Amyloid-like Aggregates Sequester

Numerous Metastable Proteins

with Essential Cellular Functions
Heidi Olzscha,1,4 Sonya M. Schermann,1,4 Andreas C. Woerner,1 Stefan Pinkert,1 Michael H. Hecht,2 Gian G. Tartaglia,3,5
Michele Vendruscolo,3 Manajit Hayer-Hartl,1,* F. Ulrich Hartl,1,* and R. Martin Vabulas1,*
1Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82159 Martinsried, Germany
2Department of Chemistry, Princeton University, Princeton, NJ 08544, USA
3Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK
4These authors contributed equally to this work
5Present address: Bioinformatics & Genomics Program, CRG Centre for Genomic Regulation, Dr. Aiguader 88, Barcelona 08003, Spain

*Correspondence: mhartl@biochem.mpg.de (M.H-H.), uhartl@biochem.mpg.de (F.U.H.), vabulas@biochem.mpg.de (R.M.V.)

DOI 10.1016/j.cell.2010.11.050

SUMMARY and may cause the loss of critical functions. However, in a

Protein aggregation is linked with neurodegeneration
and numerous other diseases by mechanisms that
are not well understood. Here, we have analyzed
the gain-of-function toxicity of artificial b sheet
proteins that were designed to form amyloid-like
fibrils. Using quantitative proteomics, we found that
the toxicity of these proteins in human cells corre-
lates with the capacity of their aggregates to promote
aberrant protein interactions and to deregulate the
cytosolic stress response. The endogenous proteins
that are sequestered by the aggregates share
distinct physicochemical properties: They are rela-
tively large in size and significantly enriched in
predicted unstructured regions, features that are
strongly linked with multifunctionality. Many of the
interacting proteins occupy essential hub positions
in cellular protein networks, with key roles in
chromatin organization, transcription, translation,
maintenance of cell architecture and protein quality
control. We suggest that amyloidogenic aggregation
targets a metastable subproteome, thereby causing
multifactorial toxicity and, eventually, the collapse
of essential cellular functions.
INTRODUCTION
The majority of proteins must fold into well-defined three-dimen-
sional structures in order to fulfill their biological functions. This
fundamental process is aided by a complex cellular machinery
of molecular chaperones, which act to prevent misfolding and
aggregation (Frydman, 2001; Hartl and Hayer-Hartl, 2002; Mori-
moto, 2008). Failure of a protein to fold properly, or to retain its
folded state, has emerged as the cause of numerous diseases.
Aberrant folding is often the result of destabilizing mutations
growing number of diseases, misfolding and aggregation results
predominantly in a toxic gain of function (Stefani and Dobson,
2003; Winklhofer et al., 2008). In these disorders, specific
proteins, differing substantially in size and sequence, typically
self-assemble into amyloid-like fibrils with cross-b structure
which are deposited within or outside of cells. This phenomenon
underlies some of the most debilitating neurodegenerative
disorders, including Parkinson’s, Huntington’s, and Alzheimer’s
disease.
Amyloidogenic aggregation is observed with many protein
sequences (Chiti and Dobson, 2006; Goldschmidt et al., 2010)
and is often associated with the accumulation of soluble, oligo-
meric species that precede fibril formation and are thought to
be responsible for toxicity (Campioni et al., 2010; Chiti and
Dobson, 2006; Jahn and Radford, 2008). The underlying mech-
anisms are only poorly understood but a prominent hypothesis
suggests that the aggregates, in particular the more heteroge-
neous oligomers, may expose flexible hydrophobic surfaces
that can mediate aberrant interactions with other proteins,
resulting in their functional impairment and sequestration
(Bolognesi et al., 2010; Chiti and Dobson, 2006). In another
model, misfolding proteins, by engaging the chaperone
machinery, are thought to interfere with central protein quality
control and clearance mechanisms, possibly resulting in a prop-
agation of folding defects (Balch et al., 2008; Bence et al., 2001;
Gidalevitz et al., 2006). Finally, based on experiments with model
membranes, oligomeric aggregation intermediates can compro-
mise the integrity of lipid membranes (Lashuel and Lansbury,
2006). Importantly, these different routes of toxic action are not
mutually exclusive but may operate in parallel.
To investigate the toxicity mechanisms of amyloid-like aggre-
gation, we have established a cellular model based on the
expression of artificial proteins that were designed to form
b sheet structures, and shown previously to self-assemble into
fibrils in vitro (West et al., 1999). The sequences of these proteins
were explicitly designed to contain b strands with an alternating
pattern of polar and nonpolar residues, while the exact identities
of the side chains were varied combinatorially. Similar bipolar

Cell 144, 67–78, January 7, 2011 ª2011 Elsevier Inc. 67

segments occur in 30% of human proteins, including several
neurodegenerative disease proteins, but are usually buried
within the folded structure (Tartaglia et al., 2008). Because the
model proteins were designed de novo, they are not biased by
the evolutionary burden of natural proteins and thus allowed us
to study the gain-of-function toxicity caused by aggregation
without interference either from loss-of-function alterations or
from an augmentation of the biological activities of natural
disease proteins (Cooper et al., 2006; Lam et al., 2006).
Here, we tested specifically the hypothesis that the aggre-
gates engage in widespread aberrant protein interactions. We
found that expression of the model proteins in human cells
results in aggregate formation and toxicity. Quantitative proteo-
mic analysis reveals that the aggregates interact with and
sequester multiple preexistent and newly synthesized proteins.
Interestingly, these interactions can be explained in terms of
specific sequence features of the coaggregating proteins, such
as their multidomain character and their enrichment in disor-
dered regions, properties that are strongly linked with multifunc-
tionality and the occupancy of hub positions in the cellular
protein network. We suggest that aberrant interactions with
numerous proteins having key cellular functions contribute to
aggregate toxicity.
RESULTS
Designed b Sheet Proteins Are Cytotoxic
To investigate the gain-of-function cytotoxicity associated with
amyloid-like aggregation, we used several model polypeptides
from a combinatorial library rationally designed to form cross-
b fibrils (West et al., 1999). These proteins, henceforth desig-
nated as b proteins, contain six b strands connected by 4-amino
acid linker segments, with each strand comprising seven amino
acids in a polar-nonpolar alternating pattern. An N-terminal
c-Myc-epitope was attached to facilitate detection (Figure 1A).
The three proteins chosen for analysis, b4, b17, and b23, differ
in sequence (pairwise identities of b strands 35%), with b23
having the highest hydrophobic volume and b sheet propensity,
due to its higher isoleucine content (Figure 1A) (Tartaglia et al.,
2008). As a control, we used the designed a-helical protein,
a-S824, which is similar to the b proteins in amino acid compo-
sition but folds into a 4-helix bundle structure (Wei et al., 2003)
(Figure 1A).
Upon dilution from denaturant into physiological buffer, the
purified b proteins adopted b sheet conformation as determined
by CD and rapidly assembled into aggregates detectable with
the amyloid-binding dyes thioflavin T (ThT) and NIAD-4 (Nesterov
et al., 2005) (Figures 1B, 1C, and Figures S1A and S1B available
online). The intensity of ThT and NIAD-4 binding was highest for
b23, followed by b17 and b4 (Figures 1B and 1C), consistent with
the relative b aggregation propensity of these proteins calcu-
lated with the sequence-based Zagg method (Tartaglia et al.,
2008) (Zagg scores are: b4, 0.79; b17, 0.83; b23, 0.93; a-S824,
and 0.30). ANS fluorescence, a probe for exposed hydrophobic
regions, suggested the presence of hydrophobic surfaces on the
aggregates, in particular for b23 and b17 (Figure 1D). As shown
via electron microscopy, the b proteins formed mostly relatively
short protofilaments (2–3 nm in diameter) as well as more
68 Cell 144, 67–78, January 7, 2011 ª2011 Elsevier Inc.
heterogeneous, globular aggregates (Figure 1E). Formation of
globular species was most pronounced with b23 (Figure 1E).
Similar prefibrillar aggregates are also observed with natural
amyloidogenic proteins and correlate with cytotoxicity (Bolog-
nesi et al., 2010; Campioni et al., 2010; Chiti and Dobson,
2006). In contrast, in low-salt buffer (pH 6), the b proteins formed
thicker (10–12 nm diameter) and longer fibrils (Figure 1F) with
FTIR spectroscopic properties characteristic of amyloid (Fig-
ure S1C). Thus, the model proteins undergo amyloid-like aggre-
gation in vitro, and in physiological buffer populate prefibrillar
species.
Upon expression in HEK293T cells for 3 days, the b proteins,
but not a-S824, reduced cell viability substantially, as measured
by the MTT assay (Figure 2A), and induced cell death in the order
b23 > b17 > b4 (Figures S2A and S2B). Cell viability was less
impaired after 24 hr of expression, without a significant differ-
ence in toxicity between the three b proteins (Figure 2A). Upon
cell fractionation, the b proteins were largely recovered in the
insoluble fraction, whereas a-S824 was soluble (Figure 2B).
Note that b4 and b17 migrated on SDS-PAGE more slowly
than expected, but this difference was not observed in urea/
SDS gels (Figure S1A). Confocal immunofluorescence micros-
copy with anti-Myc antibody showed that the b-protein-express-
ing cells adopted a collapsed shape lacking filopodia (Figure 2C).
Aggregates accumulated mostly in the perinuclear space and
the nuclei were often deformed. The aggregates were NIAD-4
positive (Figure 2D), suggesting the presence of amyloid-like
material. To detect oligomeric aggregation intermediates, cell
extracts were fractionated by size exclusion chromatography
followed by dot blot analysis with the A11 antibody, which was
raised against the Alzheimer Ab peptide and preferentially
recognizes amyloid oligomers associated with cytotoxicity, inde-
pendent of amino acid sequence (Kayed et al., 2003). b23
expression generated substantially higher levels of A11 reactive
material than expression of b4 and b17 (Figure 2E and Fig-
ure S2C), consistent with the greater toxicity of b23 and its
pronounced tendency to form prefibrillar aggregates in vitro
(Figures 1E and 1F).
In summary, the designed b proteins resemble amyloidogenic
disease proteins in terms of aggregation properties and toxicity
and allow us to investigate the mechanism of gain-of-function
toxicity independently from evolved biological interactions.
Identification of the b Protein Interactome
Gain-of-function toxicity of aggregation may arise, at least in
part, from aberrant interactions of the aggregates with cellular
proteins. To test this hypothesis, we performed a sensitive,
quantitative proteomic analysis of the b protein interactome
using SILAC (stable isotope labeling with amino acids in cell
culture) (Ong and Mann, 2006) and peptide identification by
tandem mass spectrometry (LC-MS/MS). These experiments
were performed at 24 hr after b protein transfection when cell
viability was not yet severely impaired (Figure 2A). In one set of
experiments, cells labeled with light (L), medium (M) or heavy
(H) arginine and lysine isotopes were transfected with empty
vector, a-S824 and b23, respectively. In another set-up,
a-S824, b4, and b17 were expressed in L-, M-, and H-labeled
cells, respectively (Figure 3A). Preferential interactions with b4,
 A Myc epitope
1 80
β4: MCEQKLISEEDL GMQISMDYQLEIEGNDNKVELQLNDSGGEVKLQIRGPGGRVHFNVHSSGSNLEVNFNNDGGEVQFHMH
β17: MCEQKLISEEDL GMQISMDYEIKFHGDGDNFDLNLDDSGGDLQLQIRGPGGRVHVHIHSSSGKVDFHVNNDGGDVEVKMH
β23: MCEQKLISEEDL GMQISMDYNIQFHNNGNEIQFEIDDSGGDIEIEIRGPGGRVHIQLNDGHGHIKVDFNNDGGELQIDMH

1 64
α-S824: MCEQKLISEEDL GMYGKLNDLLEDLQEVLKHVNQHWQGGQKNMNKVDHHLQNVIEDIHDFMQGG
65 GSGGKLQEMMKEFQQVLDEIKQQLQGGDNSLHNVHENIKEIFHHLEELVHR 105

