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Citation for the original published paper (version of record):
Eremenko, E., Ben-Zvi, A., Morozova-Roche, L., Raveh, D. (2013)
Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a
Yeast Model.
PLoS ONE, 8(3): e58218
http://dx.doi.org/10.1371/journal.pone.0058218
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Aggregation of Human S100A8 and S100A9
Amyloidogenic Proteins Perturbs Proteostasis in a Yeast
Model
Ekaterina Eremenko1, Anat Ben-Zvi1,2*, Ludmilla A. Morozova-Roche3, Dina Raveh1*
1 Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel, 2 National Institute for Biotechnology in the Negev, Ben-Gurion University of the
Negev, Beer-Sheva, Israel, 3 Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden
Abstract
Amyloid aggregates of the calcium-binding EF-hand proteins, S100A8 and S100A9, have been found in the corpora
amylacea of patients with prostate cancer and may play a role in carcinogenesis. Here we present a novel model system
using the yeast Saccharomyces cerevisiae to study human S100A8 and S100A9 aggregation and toxicity. We found that
S100A8, S100A9 and S100A8/9 cotransfomants form SDS-resistant non-toxic aggregates in yeast cells. Using fluorescently
tagged proteins, we showed that S100A8 and S100A9 accumulate in foci. After prolonged induction, S100A8 foci localized
to the cell vacuole, whereas the S100A9 foci remained in the cytoplasm when present alone, but entered the vacuole in
cotransformants. Biochemical analysis of the proteins indicated that S100A8 and S100A9 alone or coexpressed together
form amyloid-like aggregates in yeast. Expression of S100A8 and S100A9 in wild type yeast did not affect cell viability, but
these proteins were toxic when expressed on a background of unrelated metastable temperature-sensitive mutant proteins,
Cdc53-1p, Cdc34-2p, Srp1-31p and Sec27-1p. This finding suggests that the expression and aggregation of S100A8 and
S100A9 may limit the capacity of the cellular proteostasis machinery. To test this hypothesis, we screened a set of
chaperone deletion mutants and found that reducing the levels of the heat-shock proteins Hsp104p and Hsp70p was
sufficient to induce S100A8 and S100A9 toxicity. This result indicates that the chaperone activity of the Hsp104/Hsp70 bichaperone system in wild type cells is sufficient to reduce S100A8 and S100A9 amyloid toxicity and preserve cellular
proteostasis. Expression of human S100A8 and S100A9 in yeast thus provides a novel model system for the study of the
interaction of amyloid deposits with the proteostasis machinery.
Citation: Eremenko E, Ben-Zvi A, Morozova-Roche LA, Raveh D (2013) Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis
in a Yeast Model. PLoS ONE 8(3): e58218. doi:10.1371/journal.pone.0058218
Editor: Harm H. Kampinga, UMCG, The Netherlands
Received September 10, 2012; Accepted February 1, 2013; Published March 6, 2013
Copyright: ß 2013 Eremenko et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by FP7 EU contract PITN-GA-2008-215148, http://cordis.europa.eu/fp7/home_en.html. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected] (AB); [email protected] (DR)
modulate the functional properties of S100A8 and S100A9 and
their interactions with various molecular targets. Binding of Zn2+
to S100A8 and S100A9 leads to fine-tuning of their folding and
may affect their function. The assembly of S100A8 and S100A9
into multiple hetero-dimeric and hetero-tetrameric complexes is
considered to be a generic mechanism of protein functional
diversification through variation of their conformational states and
may determine their association with different ligands.
Nine different S100A proteins (A1–A6, A8, A9 and A12) appear
in corpora amylacea inclusions in the brain during normal aging
[9]. Recently, S100A8 and S100A9 were identified in amyloid
aggregates in corpora amylacea of prostate cancer patients. That
study was the first report of amyloid fibril formation by members
of the S100 protein family. S100A8/9 inclusions were found in the
corpora amylacea together with bacterial DNA and proteins that
were surrounded by inflamed tissues infiltrated by neutrophils
[10], [11]. The findings that under inflammatory conditions the
S100A8/9 complex accounted for up to 40% of the total cytosolic
proteins in neutrophils and that secreted S100A8/9 was found at
high concentrations in inflamed tissues [12] has led to the
hypothesis that S100A8/9 amyloids may be formed in response to
Introduction
S100 proteins are a family of 10- to 14-kDa EF-hand calciumbinding proteins that regulate diverse cellular processes affecting
cell survival, proliferation, differentiation, and motility [1].
Marked changes have been observed in the expression levels of
many S100 proteins in different types of cancer, neurodegenerative disorders, and inflammatory and autoimmune diseases.
Among these proteins, S100A8 and S100A9, in particular, are
involved in inflammation and cancer [2]; these two proteins
activate the MAP kinase and NF-kB signaling pathways, trigger
translocation of the RAGE receptor in human prostate cancer cells
[3], and activate Toll-like receptor 4 [4], [5]. The secretion of
S100A8 and S100A9 in response to cell damage or immune
response activation constitutes a danger signal that activates
immune and endothelial cells. Consequently, S100A8 and S100A9
are defined as damage-associated molecular pattern proteins in
innate immunity [6], [7].
Structural studies indicate that in vitro S100A8 and S100A9
form homo- and hetero-dimers and heterotetramers [8]. This
oligomerization is calcium dependent, and the binding of Ca2+ to
the EF-hand domain triggers conformational changes that
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S100A8/A9 Aggregation Perturbs Proteostasis
transformed with pGFP-S100A8, pGFP-S100A9 or with both
pmCherry-S100A8 and pGFP-S100A9 showed diffuse fluorescence
throughout the cytoplasm (Figure 1A and 1B). Western blot
analysis of yeast that produced either one or both of the S100A
proteins showed a protein band of the expected size in the induced
cells but not in an empty vector control (Figure 1C).
GFP
S100A8 and GFPS100A9 aggregates revealed an annular or
punctate localization after two days of induction in wild type yeast
(Figure 2A). Prolonged induction resulted in accumulation of
S100A8 foci, specifically in the vacuole, as visualized by the FM464 lipophilic fluorescent dye [29] (34.464.5% compared with
16.760.7% for the GFP control, p,0.05). In contrast, GFPS100A9
aggregates were observed throughout the cell after four days of
induction (Figures 2B and 2C). Cotransformants, mCherryS100A8/GFPS100A9, showed early formation of bright foci that
were localized inside the vacuole of the cells (24.467.7% vacuolar
compared with 9.764.8% cytoplasmic) after two days of
induction, suggesting that S100A8 affected localization of the foci
in the cotransformants (Figure 3 and data not shown). To support
our observation that GFPS100A8 foci accumulate in the vacuole,
we produced GFPS100A proteins in a pep4D deletion strain that
lacks the vacuolar protease A [30] and examined the formation of
foci. This treatment resulted in a sharp increase in GFPS100A8 foci
in the vacuole (80.964.1%) compared with the GFP control or
with GFPS100A9 (39.966.9% and 43.660.8%, respectively,
p,0.005) (Figure 4). Thus, GFPS100A8 and GFPS100A9 transformed separately or together resulted in the formation of foci over
time; both foci containing GFPS100A8 alone and GFPS100A8 cotransformed with GFPS100A9 showed specific accumulation in the
vacuole.
