FXTAS syndrome?Ljiljana Sjekloc´a*, Kris Pauwels and Annalisa Pastore MRC National Institute for Medical Research, London, UK Introduction The fragile X mental retardation protein FMRP i
Trang 1FXTAS syndrome?
Ljiljana Sjekloc´a*, Kris Pauwels and Annalisa Pastore
MRC National Institute for Medical Research, London, UK
Introduction
The fragile X mental retardation protein (FMRP) is
an 70 kDa human protein encoded by the X-linked
gene FMR1 which is expressed in different organs,
most prominently in brain and gonads [1–3] FMRP is
a multi-domain protein which contains two Tudor
domains connected to two protein K homology (KH)
domains by an ca 80 amino acids residue long linker
This region is followed by a putative intrinsically
unstructured region which contains also arginine- and
glycine-rich (RGG) motifs [4,5] Co-presence of
differ-ent nucleic acid binding domains in FMRP suggests
that the protein has a prominent capacity to bind nucleic acids, in particular RNA, as experimentally confirmed both in vitro and in vivo [6,7]
The cellular role of FMRP is not well understood Experimental evidence shows that FMRP binds co-transcriptionally to certain messenger RNAs forming messenger ribonucleoprotein (mRNP) particles, which are exported from the nucleus to the cytoplasm [8] In the cytoplasm FMRP associates to microtubules, to polysomes and to mRNPs and permits the mRNP par-ticles to be delivered to distal dendrite sites [9] It has
Keywords
aggregation; fragile X mental retardation
syndrome; fragile X related tremor ataxia
syndrome (FXTAS); protein misfolding;
structure
Correspondence
A Pastore, MRC National Institute for
Medical Research, The Ridgeway, London
NW7 1AA, UK
Fax: +44 20 8905 4477
Tel: +44 20 8816 2630
E-mail: apastor@nimr.mrc.ac.uk
*Present address
Department of Cell and Molecular Biology,
Karolinska Institutet, Stockholm SE-171 77,
Sweden
(Received 2 February 2011, revised 20
March 2011, accepted 25 March 2011)
doi:10.1111/j.1742-4658.2011.08108.x
Fragile X mental retardation protein (FMRP) is an RNA binding protein necessary for correct spatiotemporal control of neuronal gene expression in humans Lack of functional FMRP causes fragile X mental retardation, which is the most common inherited neurodevelopmental disorder in humans In a previous study, we described the biochemical and biophysical aggregation properties of constructs spanning the conserved region of FMRP and of two other human fragile X related (FXR) proteins, FXR1P and FXR2P Here, we show that the same regions have an intrinsic ten-dency to aggregate and spontaneously misfold towards b-rich structures, also under non-destabilizing conditions These findings pave the way to future studies of the mechanism of formation of FXR-containing ribonu-cleoprotein granules and suggest a possible link with the as yet poorly understood FXR proteins’ associated pathologies
Structured digital abstract
l FXR2P binds to FXR2P by fluorescence technology (View interaction)
l FMRP binds to FMRP by electron microscopy (View interaction)
l FXR1P binds to FXR1P by electron microscopy (View interaction)
Abbreviations
CD, circular dichroism; FMRP, fragile X mental retardation protein; FXR, fragile X related; FXS, fragile X syndrome; FXTAS, fragile X associated tremor ataxia syndrome; KH, K homology; mRNP, messenger ribonucleoprotein; NDF, N-terminal domain; NES, nuclear export signal; rCGG, cytosine guanine triribonucleotide; ThT, thioflavin T.