B ThT C NIAD-4 D ANS

20 120
10
Fluorescence (AU)

Fluorescence (AU)

Fluorescence (AU)
100
15 8
80
10 6
60

40 4
5
20 2
0
0 0
460 480 500 520 540 500 550 600 650 700 400 450 500 550 600
Wavelength (nm) Wavelength (nm) Wavelength (nm)
α-S824 β4 β17 β23

E
β4 β17 β23

100 nm 100 nm 100 nm

F
β4 β17 β23

100 nm 100 nm 100 nm

Figure 1. Amyloidogenic Aggregation of Model Proteins In Vitro

(A) Sequences of the model proteins, b4, b17, and b23, designed to form b-sheet fibrils, and a-S824 designed to form a 4-helical bundle. Polar and nonpolar
amino acids are indicated in gray and yellow, respectively, b strands and a helices by blue arrows and rods respectively. N-terminal c-Myc tags are shown in red.
(B–D) Tinctorial properties of b protein aggregates. The purified proteins indicated (3 mM) were analyzed in 25 mM HEPES buffer (pH 7.5), 150 mM KCl, 0.5 mM
MgCl2-containing 20 mM Thioflavin T (B), 1 mM NIAD-4 (C), or 20 mM ANS (D). Fluorescence was recorded as described in Experimental Procedures.
(E and F) Transmission electron microscopy of aggregates formed by b4, b17 and b23, as above, at pH 7.5 (E) or 10 mM potassium phosphate (pH 6.0) (F).
Proteins were negatively stained and observed at a magnification of 55,0003.
See also Figure S1.

b17, or b23 were explored in a third type of experiment. Total cell
lysates were prepared essentially without removal of aggregate
material and combined 1:1:1 (Figure 3A and Figure S3A). The
expressed proteins were quantitatively isolated using anti-Myc
antibody coupled to magnetic beads, followed by SDS-PAGE,
in-gel digestion, and LC-MS/MS analysis.
It seemed plausible that initial coaggregation may be driven by
relatively weak interactions, which might introduce a stochastic
element in the proteomic analysis. To overcome this problem
we based our analysis on extensive biological repetitions of the
experiments. Three proteomic experiments were performed,
each consisting of three biological repeats (independent trans-
fections). A protein was identified as b protein interactor
when its isotope-labeled peptides were either enriched relative
to the a-S824 control or relative to one of the other b proteins
with > 95% confidence in at least two of the three repeats of
a set (see Extended Experimental Procedures and Figures
S3B–S3D). A total of 94 interactors of b23, 73, of b17 and 57 of
b4 were identified in experiments of equivalent sampling size,
consistent with the relative toxicity of the proteins (Figure 3B

Cell 144, 67–78, January 7, 2011 ª2011 Elsevier Inc. 69

A 24 h B Figure 2. Cytotoxicity and Aggregation of
72 h
100
Viability (% of control)
80 C β4
60 kDa T T S P T S P T S P T S P
17
40
10
20
0 transfection by centrifugation and immunoblotting
C β4 β17 β23 α-S824
C α-S824 β4 β17 β23
DIC
Myc
D represents 20 mm. Representative images of three
NIAD-4
fractions from size exclusion chromatography was corrected for the cumulative anti-Myc signal, indicating the amounts of a-S824, b4, b17, and b23, and
expressed relative to the A11 reactivity in a-S824 expressing cells (set to 1) (see Figure S2C for original data). Averages and standard deviations represent at least
three independent experiments.
See also Figure S2.
and Tables S1–S3). Only four proteins were marginally enriched
on a-S824 relative to the vector only control, including two ribo-
somal proteins (Figure S3B). Approximately 60% of the b4 and
b17 interactors were also found to interact with b23, indicating
a high degree of overlap in interaction profiles (Figure 3B).
Western blotting of pulldowns and immunofluorescence analysis
of cells confirmed the results from SILAC/MS for several interac-
tors (Figures S3E and S3F). Thus, interactions of the b protein
aggregates with multiple endogenous proteins precede the
strong decrease in cell viability observed at 72 hr after transfec-
tion (Figure 2A).
As summarized for b23, most of the proteins associated with
the aggregates have their primary location in the cytoplasm,
nucleus and mitochondria (Figure 3C and Table S1). Proteins
involved in chromatin regulation, RNA processing, transcrip-
tion, translation, cytoskeletal function, vesicle transport, and
protein quality control were highly represented. These proteins
are generally of average cellular abundance (Su et al., 2002)
and for several of them between 10% and 45% of total was
associated with the aggregates, based on depletion from
supernatant fractions after pulldown as measured by SILAC/
MS (Table S1 and Extended Experimental Procedures). Note
that this analysis probably underestimates the extent of
sequestration, since coaggregates may partially dissociate
during isolation. Interestingly, 12 different translation initiation
factors interacted directly or indirectly with the aggregates,
including 9 of the 13 subunits of the eIF3 complex and 3
subunits of eIF4 (Figure 3C). b17 aggregates contained 10
and b4 aggregates 9 of these proteins (Tables S2 and S3).
70 Cell 144, 67–78, January 7, 2011 ª2011 Elsevier Inc.
Model Proteins in HEK293T Cells
(A) Viability of HEK293T cells expressing b4, b17,
b23, and a-S824 measured by MTT assay 24 hr
β17 β23 α-S824
and 72 hr after transfection. MTT reduction by
control cells transfected with empty vector, C, was
set to 100%. Standard deviations were derived
from at least three independent experiments.
(B) Solubility of b4, b17, b23, and a-S824 analyzed
by fractionation of lysates from cells 24 hr after
with anti-Myc antibody. T, total lysate; S, soluble
fraction; P, pellet fraction; C, empty vector control.
E Representative results from at least three inde-
pendent experiments.
(C and D) Protein distribution and aggregation in
5 intact cells expressing a-S824, b4, b17, and b23.
A11 reactivity (AU)
After 24 hr, proteins were detected by immuno-
4
fluorescence with anti-Myc antibodies (C). DIC,
3 differential interference contrast images. Amyloid-
like aggregates were detected by staining with
2 NIAD-4 (D) (see Experimental Procedures). Nuclei
were counterstained with DAPI. The scale bar
1
0 independent experiments.
α-S824 β4 β17 β23
(E) Quantification of A11 antibody reactivity in
extracts from b-protein-expressing cells 24 hr
after transfection. The cumulative dot blot signal of
Immunofluorescence analysis demonstrated extensive co-
localization of eIF3D and eIF4GII with the aggregates and
western blotting of pulldowns confirmed that at least 10%
of cellular eIF3D coaggregated with b23 and b17, compared
to 6% with b4 (Figures S3E and S3F, and data not shown).
Indeed, labeling experiments showed that cells expressing
b23 for 24 hr had a 35% reduced protein synthesis capacity
(Figure S3G). Similarly, the altered morphology of b-protein-
expressing cells revealed by actin staining (Figure S3H) may be
attributed to the association of filamins A, B, C, (FLNA, FLNB,
FLNC), and the giant protein plectin-1 (PLEC1) (500 kDa)
with the aggregates (Figure 3C), proteins that are critical for
the formation and maintenance of cytoskeletal architecture.
These results show that many different proteins, involved in
a range of essential cellular functions, are affected by the
b protein aggregates.
Aberrant Stress Response in b-Protein-Expressing Cells
The proteomic analysis identified several cytosolic chaperones
and chaperone regulators to be associated with the aggregates,
including Hsc70 (Hsc71) and its cochaperones Hsp110
(Hsp105), Hdj1/2 and Bag2, as well as the nascent chain associ-
ated complex, NAC (Figure 3C). Hsp110 was enriched in the
aggregates in a manner correlating with the relative toxicity of
b4, b17 and b23, as confirmed by western blotting and immuno-
fluorescence (Figures 4A and 4B). Indeed, overexpression of
Hsp110 (Figure S4A) partially suppressed b4 and b17 aggrega-
tion and toxicity but was inefficient in mitigating the toxic effects
of b23 (Figures 4C and Figures S4B and S4C).
A L: Arg0,Lys0 M: Arg6,Lys4 H: Arg10,Lys8

Expt. I Expt. II Expt. III

β23 β23
Expt. I: Vector only α-S824 β23

Intensity
mix β17 β17
Expt. II: α-S824 β4 β17 lysates IP LC-MS/MS
α-S α-S β4 β4
1:1:1 C 824 824
Expt. III: β4 β17 β23
m/z m/z m/z

B C Miscellaneous
β4 ABCE1 OGT
Molecular chaperones
BAG2 HSPA8 Translation
FAM120A SBDS
(57) Protein degradation
CHORDC1
DNAJA1
HSPH1
NACA
EEF1A1
EIF3A
EIF3I
EIF3L
CACYBP UBC DNAJB1 NACB EIF3C EIF3M
ERLIN2 UBR4
EIF3D EIF4A1
SUMO2 Mitochondria
EIF3E EIF4A3
Vesicle transport EIF3F EIF4G3 SLC25A6 VDAC1
21 AP1B1 CLTC tRNA synthetases EIF3G IGF2BP3 CHCHD3 VDAC2
AP1G1 SEC16A EPRS WARS IMMT VDAC3
AP1M1 VAPA YARS SSBP1

1 4
Metabolism
31 ALDH18A1 Ribosome biogenesis
RNA processing
BAT1 RBM8A
CAD DIMT1L GEMIN4 SMN1
CHERP NVL GEMIN5 SR140
47 23 HDLBP PDCD11 STRAP
15 Nuclear structure WDR3
AHNAK
Cytoskeleton Chromatin structure
LMNB1 Transcription
AKAP12 PLEC1 CHD4 RUVBL2
NUMA1
Nuclear transport H1F0 SMC4 CAND1 PNN
ARHGDIA SEPT2
KPNA2 RBBP4 SMARCA4 CNOT1 PURA
CCDC88A SEPT7
GTF2I SND1

β17
DIAPH1 SEPT9 RANBP1 RUVBL1 SMARCA5

β23 FLNA TUBA1A THOC2 SMARCC1

(94) (73) FLNB

FLNC
TUBB2C
VIM Unspecified function PCNP
KIF5B ZYX
BAT2D1 DNA maintenance PRKDC
MARCKS
HCFC1 TNKS1BP1

Figure 3. Interactome Analysis of b proteins

(A) Design of SILAC experiments to identify b protein interactors by LC-MS/MS. L, M, and H, light, medium, and heavy isotope media. C, vector only control.
(B) Overlap between the interactor sets of b4, b17, and b23. Total numbers of identified interactors are given in parentheses.
(C) The b23 interacting proteins are grouped according to cellular location and function.
See also Figure S3 and Tables S1–S3.