The formation of very bright foci or ring-like structures is
known to be strongly associated with ordered amyloid-like protein
aggregation [25]. Given that S100A8 and S100A9 proteins form
oligomeric and fibrillar structures [6], [8], [10], we examined their
aggregation by native gel analysis. After two and four days’
induction S100A8 and S100A9 proteins formed insoluble high
molecular weight (MW) structures that were retained in the well of
the gel, indicative of aggregate formation (Figure 5A). Similar
behavior was observed for the S100A8/9 co-transformation. High
MW species were also detected upon semi-denaturing detergentagarose gel electrophoresis (SDD-AGE) [31], [32]. After two days
of incubation, GFPS100A8 and GFPS100A9 and mCherryS100A8/GFPS100A9 proteins formed SDS-resistant aggregates
that could be dissolved only by boiling (Figure 5B). This behavior
was not related to the fluorescent tag, since non-tagged S100A8
and S100A9 proteins also formed insoluble aggregates on SDDAGE gels (Figure S1) or in the filter-trap assay (Figure 5C). To
further characterize the S100A aggregates, we used thioflavin T
(ThT), a benzothiazole dye that exhibits enhanced fluorescence
upon binding to b-sheets of protein amyloids both in vivo and
in vitro [33]. After ThT staining of spheroplasts prepared from cells
that expressed single non-tagged S100A8, S100A9 or both
proteins, the S100A8 and S100A9 transformants exhibited bright
fluorescence in addition to stained foci, similar to those observed
with the fluorescently tagged proteins (Figure 5D). Thus, S100A8
and S100A9 protein expression in the yeast S. cerevisiae results in
the formation of amyloid-like aggregates.
chronic inflammation [10] and consequently may enhance the risk
of cancer.
Accumulation of amyloid aggregates in cells is a common
molecular event in a large number of human diseases [13]. Such
diseases include Alzheimer’s disease, which is associated with the
polymerization of amyloid b-peptide, and polyglutamine diseases,
such as Huntington’s disease, which is characterized by the
presence of extensions of the polyQ stretches in certain proteins
[14]. Accumulation of misfolded proteins in the cell disrupts
cellular homeostasis and can lead to toxicity and cell death.
Cells have developed an elaborate machinery to preserve
protein homeostasis that involves several strategies aimed at either
refolding, degrading, or sequestering misfolded proteins [15]. The
cellular proteostasis machinery consists of molecular chaperones
and of the cellular protein degradation machineries, such as the
ubiquitin-proteasome system and autophagy pathways. Under
optimal conditions, these mechanisms balance the cellular load of
metastable and misfolded proteins [16], [17]. However, the
expression of aggregation-prone proteins can interfere with the
degradation of proteasome substrates, alter the subcellular
distribution of essential proteins such as chaperones, and have
deleterious effects on folding of other proteins [18], [19], [20],
[21], [22]. It has been proposed that protein-misfolding diseases
are initiated by the global disruption of cellular proteostasis due to
the depletion and redistribution of essential components of the
proteostasis network, resulting in a reduced protein folding
capacity [23], [24].
The development of non-mammalian models to study protein
aggregation diseases has been invaluable for the discovery of
pathways and modifiers and for the elucidation of the underlying
mechanism of toxicity [22]. Yeast has emerged as a simple
eukaryote model for the characterization of amyloidogenic
proteins and their interactions with cellular defense mechanisms
[25], [26], [27], [28]. To examine the interactions of the
aggregation-prone human S100A8 and S100A9 proteins with
the proteostasis network, we established a novel model system by
expressing them in the yeast, S. cerevisiae. Our current study showed
that expression of the amyloidogenic human proteins, S100A8 and
S100A9, in yeast does not affect the viability of wild type cells.
Yeast, therefore, provides an excellent cellular model to specifically
study the effect of aggregation of S100A8 and S100A9 proteins on
the vital components of the cell proteostasis machinery. Indeed, we
found that expression of S100A8 and S100A9 exposed unrelated
metastable proteins in the background, which suggests that the
expression of aggregating proteins significantly burdens the cell
proteostasis mechanisms and can be a critical factor in their
survival under stress conditions. We attribute increased toxicity of
metastable proteins to depletion of molecular chaperones required
for stabilization of the endogenous mutant protein.
Results
A Yeast Model for Investigating Human S100A8 and
S100A9 Amyloidogenic Proteins
We established a S. cerevisiae model system to study human
S100A8 and S100A9 protein aggregation and potential toxicity by
expressing S100A8 and S100A9 from the inducible GAL promoter.
S100A8 and S100A9 proteins were produced either as fluorescently tagged (pmCherry-S100A8 or pGFP-S100A8, and pGFPS100A9, respectively) or as non-tagged proteins. The plasmids
were transformed separately (GFP-tagged proteins) or together
(pmCherry-S100A8 and pGFP-S100A9) into W303 wild type yeast
and plated on either glucose (non-inducing conditions) or galactose
(inducing conditions) plates. After overnight induction, cells
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S100A8 and A9 Aggregates are not Toxic in Yeast
Protein aggregation is often associated with growth arrest and
cell death [25]. We therefore tested whether S100A8 and S100A9
are toxic to yeast. Serial ten-fold dilutions of cells expressing pGFPS100A8, pGFP-S100A9 or cotransformants with pmCherry-S100A8
and pGFP-S100A9 were plated on SD plates with either glucose or
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Figure 1. Overnight induction of pGFP-S100A8 or pGFP-S100A9 in yeast. (A) Single S100A8 or S100A9 yeast transformants or (B)
cotransformants with plasmids mCherryS100A8/GFPS100A9 were grown overnight in SG, and images were obtained with a fluorescence microscope. (C)
TCA precipitates of extracts from cells growing on glucose or galactose medium were separated by 10% SDS-PAGE and analyzed by Western blot.
doi:10.1371/journal.pone.0058218.g001
affect S100A aggregation, we monitored the formation of high
MW species using SDD-AGE. We found that at 30uC, a
temperature at which no effect on cell viability was observed,
expression of S100A8 or S100A9 in the cdc53-1 mutant resulted in
the accumulation of more high MW species than in the wild type
strain (Figures 7B and S3).
cdc53-1 mutant cells transformed with an empty vector alone
showed a mild decrease in viability on galactose plates compared
with glucose plates and this could have contributed to the
reduction in viability observed when we induced the S100A8 and
S100A9 genes with galactose. We therefore examined the toxicity
of the ts mutant using an alternative inducible system in which
S100A8 and S100A9 were regulated by the TET on-off promoter.
There was no change in cell viability when wild type cells
expressing pTET-S100A8 or pTET-S100A9 were grown on regular
(inducing) or doxycycline-supplemented (non-inducing) plates
(Figure S2A), although the proteins were expressed and formed
aggregates, as visualized by ThT staining (Figure S2B). In contrast,
expression of pTET-S100A8 or pTET-S100A9 in the cdc53-1
mutant under permissive conditions (32uC) resulted in decreased
viability (Figure 7C).
To extend our observations, we expressed S100A8 and S100A9
in three additional ts mutant strains: cdc34-2, a G58R mutant of
the SCF-related ubiquitin-conjugating enzyme (E2) [35]; srp1-31, a
S116F mutant of the importin-a ortholog that functions as a
nuclear import receptor for proteins with a classic nuclear
localization signal [36]; and sec27-1, a G688D mutant in an
essential coat protein, involved in endoplasmic reticulum (ER)-toGolgi and Golgi-to-ER transport [37]. Production of S100A8 or
S100A9 in the cdc34-2 mutant resulted in growth inhibition at
33uC, compared with cells transformed with an empty vector.
Likewise, expression of S100A8 or S100A9 in the srp1-31 or sec27-1
ts strains resulted in S100A8- and S100A9-dependent toxicity at
galactose. No marked effects on the viability of the cells expressing
these two S100A proteins compared with those expressing an
empty vector were observed (Figure 6A). Similar findings were
obtained for non-tagged S100A8 and S100A9 (Figure 6B).
