Báo cáo khoa học: Formation of highly toxic soluble amyloid beta oligomers by the molecular chaperone prefoldin pptx

Formation of highly toxic soluble amyloid beta oligomers
by the molecular chaperone prefoldin
Masafumi Sakono
1,
*, Tamotsu Zako
1
, Hiroshi Ueda
2
, Masafumi Yohda
3
and Mizuo Maeda
1
1 Bioengineering Laboratory, RIKEN Institute, Saitama, Japan
2 Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Japan
3 Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Japan
The neuropathology of Alzheimer’s disease (AD) is
characterized by loss of synapses and neurons in the
brain and the accumulation of senile plaques and
neurofibrillary tangles [1]. The 39–43 amino acid Ab
peptides represent the principal components of
plaques, and are cleaved by secretases from parental
amyloid precursor protein localized to the plasma
membrane. Synthetic Ab peptides have been shown to
spontaneously aggregate into b-sheet-rich fibrils resem-
bling those found in plaques. These insoluble fibrillar
forms were thought to cause neurotoxicity through
oxidative stress both in vivo and in vitro. However, the
relevance of these plaques to AD pathogenesis remains
unclear and is even questionable as there is no clear
correlation between the number of amyloid plaque and
the severity of dementia [2–5].

It has recently been suggested that soluble Ab
species cause AD as the levels of these species correlate
Keywords
Alzheimer’s disease; amyloid b; molecular
chaperone; prefoldin; soluble oligomers
Correspondence
T. Zako, Bioengineering Laboratory, RIKEN
Institute, 2-1 Hirosawa, Wako, Saitama
351 0198, Japan
Fax: +81 48 462 4658
Tel: +81 48 467 9312
E-mail:
M. Maeda, Bioengineering Laboratory,
RIKEN Institute, 2-1 Hirosawa, Wako,
Saitama 351 0198, Japan
Fax: +81 48 462 4658
Tel: +81 48 467 9312
E-mail:
*Present address
PRESTO, Japan Science and Technology
Agency, Saitama, Japan
(Received 16 June 2008, revised
5 September 2008, accepted 3 October
2008)
doi:10.1111/j.1742-4658.2008.06727.x
Alzheimer’s disease (AD) is a neurological disorder characterized by the
presence of amyloid b (Ab) peptide fibrils and oligomers in the brain. It
has been suggested that soluble Ab oligomers, rather than Ab fibrils,
contribute to neurodegeneration and dementia due to their higher level of
toxicity. Recent studies have shown that Ab is also generated intracellu-

larly, where it can subsequently accumulate. The observed inhibition of
cytosolic proteasome by Ab suggests that Ab is located within the cytosolic
compartment. To date, although several proteins have been identified that
are involved in the formation of soluble Ab oligomers, none of these have
been shown to induce in vitro formation of the high-molecular-mass
(> 50 kDa) oligomers found in AD brains. Here, we examine the effects
of the jellyfish-shaped molecular chaperone prefoldin (PFD) on Ab(1–42)
peptide aggregation in vitro. PFD is thought to play a general role in
de novo protein folding in archaea, and in the biogenesis of actin, tubulin
and possibly other proteins in the cytosol of eukaryotes. We found that
recombinant Pyrococcus PFD produced high-molecular-mass (50–250 kDa)
soluble Ab oligomers, as opposed to Ab fibrils. We also demonstrated that
the soluble Ab oligomers were more toxic than Ab fibrils, and were capable
of inducing apoptosis. As Pyrococcus PFD shares high sequence identity to
human PFD and the PFD-homolog protein found in human brains, these
results suggest that PFD may be involved in the formation of toxic soluble
Ab oligomers in the cytosolic compartment in vivo.
Abbreviations
Ab, amyloid b; AD, Alzheimer’s disease; ADDL, Ab-derived diffusible ligand; HFIP, 1,1,1,3,3,3-hexa-fluoro-2-propanol; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PFD, prefoldin; PI, propidium iodide; PVDF, poly(vinylidene difluoride);
TEM, transmission electron microscopy; ThT, thioflavin T; TUNEL, terminal deoxynucletidyl transferase-mediated biotin-dUTP nick
end labeling.
5982 FEBS Journal 275 (2008) 5982–5993 ª 2008 The Authors Journal compilation ª 2008 FEBS
well with the extent of synaptic loss and severity of
cognitive impairment [3–9]. The higher cytotoxicity of
soluble Ab species compared with Ab fibrillar aggre-
gates supports a casual relationship between the pres-
ence of soluble Ab species and AD. It has been
demonstrated that soluble Ab oligomers inhibit many
critical neuronal activities, including long-term potenti-

ation – a classic model for synaptic plasticity and
memory loss in vivo and in culture [10–12].
Numerous experiments have demonstrated that Ab
generation and oligomerization occur intracellularly
[13–19]. While intracellular accumulation of Ab occurs
in the mitochondria, ER and Golgi, it is predominant
in multivesicular bodies and lysosomes [13]. The pres-
ence of intracellular Ab within multivesicular bodies
has been shown to be linked to cytosolic proteasome
inhibition [20–22]. Furthermore, it has been shown
that proteasome inhibition, both in vivo and in vitro,
leads to higher Ab levels [23]. As the proteasome is
primarily located within the cytosol, these findings
strongly support the notion that Ab is also located
within the cytosolic compartment.
Molecular chaperones are proteins that selectively
recognize and bind to exposed hydrophobic surfaces of
non-native proteins, subsequently preventing protein
aggregation and facilitating correct folding of non-
native proteins in vivo [24]. Molecular chaperones are
also involved in many important aspects of protein
homeostasis, degradation and subcellular trafficking
[25]. Consistent with this activity, it has been shown
that molecular chaperones, including heat-shock pro-
teins Hsp20, Hsp70 and Hsp90, prevent Ab aggrega-
tion [18,26–28]. Several molecular chaperones are also
known to be involved in the formation of toxic Ab
species. Ab oligomers with low molecular mass
(< 30 kDa) have been shown to form in vitro during
incubation of Ab and the molecular chaperone apoli-