Remarkably, expression of the b proteins did not induce the
cytosolic stress response or heat-shock response (HSR), as no
increase in the levels of Hsp110, Hsp70, or Hsp27 was observed
(Figures 4A and data not shown). A possible defect in the HSR
was further analyzed using a luciferase reporter gene under
control of the HSF1-dependent Hsp70 promoter (Williams
et al., 1989). While inhibition of proteasome function by MG132
in control cells resulted in a 5-fold induction of the reporter,
this induction was completely abolished in cells expressing the
b proteins for 24 hr (Figure 4D). The phorbol-12-myristate-13-
acetate (PMA)-mediated induction of a luciferase reporter under
the NF-kB promoter was also impaired, but to a lesser extent
than the inhibition of the stress response (Figure 4E). Thus,
expression of the model b sheet proteins leads to a deficiency
of the normal cytosolic stress response, thereby limiting the
capacity of cells to mount an effective defense.
Structural Features of b Protein Interactors
A bioinformatic analysis of the physicochemical properties of the
b protein interactors was conducted to see whether these
proteins share certain structural features. We focused our initial
analysis on the interactors of b23 (Table S1). Compared to a set
of 3055 control proteins identified by LC-MS/MS in a total cell
lysate (Table S4), the b23 interactors are shifted to higher molec-
ular weight, with a significantly greater fraction of proteins above
150 kDa (p < 0.005) (Figure 5A). In addition, the interactors have
a lower average hydrophobicity and a bimodal hydrophobicity
distribution (Kyte and Doolittle, 1982) (p < 0.005) (Figure 5B).
The occurrence of domain folds among the b23 interactors, as
classified in SCOP, was generally similar to that of lysate
proteins. However, the b23 interactors contained significantly
more proteins with all beta domains (SCOP class b) (p < 0.05)
(Figure S5A), including the beta-barrel VDAC proteins of the

Cell 144, 67–78, January 7, 2011 ª2011 Elsevier Inc. 71

A C
Viability (% of control)
C β4 β17 β23 α-S824
Hsp110
IP
Lysate
Immunoblot
B Myc Hsp110 Merge
C
D 10
α-S824
β4
E NF-κB-Luc
β17
β23
outer mitochondrial membrane and the filamins which have
Ig-domain repeats (Table S1 and Figure S3F).
The lower hydrophobicity of many interactors suggested that
these proteins may be rich in intrinsically unstructured regions
(IURs). Indeed, compared to lysate proteins, the b23 interactors
have a significantly greater fraction of total amino acids in IURs
(p < 0.05), based on the DisoDB database (Pentony and Jones,
2009) and the DisEMBL and IUPred prediction tools for unstruc-
tured regions (Dosztanyi et al., 2005; Linding et al., 2003) (Fig-
ure 5C, Tables S1 and S4, and data not shown). Pronounced
differences emerged when considering the fraction of proteins
with IURs longer than 30 or 50 residues (Figure 5D). For example,
60% of the b23 interactors are predicted to contain at least one
unstructured segment of 30 amino acids, compared to 45% in
lysate proteins or the complete proteome (p < 0.005) (Figure 5D).
The corresponding numbers for IURs > 50 amino acids are
40% and 25% (p < 0.005), respectively. Moreover, the b23
interactors contain on average 3 disordered segments of 30
amino acids (compared to 1.8 for the lysate proteins, p <
0.005). The predicted IURs of the interactors are shifted to
greater length (p < 0.005) (Figure S5B and Tables S1 and S4),
with 21% of the proteins containing IURs > 80 amino acids
72 Cell 144, 67–78, January 7, 2011 ª2011 Elsevier Inc.
Figure 4. Impairment of the Stress
120 Response in b-Protein-Expressing Cells
100 (A and B) Association of Hsp110 with b protein
aggregates. HEK293T cells were transfected as
80 indicated (C, empty vector control). 24 hr after
60 transfection, Myc-tagged proteins b4, b17, b23,
and a-S824 were immunoprecipitated from cell
40
lysates and analyzed by immunoblotting with anti-
20 HSP110 antibody (A). Lysate samples correspond
0 to 8% of input used for IP. b4, b17, and b23 in
C β4 β17 β23 α-S824 pulldowns was associated with 5%, 9%, and
− + − + − + − + − +
16% of total cellular Hsp110, respectively (also
Hsp110 overexpression see Figure S3F). For immunofluorescence analysis
(B), cells were fixed and costained with anti-Myc
HSP70-Luc antibodies and anti-Hsp110 antibodies. Nuclei
Promoter activity were stained with DAPI. Representative examples
(fold induction) of three independent experiments are shown.
(C) Partial rescue of b protein toxicity by Hsp110
overexpression. Cells were transfected with
empty vector or the expression vector for human
1 Hsp110. 24 hr later, cells were electroporated with
empty vector, C, or expression vectors for b4, b17,
C β4 β17 β23 α-S824 b23, and a-S824. Three days after the second
transfection, MTT assays were performed. Empty
vector control was set to 100% viability. Standard
100
deviations of three independent experiments are
Promoter activity
(fold induction)
shown.
(D and E) Inhibitory effect of b proteins on
10 cellular stress response pathways. Cells were
cotransfected with HSP70-luciferase reporter (D)
or NF-kB-luciferase reporter constructs (E) and
1 the b-protein-expressing plasmids. 6 hr later, 5 mM
MG132 (D) or 16 mM PMA (E) were added to induce
C β4 β17 β23 α-S824 the respective promoter. Luciferase activity was
measured 24 hr after transfection. The promoter
activity in cells transfected with control vector, C,
without inducer was set to 1. Standard deviations
of three independent experiments are shown.
See also Figure S4.
and 10% of 100 to 429 residues. The IURs are enriched in polar
amino acids and in amino acids that have a high propensity to
form coil and turn regions, such as M, K, R, E, S, Q, and P,
and are depleted in aromatic and hydrophobic amino acids W,
Y, F, C, I, and V (Dunker et al., 2008). Such sequences are struc-
turally flexible and populate a range of conformational states
from extended disordered to collapsed, molten globule-like
structures (Dunker et al., 2008; Pentony and Jones, 2009). Using
the Zagg algorithm to predict aggregation propensities, the b23
interactors have higher aggregation scores than lysate proteins
(see Figure S6B below).
A comparison of the proteins that interact preferentially with
b4, b17, and b23 (18, 28, and 27 proteins, respectively), as
defined by the SILAC experiments (Figure S3D and Extended
Experimental Procedures), revealed that a gradual increase in
molecular weight and decrease in hydrophobicity of the interac-
tors, along with a slight increase in their disorder, correlated with
the differential cytotoxicity of the three b proteins (Figure 5E).
This trend was also observed when comparing the complete
interactor sets of the three b proteins (Tables S1–S3).
The prominent association of proteins with low hydropho-
bicity and high intrinsic disorder with the aggregates was
A p<0.005 Figure 5. Structural Properties of the
30 350 b Protein Interactors

Molecular weight (kDa)

300 (A and B) Distribution of molecular weight (A) and
% of identified proteins

average hydrophobicity (B) of lysate proteins

250
(gray) and b23 interactors (red) (Tables S1 and S4).
20 200 Box plots (insets) indicate the distribution of the
150 data. Dashed horizontal line indicates the median,
whisker caps and circles indicate 10th/90th and
100
5th/95th percentiles, respectively. P values are
10 50 based on Mann-Whitney test.
0 (C and D) Disorder analysis of b protein interactors.
Lysate β23 Percentage of disordered residues in interactor
proteins interactors
sequences (C). p value based on Mann-Whitney
0 test. Fraction of proteins with disordered stretches
longer than 30 or 50 amino acids (D). **p < 0.005
0

40

80

120

160

200

240

280

320

480

520

560

600

640
based on a chi-square test. Disorder was deter-
mined using DisoDB.
Molecular weight (kDa)
(E) Structural properties of lysate proteins and of
B p<0.005 proteins interacting preferentially with b4, b17, or
14 0.2
b23.
0.0
% of identified proteins

12 See also Figure S5 and Tables S1–S6.

Hydrophobicity

-0.2
10
-0.4
8 -0.6

6 (Lee et al., 2006). The Arctic mutant -0.8

formed visible aggregates more readily -1.0
4
-1.2 and showed substantially greater toxicity
Lysate β23
2 proteins interactors than WT Ab1 42 (Figures S5D and S5E).
Analysis by SILAC/MS revealed that
0 the Ab interactome is comparable in
complexity to that of the b proteins,
0.0

0.2

0.4

0.6

0.8

1.0
-1.6

-1.4

-1.2

-1.0

-0.8

-0.6

-0.4

-0.2

with a direct overlap of 25%, promi-

Erlin Hydrophobicity
nently including translation initiation
C D E Preferred interactors factors, chromatin regulators, RNA pro-
p<0.05 cessing proteins, mitochondrial mem-
24
Disordered residues (%)

Proteins with disordered

60 ** brane proteins and chaperones (Table

Lysate proteins
(kDa)

20 70 S5). We also identified 31 proteins which

50 β4 interactors
stretches (%)

60
16
** β17 interactors were enriched on the Arctic mutant rela-
t

40 50
Molecular weigh

12 30
40 β23 interactors tive to the less toxic Ab1 42 WT (Table
30 S6). Notably, these proteins resemble
8 20 20
-0.55 the b protein interactors in physico-
y

10 -0.50
cit

4 10
bi

0 -0.45 chemical properties and are significantly

ho

20 -0.40
op

0 0 40 60
Disord -0.35 enriched in IURs (p < 0.05) (Figures
dr

a.a. >30 >50 er 80 100

Hy

[prote >30 a.a. S5F–S5I).

Lysate proteins in s (%)]
β23 interactors From these results, we conclude that
cells contain a subpopulation of meta-
stable proteins that are prone to interact
with and potentially become sequestered
unexpected. To test whether such proteins are targeted more by toxic species populated in the process of amyloid-like
generally by amyloid-like aggregation, we performed an initial aggregation.
analysis of interactors of wild-type Ab1-42 and its Arctic mutant
(E22G), which causes early-onset Alzheimer’s disease. The b Protein Interactors Include Pre-Existent and Newly
latter was included because it is known to populate higher Synthesized Proteins
levels of prefibrillar aggregates and toxic oligomers exposing While the results above suggested that structural flexibility is crit-
hydrophobic surfaces (Bolognesi et al., 2010; Nilsberth et al., ical in facilitating the interaction of endogenous proteins with the
2001). To allow a comparison with the model b aggregates, b aggregates, we noted that for 40% of the b23 interactors no
the Ab proteins were also expressed in the cytosol, using IURs > 30 amino acids are predicted (Figure 5D and Table S1).
GFP fusions (Kim et al., 2006). In contrast to the artificial We therefore considered the possibility that some of these
b proteins, the Ab constructs were degraded but accumulated proteins may succumb to coaggregation upon synthesis
upon partial proteasome inhibition with MG132 (Figure S5C) before adopting stably folded structures. To test this idea, we

Cell 144, 67–78, January 7, 2011 ª2011 Elsevier Inc. 73

A L: Arg0,Lys0 M: Arg6,Lys4 B 50 Figure 6. Newly Synthesized and Pre-Exis-
tent b Protein Interactors
(A) Design of SILAC based mass spectrometric
45
analysis to identify proteins preferentially inter-
40 acting with b23 as newly synthesized (new) or
preexistent (old) proteins (Pulse-SILAC).
Transfection : α-S824 β23 35 (B) Ratios of heavy to medium isotopes (H/M) of
b23 interactors relative to the H/M ratios for the
New
L: Arg0,Lys0 M: Arg6,Lys4 H: Arg10,Lys8 30 same proteins in the total cell lysate (see Extended
Experimental Procedures). The log of this ratio of
25 ratios increases with the tendency of a protein to
β23 interactors interact with b23 as a new protein.
20
(C and D) Disorder analysis of new and old b23
interactors. Percentage of disordered residues
15
in interactor sequences (C). p value is based
β23 β23 10 Old on Mann-Whitney test. Fraction of proteins with
Intensity

Intensity

continuous disordered stretches > 30 or > 50

5 amino acids (aa) (D). **p < 0.005 for old interactors
α-S α-S relative to lysate (see Figure 5D), based on Chi-
824 β23 824 β23
square test.
m/z m/z -0.6 -0.3 0.0 0.3 0.6 0.9 1.2 1.5 (E) Molecular weight, disorder and hydrophobicity
β23 interactor: Preexistent Newly-synthesized of old and new b23 interactors relative to lysate
(Old) (New)
Log(H/Mβ23)/(H/Mlysate)
proteins.
See also Figure S6 and Tables S4 and S7.
C D E
p<0.05
35 80 **
Disordered residues (%)

Proteins with disordered

30 70 Lysate proteins
t (kDa)

70
60 ** Old β23 interactors
stretches (%)

25 60
translation initiation factors (Table S7),
50 50 New β23 interactors
Molecular weigh

20 consistent with impairment of translation

40 40
15 efficiency being an early consequence
30
30
20
10 20 -0.7 of b protein toxicity (Figures S3E–S3G).
y

-0.6 10
cit

5 10
-0.5 Interestingly, the old and new interac-
bi

0
ho

20 40 -0.4
0 0
op

Disord 60 -0.3 tors from the ends of the distribution (15

dr

a.a. >30 >50 er 80 100

Hy

[prote >30 a.a.