Trang 2also been shown that messenger RNAs bound to
FMRP are translationally repressed and that, in
neu-rons, FMRP acts in an activity-dependent manner as
an inhibitor of translation initiation ([10] and
refer-ences therein)
Most studies on FMRP are related to its functions
in brain neurons for two reasons First, the lack of
functional FMRP, due to transcriptional silencing of
FMR1 gene, causes a neurodevelopmental disorder,
fragile X mental retardation syndrome (FXS), the most
common inherited mental disorder in humans FXS is
characterized by mild to severe mental retardation,
autistic behaviour and, in male patients,
macro-orchi-dism [11] Second, alteration of FMRP expression,
characterized by increased levels of FMR1 mRNA and
decreased protein levels, can lead to a late onset
neuro-degenerative disorder, the fragile X associated tremor
ataxia syndrome (FXTAS), with symptoms similar to
Parkinsonism, and to premature ovarian failure in
females [12,13] Two other proteins, fragile X related
(FXR) proteins 1 and 2 (FXR1P and FXR2P), can
compensate partially for lack of FMRP in some
organs of FXS patients, but not in brain and in
gonads, thus emphasizing the crucial role of FMRP in
correct spatiotemporal control of neuronal gene
expression and for normal sexual maturation FXR1P
and FXR2P are structurally and functionally related
to FMRP and they all together form the FXR protein
family which includes members from different phyla
[14] All FXR proteins show a high degree of amino
acid sequence conservation in their amino terminus
and central region comprising Tudor and KH
domains, whereas the carboxyl terminus has low
sequence similarity with the only common
denomina-tor being the presence of RGG motifs
FXR proteins are components of different types of
nuclear and cytoplasmic ribonucleoprotein granules in
which they often co-localize [1,15–19] FXR proteins
can form hetero-oligomers in vitro and when
over-expressed in cellular systems [20–22], although in
mammalian cells they are believed to preferentially
homo-oligomerize [21] The oligomerization properties
of FXR proteins are likely to have significant
impor-tance for regulation of their cellular functions as
shown in different model systems In Drosophila
neu-rons, for instance, the mobility of certain mRNAs is
controlled by FMRP in a concentration-dependent
manner [23] High levels of transfected Fmrp in mouse
embryonic Fmr1 KO STEK cells induce formation of
cytoplasmic stress granules in which mRNAs are
trapped into repressed mRNP granules [24,25] In a
recent study, we investigated the oligomerization
properties of human FXR proteins and showed that,
in vitro, multi-domain constructs from the highly con-served N-terminus have an elevated tendency to aggre-gate [26] They self-assemble not just by forming dimers but through a more complex pattern of self-association which proceeds in a continuous way from the monomer to large molecular weight aggregates via formation of dimeric species We proposed that this behaviour is typical of ‘complex-orphan proteins’, i.e proteins which exist in the cell as part of large molecu-lar assemblies When produced in isolation, they have
an elevated tendency to self-associate
To further characterize the nature of aggregation of FXR proteins, we have carried out a study of their aggregation properties using different approaches We identified by in silico analysis potential hot-spots of aggregation⁄ fibrillation and showed that they all cluster in the protein N-terminus We then studied the aggregation behaviour of various constructs from FMRP, FXR1P and FXR2P using complementary biophysical techniques We demonstrate that not only
do all constructs have an intrinsic tendency to aggre-gate but they also undergo an irreversible conforma-tional transition towards b-enriched structures which are typical of amyloidogenic diseases such as Alzhei-mer’s, Parkinson’s and Huntington’s diseases The transition occurs spontaneously also under native-like conditions without any need for fold destabilization
We propose that our findings could be relevant for understanding granule formation and could have a link with the pathogenesis of FXTAS
Results
FXR proteins present aggregation and amyloidogenic hot-spots
To understand the aggregation properties of FXR pro-teins, we first analysed their sequence for predicting potential aggregative and amyloidogenic hot-spots
in polypeptides The results all suggest the presence of several aggregation hot-spots which cluster in the highly conserved N-terminal 400 amino acids (Fig 1A) whereas no sequence was identified in the C-terminal region of any of the FXR sequences, includ-ing the most ancestral FMRP homologue dFMR1 from Drosophila melanogaster Two putative amyloidogenic regions are present in the NDF of the human FXR proteins (sequences YVIEYA and TYNEIV) Two more hot-spots amongst the nine detected in FMRP by waltz with reliability higher than 90% correspond to the residues 166–174 located in the linker region con-necting the Tudor and KH domains and to residues 303–308 (LIQEIV) in the second KH domain