poprotein J, which has been found in AD brains
[10,29]. However, Ab oligomers with a wide molecular
mass distribution (< 10 to > 100 kDa) are found in
the AD brain [30], suggesting that other factors are
involved in their formation.
Prefoldin (PFD) is a molecular chaperone that has
been proposed to play a general role in de novo protein
folding in archaea, and is known to assist in the bio-
genesis of actins, tubulins and possibly other proteins
in the cytosol of eukaryotes [24]. Eukaryotic PFD is
likely to bind to substrate proteins that exist in an
unfolded state, and transfer these to the cytosolic
chaperonin-containing TCP-1 (CCT) for functional
folding [31–33]. Archaeal PFDs from Methanobacterium
thermoautotrophicum and Pyrococcus horikoshii OT3
have also been shown to stabilize non-native proteins
and denatured actins prior to chaperonin-dependent
folding in vitro [34–38]. Eukaryotic and archaeal PFDs
possess a similar jellyfish-like structure consisting of a
double b-barrel assembly with six long and protruding
coiled coils [39,40]. Biochemical and structural studies
have indicated that these ‘tentacles’ bind to substrate
proteins [34,35,40]. In the current study, we demon-
strate that archaeal PFD from P. horikoshii OT3
produces soluble and toxic high-molecular-mass Ab
oligomers in vitro with a broad molecular mass distri-
bution (50–250 kDa) as found in AD brains [30]. As it
has been shown that eukaryotic PFD is homologous
to archaeal PFD [33,41] and is expressed in the human
brain [42], our results suggest a possible involvement

of PFD in the formation of toxic Ab oligomers in the
cytosol.
Results
Fibrillation of Ab peptide in the presence of PFD
In an effort to investigate the effects of PFD on fibril-
lation of Ab(1–42) peptide, the major factor responsi-
ble for AD [1], Ab fibrillation was examined by
monitoring levels of the fluorescent dye thioflavin T
(ThT) [43]. As shown in Fig. 1A, ThT fluorescence of
the Ab sample incubated at 50 °C in the absence of
PFD increased after a lag phase of about 1 h, and
reached a plateau within 5 h. Examination by trans-
mission electron microscopy (TEM) confirmed the for-
mation of amyloid fibrils (Fig. 2A). By contrast, when
Ab was incubated with an equimolar amount of PFD
at 50 °C, the increase in ThT fluorescence was inhib-
ited (Fig. 1A). This result suggests that Ab fibrillation
is inhibited by PFD. About a one-third molar ratio of
PFD to Ab was sufficient to inhibit Ab fibrillation
(Fig. 1B). TEM observations showed that no mature
amyloid fibrils were formed after 48 h incubation in
the presence of PFD (Fig. 2B). Intriguingly, small
particles and protofibrils were observed in samples
incubated with PFD. TEM photographs show that the
size of most particles was within 100 nm and that the
particles vary in shape (Fig. 2C). These structures
were not observed in control samples containing only
PFD (data not shown).
Incubated Ab samples were then subjected to analy-
sis by gel electrophoresis. Samples were separated by

SDS–PAGE and probed with a mouse monoclonal Ab
antibody (6E10) (Fig. 3). Most Ab aggregates that
formed in the absence of PFD were insoluble, and no
soluble oligomers were observed. On the other hand,
when Ab was incubated with PFD, high-molecular-
mass Ab oligomers with a broad range of molecular
M. Sakono et al. Formation of amyloid beta oligomers by prefoldin
FEBS Journal 275 (2008) 5982–5993 ª 2008 The Authors Journal compilation ª 2008 FEBS 5983
mass (50–250 kDa) were observed. Similar results were
obtained when Ab was incubated with a lower concen-
tration (1:10 ratio) of PFD at lower temperatures (37
and 42 °C) (data not shown). Ab oligomers formed in
the presence of PFD were also separated by native
PAGE and then subjected to western blot analysis
using Ab antibody. As shown in Fig. 4, Ab oligomers
with a broad range of molecular mass were also
detected using Ab antibody, which indicates that the
Ab oligomers were in a soluble form. The molecular
mass of Ab oligomers was greater than that deter-
mined by SDS–PAGE, possibly due to binding of
PFD molecules to Ab oligomers (as described below).
These results suggest that PFD inhibits Ab peptide
fibrillation and induces the formation of high-molecu-
lar-mass soluble Ab oligomers with a size distribution
similar to that found in AD brains [30].
Dot-blot assay
In order to examine structural characteristics of the
soluble Ab oligomers formed in the presence of PFD,
binding to A11 antibody was examined. A11 antibody
recognizes prefibrillar Ab oligomers and protofibrils

and does not react with Ab monomer or fibrils
[7,44,45]. A11-positive Ab oligomers were prepared as
previously described [44,45]. Interestingly, soluble Ab
oligomers formed in the presence of PFD were not rec-
ognized by A11 antibody (Fig. 5). Weak A11 immuno-
reactivity of the Ab ⁄ PFD sample was observed, but
this might be due weak immunoreactivity with PFD
rather than Ab, as shown in Fig. 5. This result sug-
gests that the Ab oligomer conformation is different
from that of A11-positive Ab oligomers. This is
consistent with recent results that suggest multiple Ab
intermediate conformations [44,45].
Interaction between Ab oligomers and PFD
In an effort to elucidate the molecular mechanism of
soluble Ab oligomer formation, binding of PFD with
Ab oligomers formed in the presence of PFD was
analyzed by native PAGE ⁄ western blot analysis using
Ab antibody and PFD antibody. As shown in Fig. 4,
PFD that was bound to Ab oligomers of higher molec-
ular mass was detected using PFD antibody. This
result indicates that Ab oligomers are formed as a
complex with PFD. The higher molecular mass of Ab
oligomers than that shown by SDS–PAGE also
supports formation of a complex between PFD and
Ab oligomers.
Toxicity of Ab oligomers
Soluble Ab oligomers are highly cytotoxic and are
found in AD brains, and are therefore considered to
be the causative agents of the disease [3–6]. We exam-
+ PFD

– PFD
Δ
ThT fluorescence intensity (A.U.)
Incubation time (h)
0
10
20
30
40
50
A
B
0 1020304050
Δ
ThT fluorescence
intensity (A.U.)
Incubation time (h)
0
10
20
30
40
50
0246810
0
10
20
30
40
50

60
0 0.2 0.4 0.6 0.8 1
ThT fluorescence intensity (A.U.)
[PFD]/[A
β
]
Fig. 1. Ab aggregation monitored by ThT fluorescence. (A) Time
course of Ab aggregation monitored by ThT fluorescence. Ab sam-
ples incubated in the presence (+PFD, closed circles) or absence
()PFD, open circles) of PFD were withdrawn at various time inter-
vals and added to ThT solution. Changes in the ThT fluorescence at
482 nm from the incubation time (0 h) are shown as DThT fluore-
scence. The inset is an enlargement of the curves from 0–10 h.
(B) ThT fluorescence of Ab samples incubated with various PFD
concentrations. A 30 l
M aliquot of Ab was incubated with PFD
(0, 1, 3, 5, 10, 15, 25 and 30 l
M)at50°C for 24 h, and added to
ThT solution. The x axis indicates the ratio of PFD concentration to
Ab concentration.
Formation of amyloid beta oligomers by prefoldin M. Sakono et al.
5984 FEBS Journal 275 (2008) 5982–5993 ª 2008 The Authors Journal compilation ª 2008 FEBS
ined the cytotoxicity of soluble Ab oligomers produced
by the addition of PFD. Ab aggregates of various con-
centrations were added to the culture medium of rat
pheochromocytoma PC12 cells, and cell viability was
assayed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) (Fig. 6A). Addition of up
to 1 lm of Ab fibrils formed in the absence of PFD
did not induce any major changes in cell viability.