Old β23 interactors ins (% proteins each) differ markedly in their
)]
New β23 interactors structural properties. The old interactors
contain a significantly greater fraction of
amino acids in IURs than the new interac-
tors (Figure 6C). They are strongly en-
pulse-labeled HEK293T cells expressing b23 or a-S824 with riched in continuous disordered regions (Figure 6D) and are of
35
S-methionine, followed by immunoisolation of the proteins. low average hydrophobicity (Figure 6E). In contrast, the new
Around 7% of the proteins labeled within 15 min were coisolated proteins are similar to lysate proteins in terms of hydrophobicity,
with b23, compared to only 1% with a-S824 (Figure S6A), sug- but are lower in disorder and substantially larger in size (Fig-
gesting that a substantial fraction of newly synthesized polypep- ure 6E). Their folding pathways may be complex and kinetically
tides can interact with b23. slow, possibly resulting in the prolonged exposure of hydro-
To identify such proteins, we performed pulse-SILAC experi- phobic residues during folding. Based on analysis using the
ments. Cells were cultured with medium amino acid isotopes Zagg algorithm, the new b23 interactors show high intrinsic
(M) to label preexistent proteins, followed by transfection with aggregation scores only in their unfolded states (Figure S6B).
b23. The culture was divided and one half was immediately In contrast, the old interactors are highly flexible and are unable
shifted to media containing heavy amino acid isotopes (H). to bury aggregation-prone regions in their native structures.
Control cells were cultured with light amino acids (L) and trans- Thus, these proteins have high aggregation scores both in their
fected with a-S824. After 24 hr, the cells from the three condi- unfolded and folded states (Tartaglia et al., 2008) (Figure S6B).
tions were combined and subjected to anti-Myc pulldown and Some of them may coaggregate as preexistent or newly synthe-
LC-MS/MS analysis (Figure 6A). The H/M isotope ratio of the sized proteins, consistent with their lower peak values in the
b23 interactors in the pulldown relative to their H/M ratios in isotope labeling ratios compared to the new proteins (Figure 6B).
the lysate was used to indicate whether they interact with b23 In summary, the b23 interactors can be divided into two over-
preferentially as newly synthesized (New) or pre-existent (Old) lapping subsets of relatively aggregation-prone proteins: One
proteins (Figure 6A). H/M labeling ratios were obtained for 50 group is enriched in IURs, which would be prone to aggregate
b23 interactors, and a number of these showed a clear prefer- even in their post-folding state. This group is highly represented
ence for interaction soon after synthesis (Figure 6B and Table among the old proteins. The second group contains an abun-
S7). In contrast, fewer proteins interacted preferentially as old dance of large and/or multidomain proteins, which require longer
proteins. These interactors include Hsp110 as well as several times for synthesis and may fold slowly. Consequently, in

74 Cell 144, 67–78, January 7, 2011 ª2011 Elsevier Inc.

A B Figure 7. Mechanism of b Aggregation
13 12.4 Toxicity
12
(A and B) Functional context of b protein inter-
Average interactions

11
10 80 actors within the protein interaction network.
9
β4

Disease proteins
Shown in (A) are the average number of functional
/Protein

8 7.5 7.2
7 β17
60 interactions of the b23 interactors in comparison
6 β23
4.9 to proteins in the HPRD database and in the
5
4 40
3 2.8 experimentally determined cell lysate (3000
2 1.6 1.5 proteins). These functional interactions are cate-
0.8 20
1 0.4
gorized into total interactions, interactions with
0 1000

s
β23 850

r
HPRD Lysate 0 essential proteins and interactions with proteins

to
700

ac
200
All interactions Esse 250 involved in neurodegenerative disease (Ray-

r
550

te
ntial 300

In
Essential intera 350 chaudhuri et al., 2009). In (B), the complete sets of
ctors
Disease b4, b17, and b23 interactors are compared in
terms of these functional properties.
C (C) Model for the interaction of b aggregates with
pre-existent and newly synthesized proteins. Pre-
existent proteins are structurally flexible in their
functional state and are involved in multiple
Proteins or Chaperones Chaperones Newly-
protein complexes synthesized protein-protein interactions, which may be dis-
containing rupted by their association with the b aggregates.
disordered regions
The newly synthesized proteins are structurally
co-aggregation co-aggregation Amyloidogenic vulnerable to coaggregation during folding and
regions (red)
assembly. Interaction of both the preexistent
Toxic and newly synthesized proteins with the b aggre-
β-aggregates/
Structured oligomers gates is facilitated by the limiting capacity of
proteins Proteins not yet folded
and/or assembled chaperones to shield aggregate surfaces and by
the failure of the cells to mount an efficient stress
response.

conditions of limited chaperone capacity, they would be prone to neurodegenerative disease proteins (Raychaudhuri et al., 2009)
aggregate during and shortly after synthesis. This group is (Figure 7A and Table S1).
enriched among the newly synthesized proteins. Finally, some Assuming that a disturbance of functional protein interactions
proteins occupy a transition zone, combining physicochemical contributes critically to b aggregation toxicity, b23 would be
features of both groups. expected to differ in this regard from the less cytotoxic proteins
b4 and b17. We found that the b4, b17 and b23 interactors are
Aggregate Interactors Have Critical Network Functions physically linked to a total of 600, 643, and 912 different proteins,
The structural flexibility and relatively large size of the aggregate including 216, 213, and 340 essential proteins and 53, 56, and 84
interactors suggests that these proteins may normally be proteins associated with neurodegenerative disease networks,
involved in numerous functional protein interactions. To address respectively (Figure 7B). Thus, the capacity of the b protein
this possibility, we analyzed how the b23 interactors are linked aggregates to interact with and sequester highly connected
with the cellular protein network. A query of the Human cellular proteins correlates well with their relative cytotoxicity.
Proteome Reference Data Base (HPRD) (Keshava Prasad
et al., 2009) revealed that each of these proteins functionally DISCUSSION
interacts with 12 different proteins on average, compared to
7 per lysate protein and 7.5 per protein in HPRD (19,651 Widespread Coaggregation of Metastable Proteins
entries) (Figure 7A). Notably, most of the b23 interactors have A key finding of this study is that amyloidogenic aggregation can
no or only few interactions with any of the other b23 interactors, result in the sequestration of numerous proteins that share
suggesting that coaggregation may disrupt their functional distinct physicochemical properties: They are relatively large in
complexes. For example, the microfilament protein vimentin size and exhibit high structural flexibility, with a significant
interacts with more than 100 different proteins according to enrichment in disordered regions, features that are strongly
HPRD, but only three of those are among the identified b23 inter- linked with multifunctionality (Figure 7C).
actors, although 49 potential vimentin interactors were detected The artificial b sheet proteins used as a model were designed
in the lysate or background of the pulldowns (data not shown). to assemble into fibrils (West et al., 1999). Like natural amyloido-
Essential proteins often occupy critical ‘‘hub’’ positions in the genic proteins, they populate a range of prefibrillar aggregation
network (Haynes et al., 2006; Jeong et al., 2001). Each b23 target intermediates, which are likely to represent the primary toxic
protein interacts on average with 5 different essential proteins, agents in aggregation diseases (Chiti and Dobson, 2006; Jahn
compared to only 3 per lysate protein and 1.5 per entry in and Radford, 2008). Based on recent findings, the proteotoxicity
HPRD (Figure 7A). Moreover, the b23 interactors are more of such species correlates with the exposure of ANS-binding
frequently linked than lysate proteins, through direct interac- hydrophobic surfaces (Bolognesi et al., 2010; Campioni
tions, with proteins that have been found in association with et al., 2010) and reactivity with the A11 anti-oligomer antibody

