sites 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 is subject to 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 subjecisola-ted 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
37C, 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 37C in
LB⁄ ampicillin medium and induced with 50 lm isopropyl thio-b-d-galactoside (IPTG) at A600¼ 1.0 for 2 h To obtain crude extracts, cells were frozen and thawed, incuba-ted for 1 h at 4C 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 MgCl2, 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, 1 mm EDTA, 1 mm di-thiothreitol) Removal of the GST affinity tail was achieved
by incubating the resin-bound proteins overnight at 4C in 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 24C 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
Trang 8SDS⁄ 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 25C 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-sÆcm2Ædmol)1, and calculated as
½h ¼ hobsMWR=ð10lcÞ where hobsis 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
37C 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 37C The peptides were extracted from the gel
at room temperature with 50% acetonitrile containing 0.1% trifluoroacetic acid Aliquots containing 10 lL of 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 C18 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,
a C8 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 260C 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 bioworks3.1 SR1
Trang 9We 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)
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