50 nm
100 nm
A
C
B
100 nm
Fig. 2. Morphology of Ab aggregates formed in the presence of PFD. (A) Ab fibrils formed in the absence of PFD. Scale bar = 100 nm. (B)
Ab particles and protofibrils formed in the presence of PFD. Arrows indicate Ab particles shown in (C). Scale bar = 100 nm. (C) Examples of
the Ab particles shown in (B). Scale bar = 50 nm.
M. Sakono et al. Formation of amyloid beta oligomers by prefoldin
FEBS Journal 275 (2008) 5982–5993 ª 2008 The Authors Journal compilation ª 2008 FEBS 5985
However, PC12 cell death was observed upon addition
of 5 lm Ab fibrils formed in the absence of PFD. By
contrast, addition of only 0.05 lm soluble Ab oligo-
mers formed in the presence of PFD markedly induced
PC12 cell death. The observed level of cytotoxicity was
similar to that of Ab-derived diffusible ligands
(ADDL) [46]. Control samples containing only PFD in
the same concentration range showed no detectable
cell toxicity (Fig. 6B). Taken together with our obser-
vations concerning molecular size, these results support
our hypothesis that PFD mediates formation of Ab
oligomers similar to those found in AD brains.
Apoptosis assay of cell death induced by
Ab aggregates
Ab peptides have been shown to induce apoptosis [47].
In an effort to determine whether this is also true of
soluble Ab oligomers produced in the presence of
PFD, we examined DNA fragmentation and activation
of the caspase cascade. DNA fragmentation in PC12

cells incubated with PFD-induced Ab oligomers was
observed by green fluorescence using terminal deoxy-
nucleotidyl transferase-mediated biotin-dUTP nick end
labeling (TUNEL) (Fig. 7A). By contrast, only low-
level green fluorescence was detected in PC12 cells
incubated with Ab fibrils formed in the absence of
PFD and in control cells incubated with NaCl ⁄ P
i
. This
result is consistent with the results of the MTT assay,
indicating a higher toxicity of PFD-induced Ab oligo-
mers compared with Ab fibrils (Fig. 6A).
We also examined caspase-3 activation in PC12 cells.
PC cells exposed to 1 lm Ab samples incubated in the
presence or absence of PFD were lysed and then
subjected to western blotting analysis using caspase-3
antibody and a control b-actin antibody (Fig. 7B).
Activated caspase-3 was detected within 3 h of incuba-
tion in PFD-induced Ab oligomer samples, but was
barely detected even after 9 h of incubation in samples
of Ab fibrils formed in the absence of PFD. Therefore,
we conclude that soluble Ab oligomers formed in the
presence of PFD induce PC12 cell death via apoptotic
pathways.
Discussion
Several molecular chaperones are involved in the for-
mation of the low-molecular-mass (< 30 kDa) soluble
Ab oligomers or protofibrils that have been indicated
as the causative agents of AD [10,29,48]. Here we
10

m (kDa)
– PFD
Incubation time (h)
+ PFD
0
48
0
48
20
37
150
250
100
75
50
25
15
Fig. 3. SDS–PAGE analysis of Ab aggregates. Samples incubated
for 0 or 48 h with PFD (+PFD) or without PFD ()PFD) were sepa-
rated by SDS–PAGE (10–20% gels), probed using a mouse mono-
clonal Ab antibody (6E10), and visualized by chemiluminescence.
anti-Aβ
anti-PFD
1:Aβ/PFD
3:PFD
alone
2:Aβ monomer
alone
anti-Aβ
anti-PFD

Antibody
669
440
232
140
66
m (kDa)
Fig. 4. Native PAGE analysis of soluble Ab oligomers formed in the
presence of PFD. Ab aggregates formed in the presence of PFD
(Ab ⁄ PFD) were analyzed by native PAGE, and probed using a
mouse monoclonal Ab antibody (6E10) or a mouse polyclonal PFD
antibody as indicated. PFD bound to Ab oligomers is indicated by
an arrow. The samples used comprised 5 lLof50l
M sample mix-
ture. The same amount of Ab monomer alone and PFD alone were
used as control samples. An HMW native marker kit (GE Health-
care) comprising thyroglobulin (669 kDa), ferritin (440 kDa), catalase
(232 kDa), lactate dehydrogenase (140 kDa) and albumin (66 kDa)
was used as a molecular mass marker.
Formation of amyloid beta oligomers by prefoldin M. Sakono et al.
5986 FEBS Journal 275 (2008) 5982–5993 ª 2008 The Authors Journal compilation ª 2008 FEBS
report our novel findings that the molecular chaperone
PFD induces in vitro formation of soluble Ab oligo-
mers with a high molecular mass (50–250 kDa) similar
to that found in AD brains. Soluble Ab oligomers
formed in the presence of PFD were more toxic
compared with Ab fibrils, and exhibited similar
toxicities as ADDL via apoptotic cell-death pathways
(Figs 6 and 7). These data suggest that PFD might
also participate in the in vivo formation of highly toxic

Ab oligomers that lead to AD development.
Recently, it has been reported that Ab oligomeriza-
tion also occurs intracellularly [13–19]. Takahashi et al.
reported the existence of intracellular soluble Ab oligo-
mers in Tg2576 transgenic mice [17], and Walsh et al.
showed that soluble oligomers are preferentially pro-
duced intracellularly rather than extracellularly [16].
More importantly, inhibition of cytosolic proteasomes
by Ab implies that Ab is located within the cytosolic
compartment [13,20–23]. It has been shown that a
PFD-like gene is expressed in the human brain [42].
These observations support the notion that PFD
participates in the formation of Ab oligomers within
the cytosolic compartment.
In an effort to elucidate the mechanism pertaining
to the PFD-induced formation of high-molecular-mass
soluble Ab oligomers, we examined their interaction
with PFD. As shown in Fig. 4, bound PFD was
detected in soluble Ab oligomers. Figure 8 shows a
hypothetical model relating to the PFD-induced for-
mation of soluble Ab oligomers. In this model, PFD
inhibits or slows the oligomerization of Ab peptides by
binding to the peptides in their oligomeric state.
Figure 1B suggests that the number of PFD molecules
binding to one Ab oligomer molecule is at the most
one-third the number of Ab molecules in one oligomer,
which suggests that their interaction is non-specific.
Binding of PFD to protofibrils is indirectly supported
by TEM observations indicating that no Ab fibrils
were formed in the presence of PFD (Fig. 2). It is