Cell 144, 67–78, January 7, 2011 ª2011 Elsevier Inc. 75

(Kayed et al., 2003), properties that are reproduced by the model
proteins. Flexible hydrophobic surfaces and unpaired backbone
structure that is not yet integrated into a stable cross-b core
(Mossuto et al., 2010) may endow oligomers and protofilaments
with the capacity to engage in widespread aberrant interactions
with metastable proteins. Whether oligomeric species with
similar interaction properties also occur during nonamyloido-
genic aggregation, leading to amorphous structures rather
than fibrils, remains to be determined.
The b protein aggregates were found to interact with preexis-
tent and newly synthesized polypeptides (Figure 7C). The former
are strongly enriched in intrinsically unstructured regions (IURs)
and are of lower average hydrophobicity. A similar trend was
observed for interactors of the toxic aggregates of the Arctic
mutant of Ab1-42, which is known to transiently populate high
concentrations of prefibrillar aggregates (Bolognesi et al.,
2010) (Figures S5C–S5I). Proteins rich in structural disorder are
considered to be adaptable to multiple interaction partners
(Dunker et al., 2008; Pentony and Jones, 2009). On the other
hand, local structural fluctuations in these proteins are expected
to give rise to the exposure of sequence elements with a higher
propensity to form aggregates, consistent with the relatively high
Zagg scores of the b protein interactors (Tartaglia et al., 2008).
Indeed, some of the best known neurodegenerative disease
proteins, such as a-synuclein or tau, are thought to be almost
entirely unstructured. In contrast, the proteins that interact with
the aggregates during or soon after synthesis have average
hydrophobicity and disorder. These proteins tend to be large in
size and are likely to populate nonnative states which expose
hydrophobic surfaces during their folding, assembly or transport
that must be shielded by molecular chaperones (Figure 7C). For
example, among the b protein interactors are mitochondrial
membrane proteins such as VDAC and the ADP/ATP translocase
which require chaperone protection during post-translational
sorting (Young et al., 2003).
By targeting flexible regions and hydrophobic surfaces of
preexistent and newly synthesized proteins, the b protein aggre-
gates may act in a ‘chaperone-like’ manner but cannot promote
folding through regulated release. Consequently, more and more
proteins are recruited, which in turn may generate new interac-
tion surfaces, thereby magnifying the toxic potential of the
aggregates (‘snowball effect’).
Interference with Multiple Key Cellular Functions
The b protein interactors include many proteins with key cellular
functions in transcription and translation, chromatin regulation,
vesicular transport, cell motility and architecture, as well as
protein quality control (Figure 3C). Similar proteins were also
found to interact with aggregates of Ab (Table S5), suggesting
that these pathways may be more generally at risk in aggregation
disorders. Bioinformatic analysis showed that most of the coag-
gregating proteins have numerous functional interactors, consis-
tent with their preferential role as network hubs (Haynes et al.,
2006; Jeong et al., 2001). The number of functional interactions
of the sequestered proteins correlates with the relative cytotox-
icity of the b protein aggregates (Figure 7B). It is thus likely that
the aggregates compete for binding to disordered regions with
a protein’s normal interactors and the more toxic forms may be
76 Cell 144, 67–78, January 7, 2011 ª2011 Elsevier Inc.
able to compete more effectively. Based on our proteomic
and biochemical measurements, 10%–45% of total may be
sequestered for several of the interacting proteins (Figures
S3E–S3F and Table S1). Moreover, certain proteins may misfold
upon interaction with the aggregates but remain in solution.
Thus, dependent on the interaction strength of the aggregates,
an increasing number of key functions may be affected, eventu-
ally resulting in fatal network collapse. We estimate that the
human proteome contains 2000 proteins that structurally
resemble the experimentally identified b aggregate interactors.
Dependent on cell type and the exact structural properties of
the causative aggregate, different subsets of these proteins
may be affected, which may help to explain different patterns
of pathobiology. It will be interesting to see which of these
proteins are preferentially targeted by b aggregates in neuronal
cells.
Our results also lend support to the recent view that protein
misfolding and aggregation disturbs proteostasis by compro-
mising the cellular folding environment (Morimoto, 2008). We
suggest that the association of endogenous proteins with the
aggregates is facilitated by the failure of the affected cells to
mount an efficient stress response, a phenomenon that was
previously observed during prion infection (Tatzelt et al., 1995)
and may be particularly serious in postmitotic cells, such as
neurons. Inhibition of the stress response may be due to the
sequestration by the aggregates of multiple chromatin regula-
tors, which interact with numerous transcription factors,
including HSF1 (Erkina et al., 2010; Sullivan et al., 2001). As
a consequence of limiting proteostasis capacity, newly synthe-
sized polypeptides with a high chaperone requirement for folding
may become increasingly vulnerable to sequestration by disease
protein aggregates (Figure 7C). This fatal chain of events may be
further enhanced during aging, which is associated with a decline
of proteostasis and thus would result in a reduced capacity of
cells to protect their more vulnerable proteins against coaggre-
gation (Balch et al., 2008; Morimoto, 2008).
EXPERIMENTAL PROCEDURES
Protein Purification and In Vitro Analysis of Aggregates
Proteins a-S824, b4, b17 and b23 were expressed in E. coli BL21 cells and
purified as described in Extended Experimental Procedures. Fluorescence
analysis, circular dichroism, FTIR spectroscopy and negative stain electron
microscopy of the aggregates were performed using standard methods (see
Extended Experimental Procedures).
Cell Culture, Immunoblotting and Reporter Assays
Human HEK293T cells were cultured under standard conditions (see Extended
Experimental Procedures). Transient transfections were performed by electro-
poration with 30 mg expression vector or by Lipofectamin (Invitrogen) transfec-
tion for overexpression of Hsp110. Immunoblots were developed using the
chemiluminescence kit Rodeo ECL (USB) and analyzed using a LAS-3000
image reader (Fujifilm) and the AIDA software (Raytest). For luciferase reporter
assays, cells were lysed in Lysis Buffer (Promega) and luciferase activity
measured using a Lumat LB9507 (EG&G Berthold).
Cell Viability
Cell viability was analyzed by measuring the capacity of cells to reduce 3-(4,5-
Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) to formazan
at different times after transfection with a-S824, b protein or Ab42-GFP
constructs (Shearman, 1999).
Solubility Analysis and Oligomer Quantification
Cells were lysed in Triton X-100/Na deoxycholate-containing PBS with
protease inhibitors. Benzonase was used to hydrolyze DNA. Raw debris was
removed at 2000 3 g for 5 min and the supernatant was fractionated by centri-
fugation (100.000 3 g, 30 min) into pellet and soluble fractions or by gel filtra-
tion on a Superose 6 column (Amersham Bioscience), followed by dot blot
analysis with anti-oligomer antibody A11 (Kayed et al., 2003) (see Extended
Experimental Procedures for details).
Immunofluorescence and Fluorescence Imaging
Transfected cells were fixed with paraformaldehyde, permeabilized with Triton
X-100 and stained with antibodies as indicated. Images were recorded with
a Leica TCS SP2 confocal laser scanning microscope. Protein aggregates
were analyzed by staining with NIAD-4 (ICX Nomadics) (see Extended Exper-
imental Procedures).
SILAC and Sample Preparation for LC-MS/MS Analysis
Labeling of cells was performed in custom medium supplemented with light
(L), medium (M) or heavy (H) arginine and lysine isotopes (see Extended Exper-
imental Procedures). In pulse-SILAC experiments, M-labeled cells were
shifted to H-medium, as indicated in Figure 6A. Cells were lysed and cell debris
removed by low-speed centrifugation (2000 3 g, 5 min). Lysates from L, M and
H cells were adjusted to equal protein concentration and mixed at a 1:1:1 ratio.
An aliquot of this mix was set aside as ‘‘lysate’’ control. Anti-Myc or anti-GFP
MicroBeads (Miltenyi Biotech) were used to isolate the Myc-tagged proteins
or GFP-fusion proteins and their interactors. The bound proteins were eluted
and processed as described (Ong and Mann, 2006). The spectra were
interpreted using MaxQuant version 1.0.12.31 (Cox and Mann, 2008)
combined with Mascot version 2.2 (Matrix Science, www.matrixscience.
com). See Extended Experimental Procedures for details. The raw MS data
along with a full list of identified proteins and quantitations is available at
https://proteomecommons.org/tranche, entering the following hash: +Ff0/
p8lSBrrzCKZfzAwYS3+Bqw5fonokB679f136te2iklhHtFMUpeT5SM/I3XuufTyr
Xj0ycVVC6G4Li/L02 dA4jcAAAAAAABVfg = =.
Bioinformatic Analysis
Average hydrophobicity was calculated according to Kyte and Doolittle (1982),
protein disorder using the DisoDB database (Pentony and Jones, 2009) and
aggregation propensities according to Tartaglia et al. (2008). Protein fold
prediction and the analysis of functional protein interactions are described in
Extended Experimental Procedures. Student’s t test and Mann-Whitney test
were used to compare groups. Chi-square tests were used to determine signif-
icant differences between categorical data.
SUPPLEMENTAL INFORMATION
Supplemental information includes Extended Experimental Procedures,
six figures, and seven tables and can be found with this article online at
doi:10.1016/j.cell.2010.11.050.
ACKNOWLEDGMENTS
We thank C.G. Glabe for providing the A11 antibody and H. Wagner for plas-
mids HSP70-Luc and NF-kB-Luc. We acknowledge the help of H. Engelhardt
with FTIR measurements, technical assistance by O. Mihalache and V. Marcus
with electron microscopy, and R. Zenke (MPIB core facility) for confocal
microscopy. Financial support from EU Framework 7 Integrated Project
PROSPECTS, the Deutsche Forschungsgemeinschaft (SFB 596), the Ernst-
Jung Foundation, and the Körber Foundation is acknowledged. H.O. and
A.W. have a fellowship from the Fonds der Chemischen Industrie, and H.O.
is supported by the Elite Graduate Network of Bavaria.
Received: April 15, 2010
Revised: September 6, 2010
Accepted: November 11, 2010
Published: January 6, 2011
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Supplemental Information

EXTENDED EXPERIMENTAL PROCEDURES

Plasmids
For bacterial expression, the N-terminally Myc-tagged b proteins were cloned into the vector pTrcHis (Invitrogen), excluding the His-
tag of the vector. a-S824 protein was cloned into the pProEx HT vector. For mammalian expression, N-terminally Myc-tagged
proteins were cloned into pcDNA3.1/Myc-His (Invitrogen), excluding the Myc- and His-tags of the vector. Human HSP110 was in-
serted into the vector pcDNA3.1 (Invitrogen). HSP70-luciferase (Williams et al., 1989) and NF-kB-luciferase (Heil et al., 2004) reporter
constructs were kindly provided by Dr. H. Wagner (Technical University Munich). GFP fusion constructs of wild-type (WT) Ab1-42 and
its Arctic mutant (E22G) (Wurth et al., 2002) were cloned into pcDNA3.1 using the restriction sites HindIII/NotI.

Protein Purification
Protein expression in E. coli BL21 cells (DE3) was induced with 0.5 mM IPTG at an OD600 of 1.0 for 4 hr at 37
C. The cells were
collected by centrifugation at 5000 x g for 10 min and resuspended in lysis buffer (25% sucrose, 50 mM Tris pH 8.0, 1 mM
EDTA), supplemented with complete protease inhibitor (Roche) and 1 mg/ml lysozyme (Sigma). The cells were lysed by sonication
or repeated freeze-thaw cycles and the DNA was digested by Benzonase (Novagen). Inclusion bodies were isolated by repeated
washing in 0.5% Triton X-100, 1 mM EDTA and centrifugation (20.000 x g for 15 min). The pellet was dissolved in 8 M urea and purified
on a MonoQ 10/100 HR16/10 column in 8 M urea, 25 mM Tris pH 7.5 using a gradient from 0 to 1 M NaCl. The b-protein-containing
fractions were further purified by size exclusion chromatography on Sephacryl S-300 HiPreP 26/60 in 8 M urea, 0.1 M NaCl, 25 mM
Tris pH 7.5. After dialysis overnight into 0.1 M NaCl, 25 mM sodium phosphate pH 7.5, the proteins formed soluble oligomers/aggre-
gates, which eluted in the excluded volume of Sephacryl S-300 size-exclusion chromatography. The protein concentration of the
b sheet proteins was determined by UV absorbance at 210 nm (Abs210 0.1% (= g/l) = 20). All proteins were stored at 80
C in
25 mM sodium-phosphate pH 7.5, 100 mM NaCl.
To purify a-S824 protein, cell lysate was centrifuged at 20.000 x g for 15 min, and the supernatant was applied to Ni-NTA agarose
chromatography following standard procedures. The protein concentration of the purified a-S824 protein was determined by UV
absorbance at 280 nm (3280 = 12950 M-1 cm-1). The protein was stored at 80
C in 25 mM sodium-phosphate pH 7.5, 100 mM NaCl.

Antibodies
A11 anti-oligomer antibody was kindly provided by Dr. C. Glabe (University of California, Davis). Anti-Myc (Cy3-conjugated), anti-
rabbit IgG (peroxidase-conjugated), and anti-mouse IgG (peroxidase-conjugated) were purchased from Sigma, anti-Hsp110, anti-
Myc, anti-VDAC1, anti-RanBP1, anti-eIF3D, anti-eIF4GII and anti-Vigilin from SantaCruz, anti-GAPDH from Chemicon, anti-GFP
from Roche, anti-Myc (FITC-conjugated) from Zymed, and anti-mouse IgG (FITC-conjugated) from Biosource and anti-mouse IgG
(Cy3-conjugated) anti-rabbit (Cy3-conjugated) from Dianova.

Cell Culture, Immunoblotting, and Reporter Assays

HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Biochrom KG), supplemented with 10% fetal calf
serum (FCS), 100 IU/ml penicillin G, 100 mg/ml streptomycin sulfate, 2 mM L-glutamine and nonessential amino acid cocktail (all
from GIBCO). Transient transfections were performed by electroporation with 30 mg expression vector. Lipofectamin (Invitrogen)
transfection was performed for overexpression of Hsp110. Immunoblots were developed using the chemiluminescence kit Rodeo
ECL (USB) and analyzed using a LAS-3000 image reader (Fujifilm) and the AIDA software (Raytest). For luciferase reporter assays,
cells were lysed in Lysis Buffer (Promega). After mixing 20 ml of lysate with 50 ml luciferin solution (Promega), luciferase activity was
measured using a Lumat LB9507 (EG&G Berthold).

Solubility and Detection of A11 Reactive Oligomers

HEK293T cells were lysed in PBS containing 0.5% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate and 10 mM MgCl2. In addition,
a protease inhibitor cocktail (without EDTA) containing 0.3 mg/ml Leupeptin (Roche), 0.3 mg/ml Aprotinin (Roche), 1 mM AEBSF
(Roche), 0.3 mg/ml Prestatin A (Sigma), and 1 mM PMSF (Serva) was added. After 20 min, 25 U/ml benzonase was added, and
the samples incubated with agitation for a further 40 min at 4
C.
For size fractionation, 300-450 mg total protein was loaded on a Superose 6 column using a SMART system (Amersham Biosci-
ence). The column was equilibrated and run using PBS. Lysate samples were separated into 22 fractions. 10 ml of each fraction
was dotted on a nitrocellulose membrane and allowed to dry for 10 min at room temperature. The membranes were blocked in
1% skimmed milk in Tris-buffered saline (TBS)/0.05% Tween-20 and subsequently incubated with A11 antibody (1:1000 dilution)
overnight at 4
C. Bound A11 was detected with anti-rabbit-antibody (conjugated to HRP) followed by chemiluminescence detection.
The signal was visualized and quantified as described for immunoblotting. In parallel, a dot blot of the fractions with anti-Myc anti-
bodies was performed to estimate the amount of b proteins. The cumulative A11 signal was normalized relative to the cumulative anti-
Myc signal.