Therefore, expression of S100A8 and S100A9 proteins in yeast
led to the production of amyloid protein, with no effects on cell
growth. This finding suggests that yeast cells can cope with the
expression of the aggregation-prone S100A8 and S100A9 proteins
and modulate their toxicity.
S100A8 and S100A9 Aggregates are Toxic in the
Presence of an Unrelated Metastable Protein
Protein aggregation was shown to impact the cellular proteostasis machinery by inducing misfolding of temperature-sensitive
(ts) metastable proteins under permissive conditions and exposing
their specific phenotypes in a particular genetic background. This
increase in protein misfolding load, in turn, enhanced protein
aggregation [23], [24]. The extent and specificity of the genetic
interaction varied with the characteristics of the aggregates formed
[24]. Therefore, to assess the effects of S100A8 and S100A9
aggregation on cellular proteostasis, we examined their effects on
the viability of cells with a ts mutation in an essential gene under
permissive conditions. A ts cdc53-1 mutant with a R488C
substitution in the Cullin scaffold protein of the SCF (Skp1Cullin-F-box protein) ubiquitin ligase complex required for cell
cycle progression at the G1-S phase [34] was transformed with
pYES2-S100A8 or pYES2-S100A9 (Figure 7A). Induction of
S100A8 or S100A9 in the cdc53-1 ts mutant resulted in growth
inhibition at the permissive temperature of 33uC compared with
cells transformed with an empty vector. These data indicate that
S100A8 and S100A9 can uncover cdc53-1 defects in viability at the
permissive temperature. To examine whether cdc53-1 can, in turn,
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Figure 2. S100A8 and S100A9 form visible foci in yeast cells. Fluorescent microscope images of GFPS100A8 and GFPS100A9 (green) after (A) 2
days or (B) 4 days of induction. Lipophilic dye FM4-64 was used to visualize vacuoles (red). (C) Quantification of the percent of cells with GFP,
GFP
S100A8 or GFPS100A9 foci in the vacuole or in the cytoplasm, following 2 and 4 days of induction.
doi:10.1371/journal.pone.0058218.g002
30uC (Figure 7C). These results obtained with two different
induction systems and with four different ts mutants indicate that
S100A8 and S100A9 aggregation can differentially affect the
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function of an unrelated metastable protein in the cell and thus
suggest that the capacity of the protein folding homeostasis
machinery has been substantially reduced.
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Figure 3. S100A8/9 form aggregates in yeast cells. GFP (green) and mCherry (red) fluorescent microscope images of mCherryS100A8/GFPS100A9
cotransformed cells after 2 or 4 days of induction.
doi:10.1371/journal.pone.0058218.g003
when native proteins are denatured, for example, by stress, some
chaperones, such as the heat-shock protein Hsp104, specifically
interact with protein aggregates and can either promote or prevent
amyloid formation and propagation of prions [38], [39], [40].
Yeast Hsp104p was found to act directly on protein aggregates and
to lead to their resolubilization [41]. Therefore to examine
whether Hsp104p is required for modulating S100A8 and S100A9
protein aggregation and toxicity, wild type and hsp104D mutants
Hsp104p Modulates S100A8- and S100A9-associated
Toxicity
To directly examine the impact of S100A8 and S100A9
proteins on the cellular protein quality control capacity, we
examined their genetic interactions with different components of
the protein folding machinery. Whereas most chaperones bind to
non-native or misfolded protein conformations that are generated
Figure 4. S100A8 accumulates foci in the vacuole in pep4D mutant cells. (A) Confocal images of GFP, GFPS100A8 or GFPS100A9 (green) after 2
days of induction in pep4D mutants cells. Lipophilic dye FM4-64 was used to visualize vacuoles (red). (B) Quantification of the percent of cells with
GFP, GFPS100A8 or GFPS100A9 foci in the vacuole of pep4D cells, following 2 days of induction.
doi:10.1371/journal.pone.0058218.g004
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Figure 5. Aggregate formation by yeast cells transformed with pGFP-S100A8, pGFP-S100A9 or pmCherry-S100A8/pGFP-S100A9 after
prolonged induction. (A) NI, noninduced control (Lanes 1, 4, 7, 10). Extracts of cells induced for 2 or 4 days to produce GFPS100A8 (Lanes 2 and 3),
GFP
S100A9 (Lanes 5 and 6) and mCherryS100A8/GFPS100A9 (Lanes 8, 9 and 11,12) were separated on a native gel and analyzed by Western blot. (B)
Semi-denaturing agarose detergent gel. After 2 days of induction GFPS100A8 (Lane 1), GFPS100A9 (Lane 3) or cotransformants mCherry-
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S100A8/GFPS100A9 (Lanes 7, 8 and 10, 11) formed SDS-stable aggregates in yeast cells. Boiling (+) the samples led to full soloubilization of aggregates
to the monomeric form (Lanes 2 and 4). Total cell extracts (180 mg) were resolved using SDD-AGE. Blots were probed with anti-GFP or mCherry
antibodies. Total cell extract of Q103GFP cells (90 mg) was prepared after 24 h of induction (lane 5). (C) Filter retardation assay of cells grown for 3 and
5 days under inducing conditions. Loading control was visualized by CBB staining. Empty vector-transfected cells were used as control. (D)
Spheroplasts of control and induced cells stained with ThT after 3 days of incubation on galactose plates.
doi:10.1371/journal.pone.0058218.g005
were transformed with plasmids pYES2-S100A8, pYES2-S100A9
with or without pGALSc104(WT), and the viability of the
transformants was examined after galactose induction (Figures 8A
and 8B). Induction of non-tagged S100A8 and S100A9 either
individually or together led to a decrease of viability of hsp104D
mutants compared with that of wild type cells (Figure 8A).
Elevating the levels of Hsp104p in the hsp104D mutant, by
overexpressing Hsp104p from the GAL promoter, restored viability,
thereby supporting a role for Hsp104p in modulating S100A8 and
S100A9 toxicity (Figure 8B). Both wild type cells and hsp104D
mutants that produced tagged GFPS100A8 and GFPS100A9
proteins showed foci after two days of induction, indicating that
protein aggregation was not affected (Figure 8C). To further
examine how Hsp104p influences the aggregation status of
GFP
S100A8 and GFPS100A9 proteins, we monitored the accumulation of high MW species with SDD-AGE. After two days of
induction, there were no significant changes in GFPS100A8 and
GFP
S100A9 high MW species in hsp104D mutants compared with
wild type cells (Figures 8D and S4). Thus, whereas deletion of
HSP104 resulted in increased toxicity, it had little effect on
aggregation. This finding suggests that Hsp104p is involved in
modulating S100A8 and S100A9 toxicity and is required for cell
viability.
Hsp70p Chaperones are Required for Preservation of Cell
Viability in the Presence of S100A8, S100A9, or S100A8/9
Aggregates
In addition to Hsp104p, several molecular chaperones have
been reported to be involved in the disaggregation machinery in
yeast; these include Hsp70p and Hsp40p inter alia [42], [43], [44].