plausible that soluble Ab oligomers with a wide range
of molecular mass are produced due to repeated PFD
binding and release, as the binding of PFD to sub-
strate proteins was shown to be in dynamic equilib-
rium [36]. This might also account for the fact that
PFD has not been identified as one of the proteins that
A
B
Aβ concentration (μM)
Cell viability (%)
+ PFD
– PFD
0
20
40
60
80
100
0.01 0.05 0.1 0.2 0.5 1 5
PFD concentration (μM)
Cell viability (%)
0
20
40
60
80
100
120
0.1 0.2 0.5 1 5
Fig. 6. Cytotoxicity assays of soluble Ab oligomers formed in the

presence of PFD against PC12 cells using the MTT method. (A) Ab
aggregates formed with PFD (+PFD, black bars) or without PFD
()PFD, white bars) were incubated with cells at the indicated
monomer concentrations. (B) PFD was incubated with cells at the
indicated concentrations.
Aβ/PFD
PFD
Aβ fibril
Antibody
A11
6E10
A
11-positive
Aβ oligomer
Fig. 5. Dot-blot assay of soluble Ab oligomers formed in the pres-
ence of PFD. Samples of Ab oligomers formed in the presence of
PFD (Ab ⁄ PFD), Ab fibrils formed in the absence of PFD (Ab fibril),
A11-positive Ab oligomers and PFD alone were prepared. Aliquots
were spotted onto nitrocellulose membranes and probed with A11
and 6E10 antibodies.
M. Sakono et al. Formation of amyloid beta oligomers by prefoldin
FEBS Journal 275 (2008) 5982–5993 ª 2008 The Authors Journal compilation ª 2008 FEBS 5987
bind to Ab peptides, as determined by co-immunopre-
cipitation studies [49,50]. It should be noted that PFD
does not facilitate or catalyze oligomer formation in
this model. This is supported by our observation of a
ThT fluorescence time lag, which was not shortened by
the addition of PFD (Fig. 1A). Further studies are
necessary to determine the precise mechanism of
PFD-mediated oligomer formation.

Archaeal PFD shares many biochemical and struc-
tural characteristics with eukaryotic PFD [32–41,51].
Both archaeal and eukaryotic PFDs share a jellyfish-like
structure [39,40], and can bind and stabilize newly syn-
thesized or denatured proteins, and subsequently escort
these to chaperonins for further assembly or final fold-
ing into active conformations. In addition, archaeal
PFDs are homologous to eukaryotic PFDs [33,41]. Six
distinct subunits of eukaryotic PFD can be grouped into
two separate classes corresponding to the archaeal ones,
represented by PFD3 ⁄ 5 (the a-subunit) and
PFD1 ⁄ 2 ⁄ 4 ⁄ 6 (the b-subunit). The results of secondary
structure prediction for human PFD showed that each
human PFD subunit contains central b-hairpin(s)
flanked N- and C-terminally by coiled-coil helices, simi-
lar to Pyrococcus PFD (Fig. S1). The coiled-coil helices
within each Pyrococcus PFD subunit assemble in an
antiparallel orientation [51]. The result of primary
sequence alignment also showed that Pyrococcus PFD
shares high sequence identity to human PFD (59 and
62% similarity for the a-subunit to PFD3 and PFD5,
respectively, and 62, 53, 58 and 68% similarity for the
b-subunit to PFD1, PFD2, PFD4 and PFD6, respec-
Activated caspase-3
β-actin
h3 6 9
369
+ PFD
– PFD
+ PFD

A
B
– PFD
Tunel
PI
Procaspase-3
Fig. 7. Apoptosis assays. (A) DNA cleavage analyzed by the
TUNEL assay. PC12 cells were incubated with soluble Ab oligo-
mers formed in the presence of PFD (+PFD) or Ab fibrils formed
without PFD ()PFD), and TUNEL-positive green fluorescence was
observed when DNA was cleaved into fragments (right). PI label-
ing indicates DNA in whole-cell nuclei (PI, left). (B) Time course of
caspase-3 activation. PC12 cells were exposed to Ab aggregates
formed in the presence (+PFD) or absence ()PFD) of PFD for 3, 6
or 9 h. Equal amounts of proteins were separated on SDS–PAGE
using 10–20% gradient gels and probed using rabbit polyclonal
caspase-3 antibody or mouse monoclonal b-actin antibody as a
control.
A
β monomer
PFD
Aβ fibril
Aβ soluble oligomer
LMW oligomer
HMW oligomer
Inhibit/Slow down oligomerization
Aβ protofibril
Fig. 8. Schematic model of the formation of
soluble Ab oligomers in the presence of
PFD.

Formation of amyloid beta oligomers by prefoldin M. Sakono et al.
5988 FEBS Journal 275 (2008) 5982–5993 ª 2008 The Authors Journal compilation ª 2008 FEBS
tively; Fig. S1). More interestingly, hydrophobic resi-
dues located at the first (a) and fourth (d) positions of
the heptad repeat (abcdefg) of the coiled-coil helices are
well conserved in both PFDs. It has been shown that
the partially buried hydrophobic residues in these a ⁄ d
positions, which are conserved in the coiled coils of vari-
ous archaeal PFDs, are important for interaction and
stabilization of a non-native substrate [35]. Thus it is
plausible that human PFD also utilizes these hydropho-
bic residues in the coiled coils to interact with its sub-
strate. The b-subunit of Pyrococcus PFD also shares
high sequence identity to the PFD-like protein (57%
similarity) found in the human brain [42]. It should be
noted that the isoelectric point of Ab(1–42) peptide
(5.24) calculated from the amino acid sequence is similar
to that of known substrates of eukaryotic PFD, namely
b-actin (5.18) and b-tubulin (4.64), suggesting that Ab
peptide is a potential substrate for eukaryotic PFD. This
idea is supported by the fact that there are hydrophilic
residues at the tips of the eukaryotic PFD tentacles that
appear to be important for interaction with substrate
proteins [40,52,53]. Although archaeal PFDs have been
considered to bind a wide range of substrates through a
set of hydrophobic residues located at the tips of the
tentacles [34,35,39,52], it has also been shown that there
are basic residues in the distal regions of the tentacles of
Pyrococcus PFD used in this study that might be impor-
tant for their interaction with chaperonin [37]. Thus, it