Trang 3Taken together, this in silico analysis suggests the
presence of several potential aggregation foci all
grouped in the highly conserved N-terminus
Temperature induces a conformational transition
of FMRP domains
We tested experimentally the role of the different
regions in aggregation⁄ fibrillation using single-and
multiple-domain constructs from human FXR proteins
which we knew from previous extensive
characteriza-tion are stable to degradacharacteriza-tion [26] (Fig 2) Although
monodispersed at sufficiently low concentrations, we
had previously demonstrated that they all have a
strong tendency to aggregate [26] To check whether
aggregation is associated with misfolding, we probed
their secondary structure as a function of temperature
by far-UV circular dichroism (CD) We first analysed
the secondary structure content at different
tempera-tures of FMRP Nt-KH1 (residues 1–280), the longest
of the FMRP fragments we could obtain in a stable
form (Fig 3A) The spectrum of Nt-KH1 is typical of
an a–b fold at 20C, as expected from the presence in
the construct of Tudor and KH domains [4,28] At
higher temperatures (40–45C), the conformation
starts to change (Fig 3B) At 55C, the spectrum
becomes typical of an all-b protein indicating a profound structural rearrangement with a minimum around 215 nm The transition is irreversible, as the CD spectra of samples treated at 45C remain
Fig 1 Sequence alignment of FXR proteins and indication of fibrillogenic regions The alignment was produced and colour coded according
to CLUSTALW2 [27] Extra rows were added below for the rulers relative to human FMRP, FXR1P and FXR2P The regions predicted as fibrillo-genic by the WALTZ software [40] are indicated as red crosses.
Fig 2 Summary of the modular structure of FXR proteins (A) and
of the constructs used in this study (B) using a SMART -like [41] rep-resentation NDF stands for the N-terminal domain which contains two Tudor domains KH stands for protein K homology domain Linker indicates the region between the second Tudor domain and KH1 NES stands for nuclear export signal The domain boundaries used are the same as in [26].
Trang 4unaltered after the temperature is decreased to 37 or
to 20C
The spectra of shorter FMRP fragments lacking the
KH domain, NDF (residues 1–134) and Nt (residues
1–217), were examined to assess the role of the
individual domains (Fig 3C,D) These fragments also
undergo structural rearrangements characteristic of an
irreversible increase of b content above 45C,
suggest-ing that they are individually able to misfold (Fig 3B–
D) To test whether pre-incubation at a fixed moderate
temperature could also cause the observed b-enriched
structural rearrangement, the three FMRP fragments,
at the same protein concentration (5 lm), were
inde-pendently incubated at different temperatures In a
time course measurement at 45C, NDF underwent a
conformational change after 3 h pre-incubation
(Fig 3E) For comparison, the two longer constructs
Nt and Nt-KH1 incubated at 45C did not reach,
over the same time, the minimum CD signal (Fig 3E)
observed for the corresponding samples at 55C A
similar experiment was performed at 50C and
resulted in a faster conformational transition compared
with 45C (Fig 3F) At this temperature, the intensi-ties of the NDF and Nt-KH1 spectra reached a maxi-mum after 40 and 120 min, respectively Nt underwent
a conformation transition at 50C which was not, however, complete during the time course of the exper-iment (3 h) This suggests that, under the same experi-mental conditions, the region C-terminal to the NDF, comprising the linker between NDF and KH1, has a prominent role in aggregation
Taken together, these data show that different domains of the conserved region of FXR proteins rear-range their structure upon temperature treatment In all cases examined this rearrangement occurs with very similar modalities and results in a significant enrich-ment of the b content
Tendency to misfold is a conserved feature of FXR proteins
To extend our studies to other FXR proteins, we used the human FMRP paralogues FXR1P and FXR2P The secondary structure of FXR1P Nt-NES was first
Fig 3 Spectroscopic study of temperature-induced conformational changes of FMRP (A) Far-UV CD spectra of Nt-KH1 of FMRP recorded
at 20 C (black line) or 55 C (dotted line) and expressed in molar ellipticity of Nt-KH1 (B) Temperature course at 220 nm, in molar ellipticity,
of FMRP NDF (curve a), Nt (curve b) and Nt-KH1 (curve c) The rate of temperature increase was 1 CÆmin)1 (C), (D) Far-UV CD spectra at
20 C (black lines) or 55 C (dotted lines) of NDF and Nt respectively (E), (F) Time course of the a-to-b transition of FMRP NDF (curve a),
Nt (curve b) and Nt-KH1 (curve c) induced by incubation at 45 and 50 C, respectively All spectra were recorded at protein concentrations of
5 l M.