Cell 144, 67–78, January 7, 2011 ª2011 Elsevier Inc. S1

Immunofluorescence and Fluorescence Imaging
HEK293T cells were fixed in 4% paraformaldehyde/PBS solution for 1 hr, permeabilized with 0.1% (v/v) Triton X-100 for 5 min,
washed twice with PBS, blocked with 1% BSA solution (PBS-B) and then successively treated with primary and secondary anti-
bodies (1 hr each). Fluorescent dye-conjugated primary antibodies were diluted 1:200, other primary antibodies were diluted 1:50
to 1:300 in PBS/1% BSA. The secondary antibodies were diluted 1:200. During the last incubation step with secondary antibody,
0.25 mg/ml DAPI (Invitrogen) was added. After the last incubation, cells were washed 3-times with PBS and mounted in Prolong Anti-
fade (Molecular Probes).
To detect amyloid species, cells were stained with 10 mM NIAD-4 (dissolved in 10% DMSO/90% 1,2-propanediol) for 1 hr, washed
3-times with PBS, fixed, permeabilized, blocked, treated with DAPI and mounted as described for immunofluorescence. NIAD-4 fluo-
rescence was excited at 475 nm and emission recorded at 625 nm.
To visualize the actin cytoskeleton, cells were washed, fixed, and permeabilized as described above. Cells were incubated with
165 nM Rhodamine-conjugated Phalloidin in PBS for 1 hr, washed 3-times with PBS and mounted.

Detection of Apoptotic Cells

2 3 106 of the HEK293T cells were resuspended in 100 ml PBS and added to 0.5 ml of acridine buffer I (20 mM citrate-phosphate, pH
3.0/0.1 mM EDTA/0.2 M sucrose/0.1% Triton X-100). 0.5 ml acridine buffer II (10 mM citrate-phosphate pH 3.8/0.1 M NaCl) was
added together with 20 mg/ml acridine orange (Invitrogen). Cells were resuspended and analyzed by fluorescence microscopy.
TNF/cycloheximide/staurosporine treated cells were used as a reference for apoptotic morphology. At least 200 cells were evaluated
per sample.

Radioactive Protein Labeling

Transfected HEK293T cells were plated on 6-well plates. 24 hr later, cells were washed first with PBS, then with prewarmed FCS-free,
methionine-reduced DMEM (DMEM minus). Labeling was initiated by adding 50 mCi/ml 35S-Met (EasyTag, NEN Radiochemicals) in
DMEM minus. 15 min later, translation was stopped by discarding the radioactive medium and applying cold PBS. The cells were
lysed in 1% Triton X-100/PBS with Protease inhibitor cocktail (Roche). DNA was digested by adding 2.5 mM magnesium chloride
and Benzonase. The extracts were mixed with SDS gel loading buffer, heated and separated on a NuPAGE 4%–12% Bis-Tris gel.
For immunoprecipitation, total extracts were centrifuged at 400 x g for 5 min to remove raw cellular debris, and 15 ml anti-Myc Mi-
croBeads (mMACS) were added for one hour. The unbound material was separated using MACS Separation columns (Miltenyi Bio-
tec). The beads and the associated proteins were washed three times with 0.1% Triton X-100/PBS, once with PBS and eluted with
120 ml SDS sample buffer without b-mercaptoethanol (b-ME). Subsequently, b-ME was added to eluates, the eluates were heated
and analyzed together with total lysate fractions by SDS-PAGE, Coomassie blue staining and fluorography.

Fluorescence Spectroscopy
Fluorescence measurements with purified proteins were performed on a FluoroLog-3 spectrofluorometer. The fluorescent dyes ThT
and ANS were adjusted to a final concentration of 20 mM, NIAD-4 to 1 mM. ThT fluorescence was excited at 440 nm, NIAD-4 at 475 nm
and ANS at 375 nm .

Circular Dichroism Spectroscopy

CD measurements were performed on a Jasco CD Spectrometer J-715 at 25
C in 6 mM HEPES pH 7.5/25 mM KCl, at a protein
concentration of 0.1 mg/ml. Spectra were recorded in a 0.1 mm quartz cuvette between 197 and 250 nm (with a bandwidth of
1 nm and a scanning speed of 50 nm/min). Each single spectrum was averaged from 3 accumulative scans. Secondary structure
content was analyzed with the CDSSTR algorithm (Jasco).

Fourier Transform Infrared (FTIR) Spectroscopy

b proteins (100 mM in 25 mM sodium phosphate pH 7.5, 100 mM NaCl) were dialyzed overnight against 10 mM potassium phosphate
pH 6.0/10 mM NaCl; a-S824 was dialyzed against 10 mM potassium phosphate pH 7.5/10 mM NaCl. Infrared spectra were measured
in the Vertex 70 spectrophotometer (Bruker, Germany) equipped with a TGS detector using attenuated total reflection (ATR) with
parallelogram-shaped Germanium (Ge) crystals as internal reflection plates (Korth Kristalle, Germany). A thin film of 50-100 mg of
the respective protein was dried under N2 on one side of the Ge crystal that was placed in a home-made gas-tight chamber (Heinz
et al., 2003). H-D exchange was performed by flushing D2O-saturated N2 through the chamber and monitored every 1 to 3 min until
the spectra were stable (usually after 45 min). Spectra were recorded before and after H-D exchange with a nominal resolution of
2 cm-1 in the double-sided, forward-backward mode, collecting 1024 scans per sample. Water vapor and CO2 contributions were
corrected for using the atmospheric compensation of the OPUS software (version 6.5) from Bruker. The spectral region of the amide
I band (1705 to 1595 cm-1) was extracted, corrected for the base line, and scaled so as to obtain a constant integral value for compar-
ison. Peak positions of spectral components were analyzed using Fourier-self deconvolution and the second derivative of the
spectra.

S2 Cell 144, 67–78, January 7, 2011 ª2011 Elsevier Inc.

Electron Microscopy
Carbon-coated copper grids were covered with the b protein aggregates either in 25 mM HEPES pH 7.5, 150 mM KCl, 0.5 mM MgCl2
or 10 mM potassium phosphate pH 6.0 buffer for 1 min, washed 3 times, stained for 1 min in 2% uranyl acetate and air-dried. A
CM200 FEG transmission electron microscope (FEI, Eindhoven) with a CCD-camera was used at an accelerating voltage of 160
kV and magnification of 55,000 x.

Mass Spectrometry (MS)

SILAC Medium and Sample Preparation
Custom DMEM (GIBCO) was used for SILAC labeling: originally arginine- and lysine-free DMEM was supplemented with 10% dia-
lyzed FCS, 100 IU/ml penicillin G, 100 mg/ml streptomycin sulfate, 2 mM L-glutamine and nonessential amino acid cocktail (GIBCO).
To prepare L, M, and H media, the respective amino acids were added, for L: Arg0 and Lys0 (arginine and lysine, Sigma), for M: Arg6
and Lys4 (arginine-13C6 and lysine-4,4,5,5-d4, Isotec), for H: Arg10 and Lys8 (arginine-13C6,15N4 and lysine-13C6,15N2, Isotec).
107 SILAC labeled HEK293T cells were transfected. Live cells, attached to the support, were lysed 24 hr later. To this end, cells
were collected and washed by centrifugation. 1% Triton X-100/PBS with Protease Inhibitor Cocktail was added for 20 min. 8 ml ben-
zonase were added, and the cell lysate was incubated for further 40 min. To remove cell debris, the lysate was cleared by centrifu-
gation at 2000 x g for 5 min, the supernatant was removed, and its protein concentration was determined. The lysates labeled with
different isotopes were mixed at a 1:1:1 ratio. 50 ml mMACS anti-Myc (for b proteins) or anti-GFP (for Ab1-42 and Arctic mutant
constructs) MicroBeads were used to isolate Myc-tagged and GFP-tagged proteins, respectively, and their interactome by rotating
the mixed lysates with the beads for 1 hr at 4
C. Samples were applied onto MACS columns equilibrated with 200 ml lysis buffer. The
columns were washed 4 times with 200 ml 0.1% Triton X-100/PBS and once with PBS. Bound proteins were eluted with 120 ml LDS
sample buffer (Invitrogen), followed by the addition of 2% b-mercaptoethanol and heating at 70
C for 10 min. Eluates were separated
on NuPAGE gradient gels, gels were fixed and stained with Colloidal Blue (Invitrogen), according to the manufacturer’s instructions.
Preparation of gel slices, reduction, alkylation, and in-gel protein digestion was carried out as described (Ong and Mann, 2006).
Finally, peptides were desalted, filtered, and enriched on OMIX-C18 tips (Millipore).
LC-MS/MS
Peptides were eluted from OMIX tips using 35 ml of 80% methanol/0.1% TFA. The samples were dried in a vacuum centrifuge
concentrator at 45
C until the volume was less than 5 ml (about 30 min). The volume was increased to 6 ml using 0.1% formic acid
(FA). Using an EasyLC system (Proxeon), 5 ml of sample were loaded at 0.5 ml/min in 0.1% FA onto a 15 cm long capillary column
(75 mm inner diameter) with a prepulled capillary tip (Proxeon) packed with Reprosil-Pur 3 mm C18 material (Dr. Maisch). Peptides
were eluted at 0.3 ml/min using a 120 min gradient (immunoprecipitate eluants) or 150 min gradient (lysate and flowthrough samples)
from 2%–64% acetonitrile, in 0.1% FA. In two cases, an Ultimate 3000 HPLC (Dionex) was used in place of the EasyLC; peptides
were separated as above, except that a C18 precolumn (Dionex) was used to preconcentrate the peptides before elution.
Peptides were directly injected into a Thermo LTQ-FT Ultra using a nano-electrospray ion source (Proxeon), with electrospray volt-
ages ranging from 1.5 to 2.5 kV. FT scans from m/z 400-2000 were taken at 100,000 resolution, followed by collision induced disso-
ciation (CID) scans in the LTQ of the 8 most intense ions with signal greater 2000 counts, and charge state larger than one. Dynamic
exclusion of parent masses already fragmented was enabled. CID settings were as follows: isolation width 3, normalized collision
energy 45 V, activation Q 0.220, and activation time 30 ms.

Analysis of MS Data
Generation of Ratios
MS data were analyzed with MaxQuant version 1.0.12.31 (Cox and Mann, 2008) using the following parameters: Quant; SILAC Trip-
lets, Medium Labels: Arg6, Lys4, Heavy Labels: Arg10, Lys8. Maximum labeled amino acids: 3. Variable modifications: Oxidation (M),
Acetyl (Protein N-terminus). Fixed Modifications: Carbamidomethyl (C). Database: Human IPI, released March 3 2009 with contam-
inants and a decoy database added by the SequenceReverser.exe program released with MaxQuant v. 1.0.12.4. Enzyme: Trypsin/P.
MS/MS tolerance: 0.5 Da. Maximum missed cleavages: 2. Top MS/MS peaks/100 Da: 6. Mascot version 2.2 (Matrix Sciences, www.
matrixsciences.com) was used to generate search results for MaxQuant. Identify; Peptide FDR: 0.01. Protein FDR: 0.01. Maximum
PEP: 1. Minimum unique peptides: 1. Minimum peptide length: 6. Minimum peptides: 1. Protein Quantitation based on Razor and
Unique peptides. Minimum Ratio count: 2. ‘Requantify’ and ‘Keep low-scoring versions of identified peptides’ were both enabled.
Additionally, for those proteins for which no quantitation was available, but for which at least two unique peptides were found
only in one isotope but not another isotope, arbitrary ratios of 10 or 0.1 were assigned, and Significance B was assigned the value
of ‘0’. Unless noted otherwise, normalized ratios were used. Supplemental Tables show the leading protein for each protein group;
that is, the protein which best matched all of the peptides identified. The raw MS data along with a full list of identified proteins and
quantitations is available at https://proteomecommons.org/tranche, entering the following hash: +Ff0/p8lSBrrzCKZfzAwYS3
+Bqw5fonokB679f136te2iklhHtFMUpeT5SM/I3XuufTyrXj0ycVVC6G4Li/L02 dA4jcAAAAAAABVfg = =.