is plausible that eukaryotic PFD could induce forma-
tion of Ab oligomers, as shown for archaeal PFD in this
study. This is speculative however, and further experi-
ments using eukaryotic PFD are required to clarify
possible involvement of PFD in AD pathology.
Experimental procedures
Materials
Ab(1–42), ThT, 1,1,1,3,3,3-hexa-fluoro-2-propanol (HFIP)
and RPMI-1640 medium were purchased from Sigma (St
Louis, MO, USA). P. horikoshii PFD was expressed in Esc-
herichia coli BL21 (DE3), and purified as previously
described [38]. Rabbit polyclonal caspase-3 antibody was
purchased from Calbiochem (San Diego, CA, USA). Mouse
monoclonal b-actin antibody and mouse monoclonal Ab
antibody (6E10) were purchased from Abcam (Cambridge,
UK). A11 anti-oligomer rabbit polyclonal antibody was pur-
chased from BioSource (Camarillo, CA, USA). Rat poly-
clonal antibody to Thermoccocus PFD, which is highly
similar to P. horikoshii PFD [54], was a kind gift from
T. Yoshida (Extremobiosphere Research Center, Japan
Agency for Marine-Earth Science and Technology, Kana-
gawa, Japan). Horseradish peroxidase-conjugated anti-rabbit
IgG and horseradish peroxidase-conjugated anti-mouse IgG
were purchased from R&D systems (Minneapolis, MN,
USA). Enhanced chemiluminescence and western blotting
detection systems were purchased obtained from Amersham
Biosciences (Chalfont St Giles, UK). The cell proliferation
kit (MTT) and the DeadEnd fluorometric TUNEL system
were purchased from Roche (Indianapolis, IN, USA) and
Promega (Madison, WI, USA), respectively.

Preparation of Ab aggregates
Lyophilized Ab(1–42) peptide (2 mgÆmL
)1
) was dissolved in
HFIP, dried using a spin-vacuum system, and stored at
)80 °C. HFIP-treated peptide was dissolved to 1 mm in
distilled water with vortexing and sonication, immediately
diluted to 50 lm in NaCl ⁄ P
i
with or without 50 lm PFD,
and then incubated at 50 °C for 48 h.
ThT fluorescence assay
Ab fibrillation was assessed by the ThT assay as described
previously [43]. For the time-course assay, 30 lm peptide
sample was incubated with or without 30 lm PFD in
NaCl ⁄ P
i
at 50 °C. Aliquots (2 lL) of the sample were
withdrawn from the incubation mixture at various time
intervals (0–48 h), and then added to 238 lLof50mm
glycine–NaOH (pH 8.5) buffer containing 5 lm ThT.
Changes in the ThT fluorescence from the incubation time
(0 h) are shown as DThT fluorescence. Peptide samples
(30 lm) were also incubated with PFD of various concen-
trations (0, 1, 3, 5, 10, 15, 25 and 30 lm) in NaCl ⁄ P
i
at
50 °C for 24 h. Aliquots (2 lL) of the sample were added
to 238 lLof5lm ThT solution. Each sample was excited
at 445 nm (band width 3 nm), and the emission was

recorded at 482 nm on a spectrofluorometer (FP-6500;
Jasco, Tokyo, Japan). The fluorescence intensity of 5 lm
ThT solution was used for background subtraction.
TEM
The sample incubated at 50 °C for 48 h in the presence or
absence of PFD was diluted 10-fold with distilled water
and placed on a carbon-coated copper grid and allowed to
adsorb. Excess sample was removed from the grid using
filter paper, and the grid was air-dried prior to negative
staining with uranyl acetate. Excess stain was then removed
from the grid by air drying. Samples were observed with an
excitation voltage of 100 kV using a JEM-1011 transmis-
sion electron microscope (JEOL, Tokyo, Japan).
Analysis of Ab aggregates by
SDS–PAGE/western blotting
The sample mixture (5 lL) was diluted with 5 lL SDS
loading buffer containing 10% b-mercaptoethanol and then
M. Sakono et al. Formation of amyloid beta oligomers by prefoldin
FEBS Journal 275 (2008) 5982–5993 ª 2008 The Authors Journal compilation ª 2008 FEBS 5989
denatured at 98 °C for 3 min. Following separation by
SDS–PAGE using 10–20% Tris–glycine gels for 60 min and
a constant current of 20 mA, proteins were transferred onto
poly(vinylidene difluoride) (PVDF) membranes (Millipore,
Billerica, MA, USA) for 2 h using a constant current of
140 mA. For immunoblotting, the blot was blocked over-
night in blocking reagent (Roche, Switzerland) at 4 °C.
After washing away unbound material using NaCl ⁄ Tris
containing 0.05% Tween-20 (0.05% NaCl ⁄ Tris-T), the
membrane was incubated with mouse monoclonal Ab anti-
body (6E10, 1 : 2000) for 40 min at 37 °C, followed by sec-

ondary horseradish peroxidase-conjugated anti-mouse IgG
(1 : 2000). Proteins were visualized using the ECL Plus
blotting detection system (Amersham Biosciences) accord-
ing to the manufacturer’s instructions.
Analysis of Ab aggregates by native
PAGE/western blotting
The sample mixture (5 lL) was diluted with 5 lL native
PAGE sample buffer and then subjected to native PAGE
using a Tris–glycine 10–20% gradient precast gel (Wako,
Osaka, Japan). Samples containing Ab monomer alone or
PFD alone were used as control samples. Following trans-
fer to PVDF membrane, blots were probed using mouse
monoclonal Ab antibody (6E10, 1 : 2000) or rat polyclonal
PFD antibody (1 : 2000). Bound antibodies were visualized
as described above. An HMW native marker kit (GE
Healthcare, Chalfont St Giles UK) was used as the mole-
cular mass marker.
Dot-blot assay
The dot-blot assay was performed as previously described
[7]. Peptide samples (30 l m) were incubated with or without
30 lm PFD in NaCl ⁄ P
i
at 50 °C. The sample mixture (3 lL)
was spotted onto nitrocellulose membrane (0.22 l m; What-
man, Kent, UK). After blocking with 10% skim milk and
0.01% NaCl ⁄ Tris-T for 1 h at room temperature, the mem-
brane was incubated with rabbit polyclonal antibody to the
oligomer (A11, 1 : 500) or with mouse monoclonal Ab anti-
body (6E10, 1 : 2000) for 1 h at room temperature, followed
by incubation with secondary horseradish peroxidase-conju-