Trang 5examined at different temperatures since for this
pro-tein we could obtain a long construct which spans the
whole conserved region (residues 1–380) The far-UV
CD spectrum of this construct shows that an a-to-b
conformational transition occurs under experimental
conditions similar to those used for the FMRP
domains (Fig 4A) Interestingly, at the protein
concen-trations used for the assay (5 lm), the b-enriched
con-formation of FXR1P Nt-NES persists up to 80C
This behaviour is similar to that observed for FMRP
Nt-KH1 and suggests that the KH2 and NES regions
do not significantly influence solubility Similar studies
with shorter FXR1P fragments as well as with FXR2P
fragments all underwent similar a-to-b conformational
transitions, although overall FXR2P seemed to be
more prone to aggregation (Fig 4B)
These data indicate that not only FMRP but also
the other human FXR paralogues have a strong and
well conserved tendency to misfold
Misfolding of FMRP domains occurs also under
non-destabilizing conditions
Since the duration of temperature treatment plays a
role in the observed process, we tested if prolonged
incubation could lead to a-to-b conformational
transi-tion also at 37C, i.e close to the physiological
tem-perature at which FMRP functions in human cells
Initially, incubation of FMRP Nt-KH1 (5 lm) at
37C did not cause a significant secondary structure
perturbation, but a conformational transition to a
b-enriched structure was observed after a 45-h
incuba-tion (Fig 5A) The lag time decreased to 16 h at
con-centrations threefold or sixfold higher (15 and 30 lm
respectively), as expected for a
concentration-depen-dent phenomenon such as aggregation (Fig 5B,C)
The final intensity of the recorded CD signal is very
similar to that recorded at 55C, suggesting not only that a similar process takes place at both temper-atures but also that the final states are structurally comparable
Freshly prepared FMRP Nt-KH1 samples (30 lm) are monomeric and monodispersed, and if stored at
4C they remain mainly monomeric with a small but detectable increase of dimeric species as time pro-ceeds, i.e after 16 h incubation The size exclusion chromatograms of these samples incubated at 37C over the same time show the appearance of high molecular weight species which are absent both in fresh samples and in samples incubated at 4C (Fig 5D) We can conclude that recombinant Nt-KH1 of FMRP has an intrinsic tendency to aggregate
in vitro also at physiological temperature in native-like conditions
FMRP has an intrinsic tendency to form protofibrils
To characterize the nature of the aggregates, we exam-ined the longest fragments from FMRP (Nt-KH1) and from FXR1P and FXR2P (Nt-NES) using the fluores-cence signal of thioflavin (ThT) dye that is indicative
of formation of amyloid-like structures [29] After addition of ThT to diluted FXR protein solutions (5 lm), samples were incubated for 15 min at increas-ing temperatures usincreas-ing 5C intervals and their fluores-cence was monitored No fluoresfluores-cence could be detected during incubation at temperatures below
65C At this temperature, a small increase of fluores-cence emission at 482 nm was observed Treated sam-ples were then incubated at room temperature and fluorescence was measured at different time points reaching a maximal emission at 482 nm after 12 h (Fig 6A) The following measurement after 24 h did
Fig 4 Spectroscopic studies of FXR1P and FXR2P (A) Far-UV CD spectra expressed in molar ellipticity of the FXR1P Nt-NES at 20 C (con-tinuous line) or 55 C (dotted line) using 5 l M protein concentration (B) Temperature course of FXR1P Nt-NES at 220 nm, using 5 l M con-centrations The recording rate was 1 CÆmin)1.
