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].

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-

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

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

– 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

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

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

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

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.

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.

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.

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-

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

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

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

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

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.

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.