gated anti-rabbit or anti-mouse IgG (each 1 : 2000) for 1 h
at room temperature. Proteins were visualized as described
above. To prepare A11-positive Ab oligomers as a control
sample, 45 lm Ab(1–42) peptide samples diluted from
NaOH stock (2 mm Ab dissolved in 100 mm NaOH) were
incubated in NaCl ⁄ P
i
at 25 °C for 7 days as described previ-
ously [44]. Aliquots (2 lL) were spotted onto the membrane.
Toxicity assay
Cell viability was determined by the MTT reduction assay
[55] according to the manufacturer’s instructions (Roche).
Rat PC12 cells (American Type Culture Collection,
Manassas, VA, USA) were plated on poly-d-lysine-coated
dishes in RPMI-1640 medium containing 10% heat-inacti-
vated horse serum, 5% heat-inactivated fetal bovine serum,
100 UÆmL
)1
penicillin, and 100 lgÆmL
)1
streptomycin in
humidified 5% CO
2
incubators at 37 °C. The medium was
replaced every 2 days.
PC12 cells (5 · 10
3
per well) were plated in 96-well plates
coated with poly-d-lysine, and covered with 100 lL culture
medium. Following plating, 20 lL medium was removed

from each well, and replaced with the same volume of Ab
sample diluted in NaCl ⁄ P
i
at various concentrations, which
were taken from the 50 lm Ab samples incubated with or
without PFD at 50 °C for 48 h. As a control, the culture
medium was replaced with the same volume of PFD sam-
ples in NaCl ⁄ P
i
at various concentrations instead of Ab
samples. The cultures were incubated for 24 h, and then
10 lLof5mgÆmL
)1
MTT solution was added to each well
and incubated for a further 4 h. Following incubation,
100 lL of 10% SDS in 0.01 m HCl was added to each well,
and the cultures were incubated overnight. The adsorption
values at 550 nm were determined using a model 680
microplate reader (Bio-Rad, Hercules, CA, USA).
TUNEL assay
Apoptosis was detected by performing a TUNEL assay
according to the manufacturer’s instructions (Promega).
Briefly, PC12 cells were grown in poly-d-lysine-coated slide
chambers (4 · 10
5
cellsÆmL
)1
), and 1 lm Ab aggregate was
added to the culture medium. Cultures were incubated for
24 h, fixed with 4% paraformaldehyde in NaCl ⁄ P

i
for
25 min at 4 °C, permeabilized using 0.2% Triton X-100 in
NaCl ⁄ P
i
for 5 min at room temperature, and then incubated
with propidium iodide (PI) and fluorescent-labeled
nucleotide in the presence of terminal deoxynucleotidyl
transferase. Cells were then examined using a fluorescence
microscope (IX71; Olympus, Tokyo, Japan). Fluorescein and
PI were detected using U-MGFPHQ (excitation = 460–
480 nm, emission = 495–540 nm) and U-MWIG2 (excita-
tion = 520–550 nm, emission > 580 nm) filter cubes.
Detection of activated caspase-3
PC12 cells exposed to Ab samples incubated with or with-
out PFD for predefined times (3, 6 or 9 h) were lysed in
RIPA buffer comprising 50 mm Tris ⁄ HCl (pH 7.2),
150 mm NaCl, 1% Triton X-100, 0.05% SDS, 1 mm EDTA
and 1 mm MgCl
2
. Protein concentrations were determined
by the Bradford assay using BSA as a standard. Equal
amounts of proteins were separated on a Tris–glycine 10–
20% gradient precast gel, transferred to a PVDF mem-
brane, probed using mouse monoclonal b-actin antibody
(1 : 2000) or rabbit polyclonal caspase-3 antibody
(1 : 2000), and then detected as described above.
Formation of amyloid beta oligomers by prefoldin M. Sakono et al.
5990 FEBS Journal 275 (2008) 5982–5993 ª 2008 The Authors Journal compilation ª 2008 FEBS
Acknowledgements

We thank Dr Takao Yoshida, Japan Agency for Mar-
ine-Earth Science and Technology (JAMSTEC) for
providing the antibody to Thermococcus PFD. Funds
for this research were provided by RIKEN (M.S., T.Z.
and M.M.) and the Ministry of Education, Science,
Sports, Culture and Technology of Japan (MEXT)
(T.Z., M.Y. and M.M.). M.S. is Special Postdoctoral
Researcher of RIKEN.
References
1 Selkoe DJ (2001) Alzheimer’s disease: genes, proteins,
and therapy. Physiol Rev 81, 741–766.
2 Terry RD, Maslia E & Hansen LA (1999) The neuropa-
thology of Alzheimer disease and the structural basis of
its cognitive alterations. In Alzheimer Disease (Terry
RD, Katzman R, Bick KL & Sisodia SS, eds), pp. 187–
206. Lipponcott Williams and Wilikins, Philadelphia,
PA.
3 Klein WL, Krafft GA & Finch CE (2001) Targeting
small Ab oligomers: the solution to an Alzheimer’s dis-
ease conundrum? Trends Neurosci 24, 219–224.
4 Hardy J & Selkoe DJ (2002) The amyloid hypothesis of
Alzheimer’s disease: progress and problems on the road
to therapeutics. Science 297, 353–356.
5 Haass C & Selkoe DJ (2007) Soluble protein oligomers
in neurodegeneration: lessons from the Alzheimer’s
amyloid b-peptide. Nat Rev Mol Cell Biol 8, 101–112.
6 Walsh DM & Selkoe DJ (2007) Ab oligomers – a dec-
ade of discovery. J Neurochem 101, 1172–1184.
7 Kayed R, Head E, Thompson JL, McIntire TM, Milton
SC, Cotman CW & Glabe CG (2003) Common struc-