Trang 6not show further increase and a net decrease was
observed after 96 h, probably caused by fibre
sedimen-tation (data not shown)
To verify the morphology of the end-states of aggre-gation, we used transmission electron microscopy and examined samples after the conformational transition
Fig 5 Following the conformational
transi-tion of FMRP Nt-KH1 at 37 C and different
incubation times as a function of protein
concentration (A), (B), (C) Comparison of
the FMRP Nt-KH1 CD spectra before
(con-tinuous line) and after (dotted line)
incuba-tion at 37 C using 5, 15 and 30 l M protein
concentrations, respectively (D) Size
exclu-sion chromatography elution profile of
FMRP Nt-KH1: the continuous line
chro-matogram derives from freshly prepared
Nt-KH1, the broken line chromatogram is
the profile of the same sample kept at 4 C
for 16 h, and the dotted line chromatogram
corresponds to a sample incubated at 37 C
for 16 h.
Fig 6 Testing the fibrillogenic properties of
FXR proteins (A) ThT fluorescence assay on
FXR2P Nt-NES treated over the temperature
range 20–65 C, increasing the temperature
by 5 C every 15 min and using 5 l M
protein concentration The fluorescence
observed at 20 C (black curve) decreases
at temperatures between 45 and 55 C
(red curve), and increases after exposure to
65 C (orange) The fluorescence signal
reached a maximum after 12 h (green
curve) (B) Transmission electron micrograph
of negatively stained FMRP Nt (1–217)
protofibrils pre-incubated at 50 C for 3 h.
(C) Negatively stained aggregates were
observed for the FMRP Nt-KH1 construct
(1–280) that was incubated for an extended
time at 37 C (D) Electron micrograph of
FXR1P Nt-NES aggregates obtained after
2 h incubation at 50 C The scale bars
correspond to 100 nm.
Trang 7achieved either by direct exposure to 50C or by
pro-longed incubation at 37C, at two different protein
concentrations (5 and 30 lm) Negatively stained
pro-tofibrillar and fibrillar assemblies were observed for
FMRP Nt, FMRP Nt-KH1 and FX1RP Nt-NES
(Fig 6B–D) The FMRP Nt protofibrils appeared
homogeneous, with an apparent uniform diameter
(7 nm) and variable lengths that very rarely exceeded
100 nm (Fig 6B) Interestingly, we also observed dense
networks of long linear and unbranched fibrils with a
10-nm diameter, which displayed repeating segments
and twists The FMRP Nt-KH1 samples contained
globular particles with an average diameter of 24 nm,
often decorated with stain, as well as clustered deposits
of fibrils with an average diameter of 6 nm (Fig 6C)
FX1RP Nt-NES aggregates have a curved appearance,
with an apparent average diameter of 10 nm (Fig 6D)
They also clustered together and were often found to
be decorated with the uranyl acetate stain Taken
together these results confirm a marked tendency of
FXR constructs to fibrillation
Discussion
We have shown here that different fragments of FXR
proteins not only have a strong tendency to aggregate
as previously described [26] but also undergo an
irre-versible conformational transition which leads to a
sig-nificant increase in their b-structure content Several
conserved putative aggregation and amyloidogenic
hot-spots were predicted by in silico analysis of the
FXR amino acid sequences They are all grouped in
the highly conserved (more than 70–80% identity and
80–90% similarity) N-terminal half of the proteins
which is also the region involved in most of the
inter-actions with the FXR cellular partners [30], suggesting
that the aggregation hot-spots could have a prominent
role in determining the hetero- and self-assembly
behaviours of the full-length proteins By combining
CD spectroscopy and size exclusion chromatography,
we have established a clear link between FMRP
aggre-gation and misfolding, as observed by the
concentra-tion dependence of the conformaconcentra-tional transiconcentra-tion
Relatively small variations of protein concentration
also lead to an increase of the rates at which the
con-formational transition occurs An appreciable ThT
fluorescence increase, irreversible b-enriched structural
transitions and electron microscopy analysis support
formation of ordered fibrillar aggregates We observe a
very similar behaviour for the two FMRP paralogues,
FXR1P and FXR2P, for which the conformational
transition occurs with modalities very similar to
FMRP