Determination of b Protein Interactors

Four sets of SILAC experiments were performed: I: empty vector (L) versus a-S824 (M) versus b23 (H); II: a-S824 (L) versus b4 (M)
versus b17 (H); III: b4 (L) versus b17 (M) versus b23 (H); IV: a-S824 (L) versus b23 (M) (see schematic in Figures 3A). Experiment IV

Cell 144, 67–78, January 7, 2011 ª2011 Elsevier Inc. S3

is part of the pulse SILAC analysis described below and for the purpose of interactor identification is comparable to experiment I.
Each experiment was performed 3 times independently (biological replicates). A protein was considered a b protein interactor if
an elevated ratio (95% confidence) was found in at least two of the biological replicates in any experiment. b23 interactors were deter-
mined from experiments I, III, and IV; for experiments I and IV, proteins with elevated b23/a-S824 ratios with Significance B (gener-
ated by MaxQuant) less than 0.05 were selected. For experiment III, proteins with an elevated ratio for b23/b17 or for b23/b4 with
Significance B less than 0.05 were selected. This results in 105 interactors (Table S1) which were found to interact with b23 in at least
two replicates of any one experiment (I, III or IV). In order to define more stringently identified interactors, subsets of this list were
generated in which an interactor was found in at least 1, 2 or 3 additional replicates in either of the two remaining experiments.
This is shown in the ‘confidence’ column of Table S1. Note that only a minimal further increase in the number of newly identified in-
teractors was observed after 6 biological replicates.
b17 and b4 interactors were determined from experiments II and III. Proteins which were enriched on b17 or b4 against a-S824 in at
least two replicates of experiment II were considered interactors. Additional b17 and b4 interactors were determined from Experiment
III if they were enriched to Significance B < 0.05 against one of the other two b proteins in at least two replicates, as described above
for b23. Note that each replicate of experiment III was based on the same b23 transfection used in experiment I above, and thus the
replicates were coupled for the purpose of analysis. This allowed us to calculate ratios of b17/a-S824 and b4/a-S824. As a result, 73
b17 (Table S2) and 57 b4 (Table S3) interactors were determined by requiring enrichment in at least two replicates in any one of three
experiments, analogous to what was done for b23. However, since ratios from both experiments I and III were needed to determine
b17/a-S824 and b4/a-S824 ratios, there was a reduced chance of finding b17 and b4 interactors than of finding b23 interactors from
experiment I. Therefore, in order to compare the numbers of interactors for each b protein, a list of b23 interactors was made in which
only those interactors from experiment I were considered which also had a corresponding ratio in experiment III. This results in 94
interactors of b23; proteins from the list of 105 which are not included in this list of 94 are highlighted in yellow in Table S1. Note
that these 11 proteins were removed from the list of b23 interactors only for the purposes of comparison to the b17 and b4 interactors;
they are not identified with lower confidence than the other b23 interactors.
One of the proteins identified as the leading protein was IPI00179330.6 (RPS27A). This is a fusion protein between the small ribo-
somal protein 27A and ubiquitin, which is cleaved after synthesis. Peptides from both portions were identified, however closer
inspection showed that only the peptides corresponding to the ubiquitin moiety were enriched on b23. We therefore decided to iden-
tify this protein according to the second protein in the protein group, IPI00743650.1 (UBC).

Determination of a-S824 Interactors

Three biological replicates of two independent experiments were performed in which a-S824 (M) or a-S824 (H) was analyzed relative
to empty vector (L), respectively. a-S824 interactors were determined exactly as described above for b23, namely that the interactor
must be identified with 95% confidence, according to Significance B, in at least two of the three replicates in any experiment.

Determination of Preferred Interactors

Preferred b protein interactors were considered to be those which had an enriched ratio and Significance B less than 0.05 for one
b protein compared to at least one of the other two b proteins in at least two of the three replicates of experiment III (Tables S1–
S3; proteins in bold).

Depletion of Interactors
The depletion of interactors from the total cell lysate by binding to b23 aggregates was estimated by analyzing the supernatants
(technically flow-through fractions) from the IPs in experiment I. H/M ratios were used to determine the amount of each of the inter-
actors remaining in the cell lysate after IP. The depletion was then estimated by dividing by the approximate transfection efficiency
(50%). A depletion value is reported when an H/M ratio was measured in the supernatant in at least two out of the three replicates and
when the H/M ratio is 0.95 or lower, corresponding to a mean depletion of greater than 10% (Table S1). Relative error in the SILAC
ratios prevents the calculation of reliable estimates for depletion when the depletion is very low. Therefore, for proteins where the
mean H/M ratio of at least two replicates is 0.95 or higher, a depletion value of % 10% has been entered in Table S1. Values of deple-
tion around 10% and higher were confirmed by quantitative western blotting in which protein amounts in IPs were compared with
amounts in total lysate (Figure S3F and Figure 4A, and data not shown).

Pulse-SILAC
The design of this experiment (also see experiment IV above) is shown in Figure 6B. The M-labeled cells were transfected with b23.
After the transfection, half of the cells were shifted to H medium, while the other half continued in M medium. The L-labeled cells were
transfected with a-S824, and half of them were left on L medium, as a control. Lysates were prepared, mixed and analyzed as above.
The measured H/Mlysate24h ratio of individual proteins in the lysate was used to calculate the expected H/Mlysate12h, the ratio at the
midpoint of the experiment, according to the equation:

H=Mlysate24h
H=Mlysate12h = :
2 + H=Mlysate24h

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 In addition to the predominant contribution of protein interacting directly with b23, the H/MIP measured in the pulldown also
has a small contribution from background protein. Since background proteins will have the same isotope ratios as the lysate
(H/Mlysate24h), the H/MIP was corrected according to the enrichment on b23 given by the M/LIP ratio. This generates the ratio H/
Mb23. Deviation of log of the ratio ((H/Mb23)/(H/Mlysate12h)) from 0 indicates enrichment of newly synthesized or preexistent fraction
of a protein on b23. Nonnormalized H/M ratios were used for each of these calculations.

Determination of Ab42 Interactors

Ab42 interactors were determined from experiment V: GFP (L) versus Ab42 WT (M) versus Ab42 Arctic mutant (H); three replicates were
performed. For comparison of the Ab42 interactors to the b protein interactors, all proteins which were enriched on either Ab42 WT or
Ab42 Arctic mutant compared to GFP to Significance B < 0.05 in at least two out of three of the replicates of experiment V were
included. Those proteins which are in common with the b protein interactors are shown in Table S5. In order to determine which
proteins interact preferentially with Ab42 Arctic mutant, proteins which had an elevated Ab42 Arctic/Ab42 WT ratio and Significance
B < 0.01 in any replicate of experiment V were included (Table S6).

Bioinformatic Analysis
The SUPERFAMILY predictions (http://supfam.cs.bris.ac.uk) according to SCOP 1.73 classification (http://scop.mrc-lmb.cam.ac.
uk/) where used to assign protein folds. Information on protein interactions is based on HPRD Release 9 (Keshava Prasad et al.,
2009) (www.hprd.org). This data is combined with the information about gene essentiality in mouse from MGD (Bult et al., 2008)
(http://www.informatics.jax.org/) and the compilation of proteins associated with neurodegenerative disease proteins (Raychaudhuri
et al., 2009). The cellular abundance of the b protein interactors was estimated according to HEK293T mRNA levels measured by Su
et al. (2002) (http://biogps.gnf.org).

Prediction of Intrinsic Aggregation Propensities

The intrinsic aggregation propensity, pagg
i , of an individual amino acid is defined as

pagg
i = ah phi + as psi + ahyd phyd
i + ac pci

where phi , psi , phyd

i , pci are the amino acid scales for a-helix and b sheet formation, hydrophobicity and charge and the a’s are the
corresponding weights (see below) (Tartaglia et al., 2008). An aggregation propensity profile is then defined as

1X 3
pagg
i = pagg + apat Ipat + agk Igk
7 j = 3 i + j i i

where we considered the aggregation rate of a seven-residue segment of the protein centered at position i. Ipat
i and Igk
i are included,
pat
respectively, to account for the presence of hydrophobic patterns and of gatekeeper residues. The term Ii is 1 if residue i is included
in a hydrophobic pattern over five residues and 0 otherwise, while the term Igk
i is defined as

X
10
Igk
i = ci + j
j = 10

where the sum over the charges ci of individual amino acids is made over a sliding window of 21 residues; shorter windows are
considered at the N- and C-termini. The term Igk
i is introduced to take into account the fact that, when a hydrophobic pattern is flanked
by charged residues, its contribution to the aggregation propensity is much reduced by electrostatic repulsions.
By normalizing pagg
i , the intrinsic aggregation propensity score, Ziagg , is obtained, which is used to compare the aggregation
propensity of different sequences

pagg  magg
Ziagg = i
sagg

where magg is the average value of Ziagg over a set of random polypeptides having the same length as the sequence of interest, and
agg
s is the corresponding standard deviation from the average (Tartaglia et al., 2008). The corresponding intrinsic propensity for
aggregation of the protein without structural correction, Z agg , is calculated as

1X N
Z agg = Z agg
N i=1 i

The weights a’s are determined by fitting the Z agg scores against a database of aggregation rates measured experimentally (Tar-
taglia et al., 2008).

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Prediction of Aggregation Propensities with Structural Corrections
The local stability of various regions in a protein can be investigated by using the CamP method, which uses the knowledge of the
amino acid sequence to provide a prediction of the protection factors from hydrogen exchange (Tartaglia et al., 2007). By combining
the predictions of the intrinsic aggregation propensity profiles with those for the local stability in the folded state, we account for the
influence of the structural context on the aggregation propensities. We thus define (Tartaglia et al., 2008) a sequence-dependent
agg
aggregation propensity score with the structural correction, Z~i , by modulating the intrinsic aggregation propensity profile with
the local stability score
agg
Z~i = Ziagg ð1  3lnPi Þ
agg
where 3 was fixed at 1/13 and lnPi, is the logarithm of the protection factor of residue i. The plot of Z~i versus the residue number
represents the aggregation propensity profile calculated to account for the structural protection. The corresponding propensity for
agg
aggregation of the protein, Z~ , is calculated as

P
N agg  agg agg 
Z~i w Z~i  Z~0
agg
Z~ = i = 1 N
P  ~agg ~agg 
w Zi  Z0
i=1

agg agg agg agg agg agg agg

where the function wðZ~i  Z~0 Þ is 1 for Z~i
 Z~0 >0 and 0 forZ~i
 Z~0 <0; to predict ordered aggregation we use Z~0 = 0, i.e.,
agg
only positive contributions are taken into account; to estimate the propensity of a protein to be insoluble we use Z~0 = Z~min , i.e., both
positive and negative contributions are taken into account.

SUPPLEMENTAL REFERENCES

Bult, C.J., Eppig, J.T., Kadin, J.A., Richardson, J.E., and Blake, J.A. (2008). The Mouse Genome Database (MGD): mouse biology and model systems. Nucleic
Acids Res. 36, D724–D728.
Heil, F., Hemmi, H., Hochrein, H., Ampenberger, F., Kirschning, C., Akira, S., Lipford, G., Wagner, H., and Bauer, S. (2004). Species-specific recognition of single-
stranded RNA via toll-like receptor 7 and 8. Science 303, 1526–1529.
Heinz, C., Engelhardt, H., Niederweis, M., Heinz, C., Engelhardt, H., and Niederweis, M. (2003). The core of the tetrameric mycobacterial porin MspA is an
extremely stable beta-sheet domain. J. Biol. Chem. 278, 8678–8685.
Tartaglia, G.G., Cavalli, A., and Vendruscolo, M. (2007). Prediction of local structural stabilities of proteins from their amino acid sequences. Structure 15, 139–
143.
Wurth, C., Guimard, N.K., and Hecht, M.H. (2002). Mutations that reduce aggregation of the Alzheimer’s A beta 42 peptide: an unbiased search for the sequence
determinants of A beta amyloidogenesis. J. Mol. Biol. 319, 1279–1290.
Zandomeneghi, G., Krebs, M.R., McCammon, M.G., Fandrich, M. (2004). FTIR reveals structural differences between native beta-sheet proteins and amyloid
fibrils. Protein Sci. 13, 3314–3321.