ture of soluble amyloid oligomers implies common
mechanism of pathogenesis. Science 300, 486–489.
8 Lesne S, Koh MT, Kotilinek L, Kayed R, Glabe CG,
Yang A, Gallagher M & Ashe KH (2006) A specific
amyloid-b protein assembly in the brain impairs mem-
ory. Nature 440, 352–357.
9 Caughey B & Lansbury PT (2003) Protofibrils, pores,
fibrils, and neurodegeneration: separating the responsi-
ble protein aggregates from the innocent bystanders.
Annu Rev Neurosci 26, 267–298.
10 Lambert MP, Barlow AK, Chromy BA, Edwards C,
Freed R, Liosatos M, Morgan TE, Rozovsky I,
Trommer B, Viola KL et al. (1998) Diffusible,
nonfibrillar ligands derived from Ab1–42 are potent
central nervous system neurotoxins. Proc Natl Acad
Sci USA 95, 6448–6453.
11 Wang HW, Pasternak JF, Kuo H, Ristic H, Lambert
MP, Chromy B, Viola KL, Klein WL, Stine WB, Krafft
GA et al. (2002) Soluble oligomers of b amyloid (1–42)
inhibit long-term potentiation but not long-term depres-
sion in rat dentate gyrus. Brain Res 924, 133–140.
12 Walsh DM, Klyubin I, Fadeeva JV, Cullen WK, Anwyl
R, Wolfe MS, Rowan MJ & Selkoe DJ (2002) Natu-
rally secreted oligomers of amyloid b protein potently
inhibit hippocampal long-term potentiation in vivo.
Nature 416, 535–539.
13 Laferla FM, Green KN & Oddo S (2007) Intracellular
amyloid-b in Alzheimer’s disease. Nat Rev Neurosci 8,
499–509.
14 Echeverria V & Cuello AC (2002) Intracellular A-beta

amyloid, a sign for worse things to come? Mol Neuro-
biol 26
, 299–316.
15 Tabira T, Chui DH & Kuroda S (2002) Significance
of intracellular Ab42 accumulation in Alzheimer’s dis-
ease. Front Biosci 7, a44–49.
16 Walsh DM, Tseng BP, Rydel RE, Podlisny MB &
Selkoe DJ (2000) The oligomerization of amyloid
beta-protein begins intracellularly in cells derived from
human brain. Biochemistry 39, 10831–10839.
17 Takahashi RH, Almeida CG, Kearney PF, Yu F, Lin
MT, Milner TA & Gouras GK (2004) Oligomerization
of Alzheimer’s b-amyloid within processes and synapses
of cultured neurons and brain. J Neurosci 24, 3592–
3599.
18 Magrane J, Smith RC, Walsh K & Querfurth HW
(2004) Heat shock protein 70 participates in the
neuroprotective response to intracellularly expressed
b-amyloid in neurons. J Neurosci 24, 1700–1706.
19 Wirths O, Multhaup G & Bayer TA (2004) A modified
b-amyloid hypothesis: intraneuronal accumulation
of the b-amyloid peptide – the first step of a fatal cas-
cade. J Neurochem 91 , 513–520.
20 Gregori L, Fuchs C, Figueiredo-Pereira ME, Van
Nostrand WE & Goldgaber D (1995) Amyloid b-pro-
tein inhibits ubiquitin-dependent protein degradation in
vitro. J Biol Chem 270, 19702–19708.
21 Oh S, Hong HS, Hwang E, Sim HJ, Lee W, Shin SJ &
Mook-Jung I (2005) Amyloid peptide attenuates the
proteasome activity in neuronal cells. Mech Ageing Dev

126, 1292–1299.
22 Almeida CG, Takahashi RH & Gouras GK (2006)
b-amyloid accumulation impairs multivesicular body
sorting by inhibiting the ubiquitin-proteasome system.
J Neurosci 26, 4277–4288.
23 Tseng BP, Green KN, Chan JL, Blurton-Jones M &
Laferla FM (2008) Ab inhibits the proteasome and
enhances amyloid and tau accumulation. Neurobiol
Aging 29, 1607–1618.
24 Hartl FU & Hayer-Hartl M (2002) Molecular chaper-
ones in the cytosol: from nascent chain to folded
protein. Science 295, 1852–1858.
25 Bukau B, Weissman J & Horwich A (2006) Molecular
chaperones and protein quality control. Cell 125, 443–451.
26 Muchowski PJ & Wacker JL (2005) Modulation of
neurodegeneration by molecular chaperones. Nat Rev
Neurosci 6, 11–22.
M. Sakono et al. Formation of amyloid beta oligomers by prefoldin
FEBS Journal 275 (2008) 5982–5993 ª 2008 The Authors Journal compilation ª 2008 FEBS 5991
27 Evans CG, Wisen S & Gestwicki JE (2006) Heat shock
proteins 70 and 90 inhibit early stages of amyloid
b-(1–42) aggregation in vitro. J Biol Chem 281, 33182–
33191.
28 Lee S, Carson K, Rice-Ficht A & Good T (2005)
Hsp20, a novel a-crystallin, prevents Ab fibril
formation and toxicity. Protein Sci 14, 593–601.
29 Oda T, Wals P, Osterburg HH, Johnson SA, Pasinetti
GM, Morgan TE, Rozovsky I, Stine WB, Snyder SW,
Holzman TF et al. (1995) Clusterin (apoJ) alters the
aggregation of amyloid b-peptide (Ab 1–42) and forms

slowly sedimenting Ab complexes that cause oxidative
stress. Exp Neurol 136, 22–31.
30 Kuo YM, Emmerling MR, Vigo-Pelfrey C, Kasunic
TC, Kirkpatrick JB, Murdoch GH, Ball MJ & Roher
AE (1996) Water-soluble Ab (N-40, N-42) oligomers in
normal and Alzheimer disease brains. J Biol Chem 271,
4077–4081.
31 Hansen WJ, Cowan NJ & Welch WJ (1999) Prefoldin-
nascent chain complexes in the folding of cytoskeletal
proteins. J Cell Biol 145, 265–277.
32 Siegers K, Waldmann T, Leroux MR, Grein K,
Shevchenko A, Schiebel E & Hartl FU (1999)
Compartmentation of protein folding in vivo: sequestra-
tion of non-native polypeptide by the chaperonin–GimC
system. EMBO J 18 , 75–84.
33 Vainberg IE, Lewis SA, Rommelaere H, Ampe C,
Vandekerckhove J, Klein HL & Cowan NJ (1998)
Prefoldin, a chaperone that delivers unfolded proteins
to cytosolic chaperonin. Cell 93, 863–873.
34 Okochi M, Nomura T, Zako T, Arakawa T, Iizuka R,
Ueda H, Funatsu T, Leroux M & Yohda M (2004)
Kinetics and binding sites for interaction of the prefol-
din with a group II chaperonin: contiguous non-native
substrate and chaperonin binding sites in the archaeal
prefoldin. J Biol Chem 279, 31788–31795.
35 Lundin VF, Stirling PC, Gomez-Reino J, Mwenifumbo
JC, Obst JM, Valpuesta JM & Leroux MR (2004)
Molecular clamp mechanism of substrate binding by
hydrophobic coiled-coil residues of the archaeal
chaperone prefoldin. Proc Natl Acad Sci USA 101,