Interestingly, the observed transition towards b-en-riched conformations occurs also at physiological tem-perature under non-destabilizing conditions This behaviour is very interesting for a protein such as FMRP which contains multiple globular domains While understanding how and when misfolding occurs
is easier for intrinsically unfolded proteins, such as the Alzheimer Ab peptides or a-synuclein, studies of globu-lar proteins have traditionally involved the use of
ad hoc mutations and⁄ or destabilizing conditions, such
as high temperature, molecular crowding or high pres-sure These conditions lead to destabilization of the structure and access to fibrillogenic regions normally buried in the hydrophobic core Only recently a small but steadily increasing number of examples are being described in which misfolding occurs in native-like con-ditions This is the case for instance of the globular Jo-sephin domain of ataxin-3, the protein responsible for the misfolding Machado–Joseph disease: we have recently shown that Josephin aggregation and misfold-ing is promoted by exposed hydrophobic patches involved in recognition of its natural partner ubiquitin, thus suggesting a link between normal function and misfolding [31] Likewise, the globular domain of the prion protein contains a seeding region, H2H3, which retains its fold during the early stages of unfolding [32]
It has been suggested that in many proteins related to conformational diseases aggregation⁄ amyloidogenic regions coincide with interaction surfaces [33–35] Our results bear a number of interesting conse-quences First, the strong tendency to aggregate of FXR proteins could help us to understand the driving forces that lead to granular formations and eventually understand more about their functional role The find-ings presented in this study also suggest interesting possibilities for the ability of this family of proteins to contribute to both early life syndromes such as FXS (for instance through destabilizing mutations) and aggregation-related neurodegeneration later in life; such could be the case of FXTAS The latter is a par-ticularly interesting possibility since it could shed new light onto a still poorly understood syndrome: although RNA aggregation is thought to be an impor-tant driving force for formation of the pathological neuronal intranuclear RNP inclusions observed in FXTAS patients, little is known about the factors which determine their formation and stability [36] The current view is that FXTAS is the end-point of a pro-cess that begins in early development and reaches its maximum late in life [37] rCGG expansion in the 5¢UTR region of FMR1 mRNA is required for forma-tion of neuronal inclusions in FXTAS patients, which consist also of other mRNAs and of different proteins
Trang 8amongst which FXR1P and FXR2P [38] Although
FMRP, which is expressed in FXTAS patients, has not
so far been identified amongst the components of the
inclusions, we cannot exclude at this stage that its
absence is not simply due to lack of sensitivity of the
detection methods used We suggest as a working
hypothesis that the aggregative and misfolding
ten-dency of one or more of the FXR proteins could
con-tribute to pathology, thus adding FXTAS to the
family of misfolding diseases While more work needs
to be done to test this hypothesis, we expect that
important information may come from cellular studies
of the effects of molecular crowding [39] on the FXR
folding, homo- and hetero-association when
sur-rounded by other cellular components
Experimental procedures
Bioinformatic analysis
The amino acid sequences of human (Q06787, P51114,
P51116), mouse (P35922, Q61584, Q9WVR4), chicken
(Q5F3S6), frog (P51113, Q6GLC9, P51115), zebra fish
(Q7SYM7, Q7SXA0, Q6NY99) and fruit fly (Q9NFU0)
FXR proteins were aligned using program clustalw2 [27]
These sequences were also searched for putative
determi-nants of aggregation and amyloidogenesis by the following
uab.es/aggrescan/) for prediction of hot-spots for
aggrega-tion in polypeptides; pasta (http://protein.cribi.unipd.it/
pasta/) for prediction of amyloid-like structure aggregation;
predict features related to the formation of amyloid fibrils;
sequence-dependent and mutational effects on the
aggrega-tion of the peptides and proteins; and waltz (http://
waltz.switchlab.org/) for predicting amyloidogenic regions
in protein sequences
Cloning, protein expression and purification
The constructs studied in this paper were produced