We therefore examined which other chaperones are required for
modulating S100A8 and S100A9 toxicity. Cells deleted for two of
the HSP70 chaperones (ssa1D and ssa2D) were transformed with
pYES2-S100A8, pYES2-S100A9 or both plasmids together, and cell
viability after production of S100A8 and S100A9 proteins was
examined. We found that deletion of either SSA1 or SSA2 led to a
mild decrease in viability, which was aggravated in the
cotransformants (Figure 9). This observation suggests that in
addition to Hsp104p, yeast Hsp70p is also involved in modulation
of S100A8 and S100A9 protein aggregation and toxicity. In
contrast, the viability of the mutant deleted for SSE1 or for SSE2,
that encode nucleotide exchange factors for Hsp70p proteins, was
not affected by production of S100A8 or S100A9. Similarly,
deletion of YDJ1 that encodes Hsp40p or of HSP26 did not affect
the viability of cells that produced S100A8 or S100A9 either
separately or together in cotransformants (Figure 9). These results
indicate that the Hsp104p/Hsp70p bi-chaperone system is
involved in modulating S100A8 and S100A9 toxicity.
Figure 6. Viability of yeast cells expressing S100A8 and S100A9 proteins. (A) Ten-fold dilutions of yeast cells transformed with pGFPS100A8, pGFP-S100A9 or both plasmids were plated on glucose (non-inducing) or galactose (inducing) plates and photographed after 72 h. (B) Nontagged proteins as in A.
doi:10.1371/journal.pone.0058218.g006
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Figure 7. Viability of yeast cells producing S100A8 and S100A9 on the background of a ts mutation in an essential gene. (A) wild type
and cdc53-1 ts mutant cells expressing pYES2-S100A8 or pYES2-S100A9 were spotted on glucose or galactose plates and photographed after 72 h. (B)
Semi-denaturing agarose gel. After 2 days of induction GFPS100A9 forms aggregates in wild type and ts strain cdc53-1 at 30uC and 32uC. Total cell
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extracts (180 mg) were resolved using SDD-AGE. (C) Ten-fold dilutions of cdc53-1, cdc34-2, srp1-31, and sec27-1 yeast cells transformed with pTETS100A8 or pTET-S100A9 were spotted on SD plates with (inducing) or without (non-inducing) 5 mg/ml doxycycline and photographed after 72 h.
doi:10.1371/journal.pone.0058218.g007
the general load of damaged proteins on the cell – rather than the
specific folding or clearance machinery involved – determines
cellular toxicity.
Molecular chaperones control almost all aspects of cellular
proteostasis [54], [55]. Most chaperones facilitate protein folding
and prevent protein aggregation. The influence of the chaperone
network on protein aggregation in general and of S100A8 and
S100A9 in particular are far from clear. Here, we have
demonstrated how the chaperone machinery can modulate
amyloid formation and toxicity of S100A8 and S100A9 proteins.
We examined several chaperones that play a role in other human
amyloidosis conditions and are involved in modulation of prion
propagation and toxicity [42], [56]. The effect of Hsp104p, one of
the main players in the disaggregation machinery, on protein
misfolding events has been studied in different yeast models of
amyloid diseases. Deletion of HSP104 was shown to eliminate
polyQ aggregation and toxicity [57], and inhibition or elevated
levels of Hsp104p cured several yeast prions [58], [59].
Furthermore, overexpression of HSP104 solubilized SDS-resistant
polyQ aggregates and greatly reduced toxicity. In contrast,
overexpression of HSP104 in an a-synuclein yeast model did not
show any significant effect on aggregation and toxicity [60]. Thus,
the ability of the Hsp104p chaperone to suppress toxicity of
aggregates seems to depend on the nature of the aggregated
protein. Our results indicate that in the S100A8 and S100A9
model, the Hsp104p protein modulates the toxicity of amyloid
aggregates and plays a protective role in the yeast cells. Deletion of
HSP104 may lead to formation of toxic oligomeric forms that
decrease cell viability. Expression of HSP104 in hsp104D mutants
in the presence of S100A8 and S100A9 proteins led to restoration
of viability. This finding suggests that in wild type cells the levels of
Hsp104p are sufficient to modulate S100A8 and S100A9 toxicity
and maintain cell viability.
Hsp104p usually requires other chaperones to function; in
particular, Hsp104p collaborates with Hsp70p and Hsp40p to
disaggregate protein amyloids [61]. Other small heat shock
proteins, such as Hsp26p, have been shown to contribute to the
clearance of aggregates by Hsp104p [60], [62]. Several cytosolic
forms of Hsp70p have been implicated in the disaggregation
process in yeast. Different Hsp70 proteins were shown to affect
aggregation of prion and other amyloidogenic proteins differently
in yeast models. For example, mutation in SSA2 destabilized the
[URE3] prion, whereas deletion of SSA1 did not affect prion
propagation [63], [64]. In Huntington’s disease, polyQ toxicity
was decreased in a double mutant of ssa1D, ssa2D [65]. In our
S100A8–S100A9 yeast model, members of the Hsp70p family,
Ssa1p and Ssa2p, were found to protect cells from toxicity caused
by S100A8 and S100A9 amyloids. Deletion of SSA1 and SSA2 in
the presence of S100A8 or S100A9 alone was mildly toxic to the
yeast cells. In contrast, a strong toxic effect was observed when
both S100A8/9 proteins were produced in the same mutant cell.
Our data indicate that in the absence of other single molecular
chaperones, such as Hsp26p, Ydj1p, Sse1p, and Sse2p, S100A8
and S100A9 aggregates had no effect on cell viability. However,
we cannot exclude the possibility that these chaperones may have
redundant roles in modulating S100A8 and S100A9 toxicity.
In summary, we have clearly demonstrated that S100A8 and
S100A9 proteins form non-toxic amyloid aggregates in yeast cells
when expressed either alone or together. However, cells carrying
the burden of amyloid aggregates become hypersensitive to the
Discussion
The budding yeast S. cerevisiae has been used as a model
organism for many different neurodegenerative diseases, including
Parkinson’s and Huntington’s diseases [21], [45], [27]. Here, S.
cerevisiae was used as a model system for studying human S100A8
and S100A9 aggregation. GFPS100A8 aggregates, both those
formed initially in the cytoplasm and those formed after prolonged
induction, showed pronounced vacuolar localization. In contrast,
GFP
S100A9 aggregates were found predominantly in the cytoplasm but could be localized to the vacuole when co-expressed
with S100A8, which suggests that similar proteins form distinct
aggregates that are handled differently by the cellular quality
control machinery. For example, a triple proline mutant of asynuclein displayed a vacuolar phenotype characterized by an
increased number of foci in the vacuole, whereas other a-synuclein
mutants showed a different cellular distribution [30]. We did not
detect differences in cell viability associated with localization of the
S100A8 and S100A9 aggregates, despite our hypothesis that
different folding and clearance mechanisms could be involved in
aggregate modulation in different compartments of the cell.
The relationship between amyloid formation and amyloid
toxicity in protein conformational diseases is not well understood,
and it is not clear which step of the amyloid formation cascade is
toxic. This step may vary for different amyloid diseases [46].
Sequestration of aggregates into inclusion bodies may be a cellular
defense mechanism [47], [48], [49]. For example, in amyotrophic
lateral sclerosis TDP-43 protein, a component of neuronal
aggregates, was not associated with toxicity when sequestered into
inclusion bodies, but was a strong and independent predictor of
neuron cell death when located in the cytoplasm [50]. Likewise,
the majority of neurons expressing mutant huntingtin died without
forming inclusion bodies, thus linking the formation of aggregates
with survival of neurons [51]. Here, prolonged induction and high
levels of the S100A8 and S100A9 proteins alone or together and
their subsequent aggregation did not affect the viability of wild
type yeast. Our study, therefore, supports the observation that
there is no direct correlation between protein aggregation and cell
viability.
The formation of inclusion bodies in Huntington’s disease was
shown to partially restore longevity by improving the ubiquitin
proteasome system throughput and consequently lowering the
overall cellular burden of misfolded proteins [52]. These results
suggest that the pathogenic mechanism of amyloidosis could, in
part, result from an imbalance in protein folding homeostasis [53].