4367–4372.
36 Zako T, Iizuka R, Okochi M, Nomura T, Ueno T,
Tadakuma H, Yohda M & Funatsu T (2005) Facilitated
release of substrate protein from prefoldin by chapero-
nin. FEBS Lett 579, 3718–3724.
37 Zako T, Murase Y, Iizuka R, Yoshida T, Kanzaki T,
Ide N, Maeda M, Funatsu T & Yohda M (2006) Local-
ization of prefoldin interaction sites in the hyperthermo-
philic group II chaperonin and correlations between
binding rate and protein transfer rate. J Mol Biol 364,
110–120.
38 Okochi M, Yoshida T, Maruyama T, Kawarabayasi Y,
Kikuchi H & Yohda M (2002) Pyrococcus prefoldin sta-
bilizes protein-folding intermediates and transfers them
to chaperonins for correct folding. Biochem Biophys Res
Commun 291, 769–774.
39 Siegert R, Leroux MR, Scheufler C, Hartl FU &
Moarefi I (2000) Structure of the molecular chaperone
prefoldin: unique interaction of multiple coiled coil
tentacles with unfolded proteins. Cell 103, 621–632.
40 Martin-Benito J, Boskovic J, Gomez-Puertas P, Carras-
cosa JL, Simons CT, Lewis SA, Bartolini F, Cowan NJ
& Valpuesta JM (2002) Structure of eukaryotic prefol-
din and of its complexes with unfolded actin and the
cytosolic chaperonin CCT. EMBO J 21, 6377–6386.
41 Leroux MR, Fandrich M, Klunker D, Siegers K, Lupas
AN, Brown JR, Schiebel E, Dobson CM & Hartl FU
(1999) MtGimC, a novel archaeal chaperone related to
the eukaryotic chaperonin cofactor GimC ⁄ prefoldin.
EMBO J 18, 6730–6743.

42 Zhang J, Liu L, Zhang X, Jin F, Chen J, Ji C, Gu S,
Xie Y & Mao Y (2006) Cloning and characterization of
a novel human prefoldin and SPEC domain protein
gene (PFD6L) from the fetal brain. Biochem Genet 44,
69–74.
43 LeVine H III (1993) Thioflavine T interaction with syn-
thetic Alzheimer’s disease beta-amyloid peptides: detec-
tion of amyloid aggregation in solution. Protein Sci 2,
404–410.
44 Necula M, Kayed R, Milton S & Glabe CG (2007)
Small molecule inhibitors of aggregation indicate that
amyloid b oligomerization and fibrillization pathways
are independent and distinct. J Biol Chem 282, 10311–
10324.
45 Kayed R, Head E, Sarsoza F, Saing T, Cotman CW,
Necula M, Margol L, Wu J, Breydo L, Thompson JL
et al. (2007) Fibril specific, conformation dependent
antibodies recognize a generic epitope common to
amyloid fibrils and fibrillar oligomers that is absent in
prefibrillar oligomers. Mol Neurodegener 2, 18.
46 Chromy BA, Nowak RJ, Lambert MP, Viola KL,
Chang L, Velasco PT, Jones BW, Fernandez SJ,
Lacor PN, Horowitz P et al. (2003) Self-assembly of
Ab(1–42) into globular neurotoxins. Biochemistry 42,
12749–12760.
47 Loo DT, Copani A, Pike CJ, Whittemore ER,
Walencewicz AJ & Cotman CW (1993) Apoptosis is
induced by beta-amyloid in cultured central nervous
system neurons. Proc Natl Acad Sci USA 90,
7951–7955.

48 Stege GJ, Renkawek K, Overkamp PS, Verschuure P,
van Rijk AF, Reijnen-Aalbers A, Boelens WC, Bosman
GJ & de Jong WW (1999) The molecular chaperone
aB-crystallin enhances amyloid b neurotoxicity.
Biochem Biophys Res Commun 262, 152–156.
49 Ray I, Chauhan A, Wisniewski HM, Wegiel J, Kim KS
& Chauhan VP (1998) Binding of amyloid beta-protein
to intracellular brain proteins in rat and human. Neuro-
chem Res 23, 1277–1282.
Formation of amyloid beta oligomers by prefoldin M. Sakono et al.
5992 FEBS Journal 275 (2008) 5982–5993 ª 2008 The Authors Journal compilation ª 2008 FEBS
50 Oyama R, Yamamoto H & Titani K (2000) Glutamine
synthetase, hemoglobin a-chain, and macrophage
migration inhibitory factor binding to amyloid
b-protein: their identification in rat brain by a novel
affinity chromatography and in Alzheimer’s disease
brain by immunoprecipitation. Biochim Biophys Acta
1479, 91–102.
51 Ohtaki A, Kida H, Miyata Y, Ide N, Yonezawa A,
Arakawa T, Iizuka R, Noguchi K, Kita A, Odaka M
et al. (2008) Structure and molecular dynamics simula-
tion of archaeal prefoldin: the molecular mechanism for
binding and recognition of nonnative substrate proteins.
J Mol Biol 376, 1130–1141.
52 Martin-Benito J, Gomez-Reino J, Stirling PC, Lundin
VF, Gomez-Puertas P, Boskovic J, Chacon P, Fernan-
dez JJ, Berenguer J, Leroux MR et al. (2007) Divergent
substrate-binding mechanisms reveal an evolutionary
specialization of eukaryotic prefoldin compared to its
archaeal counterpart. Structure 15, 101–110.

53 Rommelaere H, De Neve M, Neirynck K, Peelaers D,
Waterschoot D, Goethals M, Fraeyman N, Van-
dekerckhove J & Ampe C (2001) Prefoldin recognition
motifs in the nonhomologous proteins of the actin and
tubulin families. J Biol Chem 276, 41023–41028.
54 Iizuka R, Sugano Y, Ide N, Ohtaki A, Yoshida T, Fuji-
wara S, Imanaka T & Yohda M (2008) Functional
characterization of recombinant prefoldin complexes
from a hyperthermophilic archaeon, Thermococcus sp.
strain KS-1. J Mol Biol 377, 972–983.
55 Liu Y, Peterson DA, Kimura H & Schubert D (1997)
Mechanism of cellular 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) reduction.
J Neurochem 69, 581–593.
Supporting information
The following supplementary material is available:
Fig. S1. Sequence alignment of Pyrococcus prefoldin
subunits and human prefoldin subunits.
This supplementary material can be found in the
online version of this article.
Please note: Wiley-Blackwell Publishing is not
responsible for the content or functionality of any
supplementary materials supplied by the authors. Any
queries (other than missing material) should be
directed to the corresponding author for the article.
M. Sakono et al. Formation of amyloid beta oligomers by prefoldin
FEBS Journal 275 (2008) 5982–5993 ª 2008 The Authors Journal compilation ª 2008 FEBS 5993