accord-ing to procedures previously described [26] In short, clones
of human FMR1, FXR1 and FXR2 were used as templates
for DNA amplification by PCR PCR amplicons encoding
different fragments of the conserved region of FXR
pro-teins were cloned into a modified pET-24 vector (Novagen,
Gibbstown, NJ, USA) encoding an amino terminal Trx
(thioredoxin)-His6-tag and a tobacco etch virus (TEV)
pro-tease cleavage site
with plasmids encoding different FXR fragments were
grown at 37C in Luria–Bertani medium containing
appro-priate antibiotic Protein over-expression was induced with
0.2 mm isopropyl thio-b-d-galactoside after the cell culture
an additional 5 h at 28C The cells were harvested by cen-trifugation, resuspended in a lysis buffer containing 20 mm
Igepal CA-630 (Sigma–Aldrich, St Louis, MO, USA),
2 mm b-mercaptoethanol, supplemented with the Complete EDTA-free protease inhibitor cocktail (Roche, Indianapo-lis, IN, USA), and lysed by ultrasonication The recombi-nant peptides were then purified from the soluble fraction
of the centrifuged cell lysate by immobilized metal-affinity chromatography (IMAC) using Ni-NTA (Ni2+
Yokyo, Japan), and dialyzed against 50 mm Tris⁄ HCl (pH 8.0), 1 mm dithiothreitol, 0.5 mm EDTA; the recombinant Trx-His6-tag was removed by cleavage with TEV protease (Invitrogen, Carlsbad, CA, USA) The FXR peptides were further purified by IMAC, anion exchange chromatography
(Superdex 200 HR 16⁄ 60 or Superdex 200 10 ⁄ 30) using elu-tion buffer consisting of 50 mm Tris⁄ HCl (pH 8.0), 2 mm b-mercaptoethanol The purity of the recombinant peptides
mass spectrometry Protein concentration was measured by
UV absorbance at 280 nm using theoretical extinction coef-ficients calculated byProtParam
Aggregation studies by CD and size exclusion chromatography
CD spectra were recorded using a Jasco J-715 spectropola-rimeter equipped with a thermostatted cell holder controlled
by a Jasco Peltier element, at different temperatures, over a wavelength range from 260 to 190 nm in quartz cuvettes (Hellma) of path length appropriate to protein concentra-tion of the samples, i.e 1 mm for 5 lm (0.15 mgÆmL)1),
(1 mgÆmL)1) Thermally induced denaturation transitions were monitored by CD absorption at 220 nm from 10 to
inverse temperature scan The purified recombinant proteins
b-mercaptoetha-nol To monitor progression of protein aggregation over
spectra were recorded at different time points (1, 6, 16, 24,
45, 72 h, 1 week)
Analytical size exclusion chromatography was carried out
by injecting 100 lL of samples (30 lm) into a Superdex 200
10⁄ 300 GL column
ThT fluorescence assays
The ThT assays were performed by consecutively incubat-ing at 20, 30, 40, 50, 55, 60 and 65C, for 15 min at each
Trang 9temperature without shaking, the purified protein solution
diluted to 5 lm in a buffer containing 20 lm ThT, 20 mm
Tris⁄ HCl (pH 8.0), 1 mm b-mercaptoethanol After the last
heating step, at 65C, the sample was kept at room
tem-perature (20C) and spectra were recorded after 1, 2, 3, 4,
12, 24 and 96 h Fluorescence was measured using an ISS
PC1 (Interconnect Systems Solution) spectrofluorimeter All
course with excitation at 440 nm (0.4 nm slit width) and
emission at 482 nm (1.5 nm slit width) For each
measure-ment 10 scans were recorded The measuremeasure-ment of the
only a weak peak at 520 nm
Transmission electron microscopy
A sample volume of 4 lL was spotted onto freshly
pre-pared carbon-coated and glow-discharged copper grids
(FormVar) Upon adsorption to the grid surface for 30 s,
the sample was washed briefly with milli-Q water and
sub-sequently stained with 1% (w⁄ v) uranyl acetate for 30 s
Micrographs of negatively stained areas were taken with a
JEOL 1200 transmission electron microscope operating at
100 kV and at a magnification of 27 800· on electron
microscope films (Kodak) and developed with Phenisol
developer (Ilford) and Hypam fixer (Ilford) for 5 min each
Acknowledgements
We thank Steve Martin for help with CD and
fluores-cence studies, Lesley Calder for support with electron
microscopy analysis and Steve Howell for mass
spec-trometry analysis We are grateful to Cesira de Chiara
and Laura Masino for critical discussion and
assis-tance in graphic elaboration of CD results We
acknowledge support from the MRC (Grant ref
U117584256) Kris Pauwels is the recipient of an
EMBO long-term postdoctoral fellowship (ALTF
512-2008)
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