Overwhelming proteostasis, for example, in the presence of an
unrelated ts metastable protein can enhance the misfolding of
polyQ-containing proteins [23] and probably also of other
amyloid-prone proteins. Indeed expression of S100A8 and
S100A9 in mutants with metastable essential proteins, cdc53-1,
cdc34-2, srp1-31 or sec27-1 ts mutants of yeast, led to a reduction in
cell viability. We suggest that high levels of these protein
aggregates perturb the cellular protein homeostasis mechanisms
and that the molecular chaperones required for maintaining these
essential cell cycle proteins in a functional conformation are
depleted due to their recruitment to the S100A8 or S100A9
aggregates. Thus, our finding that S100A8 and S100A9 aggregation can differentially affect the function of unrelated metastable
proteins in the cell suggests that the capacity of the protein folding
homeostasis machinery has been compromised. It is possible that
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S100A8/A9 Aggregation Perturbs Proteostasis
Figure 8. HSP104 modulates S100A8- and S100A9-associated toxicity. (A) Viability of wild type or hsp104D yeast in the presence of S100A8,
S100A9 and S100A8/9 proteins. pYES2-S100A8, pYES2-S100A9 or both plasmids were expressed in wild type or hsp104D mutant cells. Viability was
monitored using the spot test assay on inducing (galactose) or noninducing (glucose) plates. (B) Ten-fold dilutions of wild type cells or hsp104D
mutants transformed with pGALSc104(WT) and with pYES2-S100A8, pYES2-S100A9 or both S100 plasmids were plated on glucose (non-inducing) or
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S100A8/A9 Aggregation Perturbs Proteostasis
galactose (inducing) plates. (C) Confocal images of GFPS100A8 and GFPS100A9 after 2 days of induction in wild type or Dhsp104 mutant cells. (D) Cell
extracts were prepared from wild type or hsp104D mutant cells expressing pGFP-S100A8 or pGFP-S100A9 after 2 days of induction. Extracts were
incubated in 2% SDS sample buffer with (+) or without (2) boiling, loaded on agarose gels, and analyzed by Western blot using anti-GFP antibodies
to detect the S100A8 and S100A9 proteins.
doi:10.1371/journal.pone.0058218.g008
presence of metastable proteins, such as mutants of Cdc53, Srp1,
Cdc34, and Sec27, because the overall protein folding machinery
becomes exhausted and compromised. The presence of S100A8
and S100A9 aggregates unmasks the weaker parts in cellular
regulation and the interconnection between vital components of
the protein network that maintains cell viability. We found that
yeast cells that produce S100A8 and S100A9 aggregates require
the Hsp104/Hsp70 bi-chaperone machinery as essential factors
for preservation of cell viability. Thus, we have developed a very
powerful and sensitive model to study the effect of S100A8 and
S100A9 aggregation on the functioning of the cell homeostasis
machinery and to examine their effect on key components of the
protein folding and aggregate clearing systems. Considering that
human S100A8 and S100A9 amyloids are broadly associated with
inflammation and also with age-dependent deposits, our cellular
model can provide a platform for further investigations to identify
other members of the protein homeostasis machinery that
determine the toxicity of these amyloids.
Materials and Methods
Yeast Strains
Wild type W303 (MATa, ade2D1, ura3-52, trp1D2, leu2-3,112,
his3-11) [66]; BY4741 (MATa, his3D1, leu2D0, met15D0,
ura3D0), (Euroscarf). cdc34-2 (MATa, ura3-52, leu2-2, bas1-2,
bas2-2, GAL2+, gcn4-1, ade8::GCN4, trp1-1, cdc34-2) is a ts
strain with a G58R substitution mutation [35]. cdc53-1 (MATa,
ura3-1, can1-100, GAL+, leu2-3,112, trp1-1, his3-11,15, ade2-1,
cdc53-1) was obtained from M. Tyers and has a R488C
substitution mutation [34]. srp1-31 (MATa, srp1-31, ura3, leu2,
trp1, his3, ade2) was obtained from J. Hood and has a S116F
substitution mutation [67]. sec27-1 (MATa, leu2-3,112, trp1,
ura3-52, sec27-1) was obtained from J. Gerst and has a G688D
substitution mutation [68]. Strains with deletion of HSP104,
HSP40, HSP26, SSE1, SSE2, SSA1, SSA2, and SSA3 were
obtained from the Euroscarf deletion library (MATa, YYYD::Kanr (KO/DAmP), his3D1, leu2D0, met15D0, ura3D0, CAN1,
LYP1).
Yeast Plasmids
Plasmids for expression of S100A8 and S100A9 with fluorescent tags:
The S100A8 and S100A9 genes were amplified from Escherichia coli
PET1120 plasmids that encoded each of these human genes [69]
using primer pair A8SalIF/A8XhoIR for S100A8 and A9BamHIF/A9HinDIIIR for S100A9 (Table S1). Each PCR product was
cloned into pGEM T-easy (Promega). pmCherry-S100A8 was
constructed by amplifying mCherry from plasmid PCS2-mCherry
[70] with primer pair mCherryEcoRIF/mCherrySalIR and
cloning the PCR product into pGEM T-easy. The mCherry
fragment was extracted with EcoRI and SalI and ligated with the
above S100A8 fragment cleaved with XhoI and EcoRI. The
mCherry-S100A8 fragment was cloned as an EcoRI fragment into
pYES2 (Invitrogen). pGFP-S100A8 was constructed by amplifying
S100A8 from Pet1120 using primer pair A8BamHIF/A8HinDIIIR, followed by insertion into pGEM T-easy and subsequent
transfer as a BamHI-HinDIII fragment into pYCPGAL-GFP [71].
Similarly, the S100A9 PCR product was cloned into pGEM T-
Figure 9. Viability of single chaperone deletion strains
producing S100A8 and S100A9 proteins. Cells expressing empty
vector (pYES2), pYES2-S100A8, pYES2-S100A9, or cotransformants with
pYES2-S100A8/pYES2-S100A9 in sse1, sse2, ssa1, ssa2, ssa3, hsp26, and
ydj1 mutants and the isogenic wild type parent were spotted on
galactose and glucose plates and photographed after 72 h.
doi:10.1371/journal.pone.0058218.g009
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S100A8/A9 Aggregation Perturbs Proteostasis
analysis of tagged proteins. For detecting non-tagged proteins, we
used monoclonal and polyclonal anti-calgranulin A (S100A8) and
anti-calgranulin B (S100A9) (Santa Cruz Biotechnology) at 1:100
dilution. Goat anti-mouse (1:2500) and anti-rabbit (1:5000)
antibodies were purchased from Sigma. As a loading control we
used anti-actin antibody (Sigma) at 1:5000 or anti-GAPDH
antibody (Abcam) at 1:1000 dilutions.
easy and then transferred as a BamHI-HinDIII fragment into
pYCPGAL-GFP.
Plasmids for expression of non-tagged S100 proteins: S100A8 and
S100A9 cloned into pGEM T-easy as above were excised with
HinDIII and XhoI (S100A8) and with BamHI and EcoRI
(S100A9) for cloning into pYES2. The plasmid marker gene,
URA3, was converted to LEU2 in vivo by homologous recombination using pYIp1392 in the case of pYES2-S100A8 to enable
cotransformation of yeast cells with both plasmids. pCM190 was
used for expression of S100A8 and A9 under the TET on-off
promoter [72]. S100A8 and S100A9 were subcloned with a pair of
restriction enzymes as a BamHI and HinDIII fragment from
pGFP-S100A8 and pGFP-S100A9, respectively.