S6 Cell 144, 67–78, January 7, 2011 ª2011 Elsevier Inc.

Figure S1. Characterization of b Proteins, Related to Figure 1
(A) Purified proteins b4, b17, b23, and a-S824. Left panel: 13.5% SDS-PAGE, Coomassie blue staining. Middle panel: Immunoblotting with anti-Myc antibodies.
Right panel: 13.5% SDS-PAGE with 4 M urea, Coomassie blue staining.
(B) Circular dichroism spectra of the purified model proteins. Spectra of 0.1 mg/ml protein solutions were recorded in 6 mM HEPES (pH 7.5)/25 mM KCl, as
detailed in Extended Experimental Procedures. The spectra of b4 (green), b17 (blue), b23 (red), and a-S824 proteins (gray) are representative scans of three
independent experiments. The table shows secondary structure content calculated from three independent experiments using the CDSSTR algorithm (Jasco).
(C) FTIR spectra of the purified model proteins. Spectra of 100 mg protein were recorded as detailed in Extended Experimental Procedures. Shown are
representative scans of 3 independent experiments. The peak at 1647 cm-1 originates from dominant a-helical structure and the band at 1624 cm-1 is
characteristic of extended b sheet structure in amyloid fibrils (Zandomeneghi et al., 2004). The small peak at 1695–1690 cm-1 seen for the b proteins is indicative of
anti-parallel b sheet structure. b4 possesses a significant spectral component at 1651 cm-1, suggesting the presence of some a-helical structure.

Cell 144, 67–78, January 7, 2011 ª2011 Elsevier Inc. S7

Figure S2. Cellular Effects of b Protein Expression in HEK293T Cells, Related to Figure 2
(A and B) b Protein expression induces cell death. HEK293T cells were transfected with the indicated proteins. After 3 days, surviving cells were counted by
Trypan Blue exclusion assay (A). The number of surviving cells after transfection with empty control vector, C, is set to 100%. The fraction of apoptotic cells was
determined by Acridine Orange staining and counting of cells displaying characteristic apoptotic morphology (B). Averages and standard deviations from at least
three experiments are shown. Statistical significance was estimated by t test: *, p < 0.05, **, p < 0.005.
(C) A11 antibody reactivity of extracts of b-protein-expressing cells. HEK293T cells were transfected with a-S824 or b23. After 24 hr, cell extracts were prepared
and separated by gel filtration on a Superose 6 column. Fractions were dot blotted and analyzed with the A11 anti-oligomer antibody. The A11 signal was
normalized to protein amounts, based on the signal obtained with anti-Myc antibody. Fraction numbers and positions of molecular weight markers are shown. A
quantification of these results is shown in Figure 2E.

S8 Cell 144, 67–78, January 7, 2011 ª2011 Elsevier Inc.

Figure S3. Interactome Analysis of b Proteins, Related to Figure 3
(A) To control the amounts of isolated aggregates, anti-Myc immunoblotting analysis of the mixed lysates was performed before and after immunoprecipitation
(IP). Note that different mobility of the proteins in SDS-PAGE allows all three b proteins to be visualized on the same gel lane. The amount of lysate analyzed
corresponds to 12% of input for IP. Approximately 85% of the b proteins was immunoisolated.
(B) b23 interactors. HEK293T cells were SILAC-labeled and transfected with b23, a-S824, or vector only and processed as detailed in Extended Experimental
Procedures and Figure 3A. Normalized protein ratios from two replicates are plotted as indicated. Red circles indicate proteins which met the criteria for inclusion
on the b23 interactor list (Table S1); white circles show proteins which are not included on the interactor list, because they did not reproducibly fulfill the criteria for
enrichment. Proteins which had intensity only in one isotope were assigned the artificial value of 0.1 or 10, resulting in proteins with a b23/a-S824 ratio of exactly
10.
(C and D) b4, b17 and b23 interactors. HEK293T cells were SILAC-labeled, transfected with b proteins and processed as detailed in Extended Experimental
Procedures and Figure 3A. Normalized protein ratios are plotted as in (B); green circles show b4 interactors, blue circles show b17 interactors. As above, red
circles indicate b23 interactors and white circles show proteins not meeting the interactor criteria.
(E and F) Validation of b protein interactors (Tables S1–S3) by immunofluorescence microscopy (E) and western blotting of pulldowns (F). The interaction of eIF3D
(preferred interactor of b23 and b17) and Vigilin (preferred interactor of b17) was analyzed by both methods. HEK293T cells were transfected as indicated (C,
vector only control). 24 hr after transfection, cells were fixed and costained with anti-Myc, anti-eIF3D or anti-Vigilin antibodies. Nuclei were stained with DAPI.
Representative examples of two independent experiments are shown. No interaction of GFP and of an actin-GFP fusion protein with the aggregates was
observed (data not shown). Western blots were also analyzed with antibodies against VDAC1 (preferred interactor of b17), RanBP1 (interactor of b17 and b23) and
eIF3E (interactor of b4, b17 and b23) (F). Western blots are representative examples of two to three independent experiments. Quantitative western blotting in
comparison to lysate samples revealed that b4, b17, and b23 interacted with 6%, 10%, and 11% of VDAC1; 0%, 1.3%, and 1.8% of RanBP1; 6%, 9%, and 10%
of eIF3D; and 7%, 8%, and 8% of eIF3E, respectively. This analysis was based on averages from two to three experiments and took into account that the
transfection efficiency for the b proteins was 50%.
(G) Impairment of protein synthesis upon b23 expression. 24 hr after transfection, HEK293T cells expressing a-S824 or b23 were pulsed-labeled with 35S-Met for
15 min to determine the amount of newly synthesized proteins (New). Radioactive proteins were normalized to total Coomassie blue stained protein (Total). The
amount of newly synthesized protein in a-S824 expressing cells was set to 100%. b23 expression inhibited protein synthesis to 63 ± 19% (standard deviation of
seven experiments).
(H) Phalloidin-rhodamine staining of the actin cytoskeleton of C, a-S824, b4, b17, and b23 expressing cells 24 hr after transfection.

Cell 144, 67–78, January 7, 2011 ª2011 Elsevier Inc. S9

Figure S4. Hsp110 Expression in b-Protein-Expressing Cells, Related to Figure 4
(A) Levels of overexpression of Hsp110 in HEK293T cells detected by immunoblotting. Cells were transfected with human Hsp110 expression plasmid using
Lipofectamine. 24 hr later they were transfected with 30 mg of the b protein expression plasmids or empty control vector by electroporation. Three days later, cells
were lysed and Hsp110 expression levels determined by immunoblotting with anti-Hsp110 antibody. GAPDH was detected with anti-GAPDH antibody as
a loading control.
(B and C) Changes in cell morphology and b protein solubility upon overexpression of Hsp110. Transfection of Hsp110 and b proteins was performed as in (A),
except that 5 mg DNA was used for b protein expression. 24 hr hours later, cell morphology and aggregation of the model proteins were evaluated by confocal
microscopy using anti-Myc and anti-Hsp110 immunostaining. Nuclei were visualized by DAPI staining. An example of a cell with Hsp110 overexpression
containing soluble b17 and having preserved normal cell shape is indicated by a white arrow (B). The fraction of cells with b protein aggregates, soluble b protein
and normal morphology was determined by evaluating at least 900 cells per condition (C). In the absence of Hsp110 overexpression (solid bars), the number of
cells expressing the b proteins was set to 100%. In case of Hsp110 overexpression (hatched bars), cells containing elevated levels of Hsp110 were set to 100%.
Averages and standard deviations from three independent experiments are shown.

S10 Cell 144, 67–78, January 7, 2011 ª2011 Elsevier Inc.

Figure S5. Structural Properties of interactors of b23 and Ab42-GFP, Related to Figure 5
(A) Domain fold distribution of lysate proteins and b23 interactors. The frequency of domain folds (SCOP fold classes) among the proteins with identified fold
topology (72 b23 interactors and 2261 lysate proteins, corresponding to 129 and 3331 folds, respectively) is shown. Repetitive domain folds are counted only
once per protein. SCOP classification (http://scop.mrc-lmb.cam.ac.uk/): a, all alpha; b, all beta; c, alpha/beta proteins (a/b); d, alpha plus beta proteins (a + b); e,
multidomain proteins (alpha and beta); f, membrane and cell surface proteins and peptides; g: small proteins; h: coiled-coil proteins. *p < 0.05 for b23 interactors
relative to lysate proteins, based on Chi-square test.
(B) Length distribution in amino acids (aa) of intrinsically unstructured regions (IURs) in lysate proteins and b23 interactors (p < 0.005 according to the Mann-
Whitney test).
(C–E) Expression of Ab142 -GFP constructs and cell viability. (C) Anti-GFP western blot of HEK293T cells expressing GFP, WT Ab142 or the Arctic mutant of
Ab142 for 24 hr in the absence or presence of 1 mM MG132 (left panel). Asterisk indicates a proteolytic fragment of WT Ab142 and the Arctic mutant. Cells
expressing b4, b17 or b23 in the absence or presence of 5 mM MG132 were analyzed by anti-Myc western blotting for comparison (right panel). (D) GFP fluo-
rescence microscopy of HEK293T cells expressing GFP, WT Ab142 or Arctic mutant in the presence of 1 mM MG132. (E) Viability of cells expressing GFP, WT
Ab142 or Arctic mutant in the absence or presence of 1 mM MG132, as measured by the MTT assay.
(F–I) Physicochemical properties of proteins that interact preferentially with the Arctic mutant of Ab142. Interactors that were enriched on the Arctic mutant
relative to WT Ab142 (31 proteins) were compared to lysate proteins in terms of molecular weight (F), hydrophobicity (G), fraction of amino acid residues in
disordered regions (H), and the presence of disordered stretches longer than 30 or 50 amino acids (I). Box plots indicate the distribution of the data. Dashed
horizontal line indicates the median, whisker caps and circles indicate 10th/90th and 5th/95th percentiles, respectively. *p < 0.05 based on the Mann-Whitney test
(H) or Chi-square test (I); **p < 0.005 based on a Chi-square test.

Cell 144, 67–78, January 7, 2011 ª2011 Elsevier Inc. S11

Figure S6. Aggregation Propensities of the b23 Interactome, Related to Figure 6
(A) Newly synthesized proteins are susceptible to b23 coaggregation. HEK293T cells were transfected and newly synthesized proteins were labeled as in Fig-
ure S3G (Total). Subsequently, an anti-Myc immunoprecipitation was performed to determine the fraction of new polypeptides interacting with the model proteins
(Bound). The average ± standard deviation from 5 independent experiments was 1 ± 0.5% for a-S824 and 7 ± 1.8% for b23. The amount of total newly
synthesized protein was set to 100%.
(B) Aggregation propensity of the unfolded polypeptides (Zagg predictor) is shown in solid bars; and aggregation propensity after correction for the local stabilities
of the folded state, Zagg + lnP predictor is shown in hatched bars (Tartaglia et al., 2008) (see Extended Experimental Procedures for details). Average scores for the
sets of proteins as indicated. Left: lysate proteins compared to b23 interactors (Tables S1 and S4); right: newly synthesized b23 interactors (New) compared to
preexistent b23 interactors (Old) (top 15 versus bottom 15 proteins in Figure 6B and Table S7).

S12 Cell 144, 67–78, January 7, 2011 ª2011 Elsevier Inc.