The plasmid used for overexpression of HSP104: pGALSc104(WT) was
purchased from Addgene (plasmid number 1146) [73], and
p103Q-GFP was as described [65]. Primer sequences appear in
Table S1. Plasmids are listed in Table S2.
Native Gel
Yeast cells were grown for two to four days on either SD or
SG selective plates. The cells were collected and washed with
phosphate-buffered saline (PBS) and adjusted to OD600 = 0.8 in
PBS. Then, 20 ml of cells were collected by centrifugation and
transferred into 200 ml PBS with proteinase inhibitor (1:1000).
Glass beads were added, and the cells were broken as above.
The cell debris and glass beads were removed by centrifugation
at 100 g for 0.5 min. The extract was clarified by centrifugation
at 650 g for 2 min and kept on ice before addition of sample
buffer 65 (50% glycerol, 0.5 Tris HCl pH 6.8, 0.02%
bromophenol blue). For Western blot analysis, 40 ml of extract
were dissolved in 10 ml of sample buffer, and 30 ml of each
fraction was separated on a 12% native-PAGE gel with a 3.5%
stacking gel, followed by Western blot analysis. The gels were
run at 60 V for approximately 4–5 h at 4uC. The gel was
transferred onto a polyvinylidene difluoride (PVDF) membrane
at 300 A for 1.2 h in transfer buffer (Tris-glycine buffer with
10% methanol and 0.1% SDS) and analyzed by Western
blotting with the antibodies described above.
Yeast Transformation and Growth Conditions
Yeast cultures were grown overnight at 30uC (28uC for ts
strains) in a rotary thermoshaker at 120 rpm in synthetic minimal
medium (SD) supplemented with the appropriate amino acids. For
constructs under the GAL promoter, SD medium and 2%
galactose (SG) medium were used for non-inducing and inducing
conditions, respectively. For aggregate detection, yeast strains were
grown at 30uC for two to six days on selective SG agar plates. For
constructs under the TET on-off promoter, SD medium with
5 mg/ml doxycycline and synthetic 2% glucose (SD) medium were
used for non-inducing and inducing conditions, respectively. For
aggregate detection, yeast strains were grown at 30uC for two to
six days on selective SD agar plates. Yeast transformation was as in
[74].
SDD-AGE
For semi-denaturing agarose gel electrophoresis, yeast cells
were broken by glass bead disruption under moderately
denaturing conditions (50 mM NaCl, 100 mM Tris pH 7.5,
10 mM b-mercaptoethanol, complete protease inhibitor cocktail
without EDTA (Roche) –1 tablet for 25 ml). Yeast extracts were
cleared by centrifugation (650 g, 2 min, 4uC). Protein concentrations of the samples were normalized at 2.0 mg/ml.
Normalized extracts (150–200 mg) were added to 4 6 SDD–
AGE buffer to a final concentration (50 mMTris, pH 6.8, 2%
(w/v) SDS, 0.025% bromophenol blue, 5% glycerol). Extracts
were incubated at room temperature for 10 min and loaded
onto a 1.5% agarose gel (prepared with 16 Tris-glycine buffer
(20 mM Tris, 200 mM glycine +0.1% SDS). Samples were run
in a regular horizontal gel chamber for DNA electrophoresis in
running buffer (20 mM Tris, 200 mM glycine +0.1% SDS) [75]
at 80 V until the bromophenol reached the bottom edge of the
gel. Gels were transferred onto a PVDF membrane for analysis
by Western blotting.
Spot Test Viability Assay
Yeast cells were grown overnight in SD medium or in synthetic
medium with 2% raffinose, diluted 1:3, and then regrown to midlog phase. Equal amounts of cells at OD600 = 0.5 were harvested
by centrifugation at room temperature for 2 min at 1500g, washed
three times in sterile water, and then resuspended in sterile water.
Ten-fold serial dilutions were made in sterile water, and 5-ml drops
were plated on SD or SG medium complemented with the
appropriate amino acids. The plates were incubated for two to
four days at 30uC. For ts strains, yeast cells were grown at 28uC
under non-inducing conditions, washed, and transferred to
appropriate medium for induction. Cells were then plated on
SD medium or SD with 5 mg/ml doxycycline medium and
incubated at 30–33uC, as indicated in the text.
TCA Precipitation
Yeast cells were resuspended in 20% TCA and incubated on
ice. Glass beads were added, and the cells were broken by vigorous
vortex for 365 min at 4uC. Precipitates were collected by
centrifugation at 14000 g for 10 min. Pellets were dissolved in
50 mL of dissociation buffer (4% sodium dodecyl sulfate, 0.1 M
Tris-HCl [pH 6.8], 20% glycerol, 2% 2-mercaptoethanol, 0.02%
bromophenol blue) and 25 ml of 1 M Tris-base. Yeast extracts
were incubated for 5 min at 100uC and separated on SDS-PAGE.
We used a nitrocellulose membrane (Biorad, 0.45 mm) for transfer
and detection.
Filter Trap Assay
Yeast cells were broken by glass bead disruption in extract buffer
(50 mM NaCl, 100 mM Tris pH 7.5, 10 mM b-mercaptoethanol,
complete protease inhibitor cocktail without EDTA (Roche) –1 tablet
for 25 ml). Yeast extracts were cleared by centrifugation (650 g,
2 min, 4uC). The extracts were diluted 1:3 in PBS buffer with 2% SDS
and incubated at room temperature for 10 min or boiled for 5 min
and then filtered through a 96-well dot blot apparatus (Bio-Rad
Laboratories, Hercules, CA) containing nitrocellulose (0,2 mm, BioRad) or cellulose acetate membranes (0,2 mm, Millipore) and
analyzed by Western blotting. Total protein load was assessed by
coomassie brilliant blue (CBB) staining of the nitrocellulose
membrane.
Antibodies
Mouse anti-GFP (Santa Cruz Biotechnology) and rabbit antimCherry (Clontech) at 1:1000 dilutions were used for Western blot
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S100A8/A9 Aggregation Perturbs Proteostasis
2% SDS sample buffer at room temperature with (+) or without
(2) boiling and then loaded on the SDD-AGE gel and analyzed by
Western blot using polyclonal anti-S100A8 antibodies.
(TIF)
ThT Binding Assay
Each culture was grown on selective plates for three to four daysand
then diluted into liquid medium and grown to OD600 = 0.5. The cells
were washed in 50 mM Phosphate buffer (pH 6.5) with 1 mM
MgCl2. A 5-ml sample of the culture was transferred into freshly
prepared 4% formaldehyde fixative in this buffer and incubated at
room temperature for 1.5–2 h with brief vortexing every 30 min. The
fixed cells were collected by centrifugation (2 min at 2000 g), and the
supernatant was carefully removed. The cells were washed in 5 ml
buffer, and the pellet was resuspended at OD600 = 10 in PMST buffer
(0.1 M H2KPO4, pH 7.5; 1 mM MgCl2; 1.2 M sorbitol; 0.1%
Tween 20; complete protease inhibitor cocktail without EDTA). For
spheroplasting, 100 ml of the cell suspension were transferred to a 0.5ml Eppendorf tube and incubated with 50–100 U Zymolyase (Zymo
Research) on a rotating wheel at room temperature for 15 min. The
spheroplasts were gently resuspended in 100 ml PMST, centrifuged
and resuspended. The cells were washed with PBS, pH 7.4, and
incubated in PBS with 0.001% ThT for 20 min, washed three times
with PBS, and then observed immediately by fluorescence microscopy.
Figure S2 Aggregation and toxicity of S100A proteins
induced by a TET on-off promoter system. (A) Ten-fold
dilutions of wild type yeast cells transformed with pCM190 (empty
vector), pTET-S100A8 or pTET-S100A9 were plated on glucose
(inducing) or glucose with 5 mg/ml doxycycline (non-inducing)
plates. (B) Spheroplasts of control and induced cells stained with
ThT after 4 days of incubation on glucose plates.
(TIF)
Figure S3 S100A8 and S100A9 protein levels in wild type
and cdc53-1 cells. Wild type or cdc53-1 mutant cells expressing
GFP
S100A8 or GFPS100A9 were grown for 2 days on glucose or
galactose plates at 30uC or 32uC. TCA precipitates of extracts
were separated by 10% SDS-PAGE and analyzed by Western
blot, using anti-actin or anti-GAPDH and anti-GFP antibodies.
(TIF)
Figure S4 S100A8 and S100A9 protein levels in wild type
and hsp104D cells. Wild type or hsp104D mutant cells
expressing GFP-S100A8 or GFP-S100A9 were grown for 2 days
on glucose or galactose plates. TCA precipitates of extracts were
separated by 10% SDS-PAGE and analyzed by Western blot,
using anti-actin and anti-GFP antibodies.
(TIF)
Microscopy
GFP-S100A8 and GFP-S100A9 and mCherry-S100A8/GFPS100A9 were induced with 2% galactose. Imaging was performed
with an Olympus FV1000 laser-scanning confocal microscope
with a x60 objective lens. Fluorescence was excited with 543 nm
for the red fluorescent markers and 488 nm for GFP.
Table S1 Primer sequences.
Staining with FM4-64
(DOC)
For staining with FM4-64 (Molecular Probes) after 2 and
4 days, cells expressing GFPS100A8, GFPS100A9 or mCherryS100A8/GFPS100A9 were collected from a SG-induced plate and
incubated with 15 mM dye (5 mM FM4-64 in dimethyl sulfoxide
was diluted with water to obtain a 150 mM stock solution) for
30 min in SG. Cells were washed three times with the same
medium and immediately observed under a microscope.
Table S2 Plasmids for expression in yeast.
(DOC)
Acknowledgments
We thank Dudy Bar-Zvi for advice and stimulating discussions and
Tommer Ravid and Maya Schuldiner for their generous help with strains.
Supporting Information
Author Contributions
Analysis of S100A8 aggregates on SDD-AGE
gels. Cell extracts were prepared from wild type cells that
produced S100A8 protein after 2, 4, and 6 days of incubation on
galactose plates. The yeast extracts were incubated for 10 min in
Figure S1
Conceived and designed the experiments: EE ABZ LMR DR. Performed
the experiments: EE. Analyzed the data: EE ABZ DR. Contributed
reagents/materials/analysis tools: LMR. Wrote the paper: EE ABZ LMR
DR.
References
1. Donato R (2001) S100: a multigenic family of calcium-modulated proteins of the
EF-hand type with intracellular and extracellular functional roles. Int J Biochem
Cell Biol 33: 637–668.
2. Gebhardt C, Nemeth J, Angel P, Hess J (2006) S100A8 and S100A9 in
inflammation and cancer. Biochem Pharmacol 72: 1622–1631.
3. Hermani A, De Servi B, Medunjanin S, Tessier PA, Mayer D (2006) S100A8
and S100A9 activate MAP kinase and NF-kappaB signaling pathways and
trigger translocation of RAGE in human prostate cancer cells. Exp Cell Res 312:
184–197.
4. Hiratsuka S, Watanabe A, Sakurai Y, Akashi-Takamura S, Ishibashi S, et al.
(2008) The S100A8-serum amyloid A3-TLR4 paracrine cascade establishes a
pre-metastatic phase. Nat Cell Biol 10: 1349–1355.
5. Loser K, Vogl T, Voskort M, Lueken A, Kupas V, et al. (2010) The Toll-like
receptor 4 ligands Mrp8 and Mrp14 are crucial in the development of
autoreactive CD8+ T cells. Nat Med 16: 713–717.
6. Ehrchen JM, Sunderkotter C, Foell D, Vogl T, Roth J (2009) The endogenous
Toll-like receptor 4 agonist S100A8/S100A9 (calprotectin) as innate amplifier of
infection, autoimmunity, and cancer. J Leukoc Biol 86: 557–566.
7. Foell D, Wittkowski H, Vogl T, Roth J (2007) S100 proteins expressed in
phagocytes: a novel group of damage-associated molecular pattern molecules.
J Leukoc Biol 81: 28–37.
8. Korndorfer IP, Brueckner F, Skerra A (2007) The crystal structure of the human
(S100A8/S100A9)2 heterotetramer, calprotectin, illustrates how conformational
PLOS ONE | www.plosone.org
9.
10.
11.
12.
13.
14.
15.
16.
17.
13
changes of interacting alpha-helices can determine specific association of two
EF-hand proteins. J Mol Biol 370: 887–898.
Hoyaux D, Decaestecker C, Heizmann CW, Vogl T, Schafer BW, et al. (2000)
S100 proteins in Corpora amylacea from normal human brain. Brain Res 867:
280–288.
Yanamandra K, Alexeyev O, Zamotin V, Srivastava V, Shchukarev A, et al.
(2009) Amyloid formation by the pro-inflammatory S100A8/A9 proteins in the
ageing prostate. PLoS One 4: e5562.
Vogl T, Gharibyan AL, Morozova-Roche LA (2012) Pro-Inflammatory S100A8
and S100A9 Proteins: Self-Assembly into Multifunctional Native and Amyloid
Complexes. Int J Mol Sci 13: 2893–2917.
Viemann D, Barczyk K, Vogl T, Fischer U, Sunderkotter C, et al. (2007)
MRP8/MRP14 impairs endothelial integrity and induces a caspase-dependent
and -independent cell death program. Blood 109: 2453–2460.
Chiti F, Dobson CM (2006) Protein misfolding, functional amyloid, and human
disease. Annu Rev Biochem 75: 333–366.
Ross CA, Poirier MA (2004) Protein aggregation and neurodegenerative disease.
Nat Med 10 Suppl: S10–17.
Chen B, Retzlaff M, Roos T, Frydman J (2011) Cellular strategies of protein
quality control. Cold Spring Harb Perspect Biol 3: a004374.
Buchberger A, Bukau B, Sommer T (2010) Protein quality control in the cytosol
and the endoplasmic reticulum: brothers in arms. Mol Cell 40: 238–252.
Su H, Wang X (2010) The ubiquitin-proteasome system in cardiac proteinopathy: a quality control perspective. Cardiovasc Res 85: 253–262.
March 2013 | Volume 8 | Issue 3 | e58218
S100A8/A9 Aggregation Perturbs Proteostasis
49. Hardy J, Selkoe DJ (2002) The amyloid hypothesis of Alzheimer’s disease:
progress and problems on the road to therapeutics. Science 297: 353–356.
50. Barmada SJ, Skibinski G, Korb E, Rao EJ, Wu JY, et al. (2010) Cytoplasmic
mislocalization of TDP-43 is toxic to neurons and enhanced by a mutation
associated with familial amyotrophic lateral sclerosis. J Neurosci 30: 639–649.
51. Arrasate M, Mitra S, Schweitzer ES, Segal MR, Finkbeiner S (2004) Inclusion
body formation reduces levels of mutant huntingtin and the risk of neuronal
death. Nature 431: 805–810.
52. Mitra S, Tsvetkov AS, Finkbeiner S (2009) Single neuron ubiquitin-proteasome
dynamics accompanying inclusion body formation in huntington disease. J Biol
Chem 284: 4398–4403.
53. Bates GP (2006) BIOMEDICINE: One Misfolded Protein Allows Others to
Sneak By. Science 311: 1385–1386.
54. Frydman J (2001) Folding of newly translated proteins in vivo: the role of
molecular chaperones. Annu Rev Biochem 70: 603–647.
55. Bukau B, Weissman J, Horwich A (2006) Molecular chaperones and protein
quality control. Cell 125: 443–451.
56. Vabulas RM, Raychaudhuri S, Hayer-Hartl M, Hartl FU (2010) Protein folding
in the cytoplasm and the heat shock response. Cold Spring Harb Perspect Biol 2:
a004390.
57. Krobitsch S, Lindquist S (2000) Aggregation of huntingtin in yeast varies with
the length of the polyglutamine expansion and the expression of chaperone
proteins. Proc Natl Acad Sci U S A 97: 1589–1594.
58. Derkatch IL, Bradley ME, Zhou P, Chernoff YO, Liebman SW (1997) Genetic
and environmental factors affecting the de novo appearance of the [PSI+] prion
in Saccharomyces cerevisiae. Genetics 147: 507–519.
59. Moriyama H, Edskes HK, Wickner RB (2000) [URE3] prion propagation in
Saccharomyces cerevisiae: requirement for chaperone Hsp104 and curing by
overexpressed chaperone Ydj1p. Mol Cell Biol 20: 8916–8922.
60. Cashikar AG, Duennwald M, Lindquist SL (2005) A chaperone pathway in
protein disaggregation. Hsp26 alters the nature of protein aggregates to facilitate
reactivation by Hsp104. J Biol Chem 280: 23869–23875.
61. Glover JR, Lindquist S (1998) Hsp104, Hsp70, and Hsp40: a novel chaperone
system that rescues previously aggregated proteins. Cell 94: 73–82.
62. Duennwald ML, Echeverria A, Shorter J (2012) Small heat shock proteins
potentiate amyloid dissolution by protein disaggregases from yeast and humans.
PLoS Biol 10: e1001346.
63. Schwimmer C, Masison DC (2002) Antagonistic interactions between yeast
[PSI(+)] and [URE3] prions and curing of [URE3] by Hsp70 protein chaperone
Ssa1p but not by Ssa2p. Mol Cell Biol 22: 3590–3598.
64. Roberts BT, Moriyama H, Wickner RB (2004) [URE3] prion propagation is
abolished by a mutation of the primary cytosolic Hsp70 of budding yeast. Yeast
21: 107–117.
65. Meriin AB, Zhang X, He X, Newnam GP, Chernoff YO, et al. (2002)
Huntington toxicity in yeast model depends on polyglutamine aggregation
mediated by a prion-like protein Rnq1. J Cell Biol 157: 997–1004.
66. Thomas BJ, Rothstein R (1989) Elevated recombination rates in transcriptionally active DNA. Cell 56: 619–630.
67. Hood JK, Casolari JM, Silver PA (2000) Nup2p is located on the nuclear side of
the nuclear pore complex and coordinates Srp1p/importin-alpha export. J Cell
Sci 113 (Pt 8): 1471–1480.
68. Gabriely G, Kama R, Gerst JE (2007) Involvement of specific COPI subunits in
protein sorting from the late endosome to the vacuole in yeast. Mol Cell Biol 27:
526–540.
69. Hunter MJ, Chazin WJ (1998) High level expression and dimer characterization
of the S100 EF-hand proteins, migration inhibitory factor-related proteins 8 and
14. J Biol Chem 273: 12427–12435.
70. Schuh M, Ellenberg J (2008) A new model for asymmetric spindle positioning in
mouse oocytes. Curr Biol 18: 1986–1992.
71. Ivantsiv Y, Kaplun L, Tzirkin-Goldin R, Shabek N, Raveh D (2006) Unique role
for the UbL-UbA protein Ddi1 in turnover of SCFUfo1 complexes. Mol Cell
Biol 26: 1579–1588.
72. Gari E, Piedrafita L, Aldea M, Herrero E (1997) A set of vectors with a
tetracycline-regulatable promoter system for modulated gene expression in
Saccharomyces cerevisiae. Yeast 13: 837–848.
73. Schirmer EC, Homann OR, Kowal AS, Lindquist S (2004) Dominant gain-offunction mutations in Hsp104p reveal crucial roles for the middle region. Mol
Biol Cell 15: 2061–2072.
74. Kaplun L, Ivantsiv Y, Bakhrat A, Raveh D (2003) DNA damage responsemediated degradation of Ho endonuclease via the ubiquitin system involves its
nuclear export. J Biol Chem 278: 48727–48734.
75. Bagriantsev S, Liebman SW (2004) Specificity of prion assembly in vivo. [PSI+]
and [PIN+] form separate structures in yeast. J Biol Chem 279: 51042–51048.
18. Bence NF, Sampat RM, Kopito RR (2001) Impairment of the ubiquitinproteasome system by protein aggregation. Science 292: 1552–1555.
19. Kim S, Nollen EA, Kitagawa K, Bindokas VP, Morimoto RI (2002)
Polyglutamine protein aggregates are dynamic. Nat Cell Biol 4: 826–831.
20. Bennett EJ, Shaler TA, Woodman B, Ryu KY, Zaitseva TS, et al. (2007) Global
changes to the ubiquitin system in Huntington’s disease. Nature 448: 704–708.
21. Outeiro TF, Lindquist S (2003) Yeast cells provide insight into alpha-synuclein
biology and pathobiology. Science 302: 1772–1775.
22. Morimoto RI (2008) Proteotoxic stress and inducible chaperone networks in
neurodegenerative disease and aging. Genes Dev 22: 1427–1438.
23. Gidalevitz T, Ben-Zvi A, Ho KH, Brignull HR, Morimoto RI (2006) Progressive
disruption of cellular protein folding in models of polyglutamine diseases.
Science 311: 1471–1474.
24. Gidalevitz T, Krupinski T, Garcia S, Morimoto RI (2009) Destabilizing protein
polymorphisms in the genetic background direct phenotypic expression of
mutant SOD1 toxicity. PLoS Genet 5: e1000399.
25. Alberti S, Halfmann R, Lindquist S (2010) Biochemical, cell biological, and
genetic assays to analyze amyloid and prion aggregation in yeast. Methods
Enzymol 470: 709–734.
26. Giorgini F, Muchowski PJ (2009) Exploiting yeast genetics to inform therapeutic
strategies for Huntington’s disease. Methods Mol Biol 548: 161–174.
27. Khurana V, Lindquist S (2010) Modelling neurodegeneration in Saccharomyces
cerevisiae: why cook with baker’s yeast? Nat Rev Neurosci 11: 436–449.
28. Tauber E, Miller-Fleming L, Mason RP, Kwan W, Clapp J, et al. (2011)
Functional gene expression profiling in yeast implicates translational dysfunction
in mutant huntingtin toxicity. J Biol Chem 286: 410–419.
29. Vida TA, Emr SD (1995) A new vital stain for visualizing vacuolar membrane
dynamics and endocytosis in yeast. J Cell Biol 128: 779–792.
30. Petroi D, Popova B, Taheri-Talesh N, Irniger S, Shahpasandzadeh H, et al.
(2012) Aggregate clearance of alpha-synuclein in Saccharomyces cerevisiae
depends more on autophagosome and vacuole function than on the proteasome.
J Biol Chem 287: 27567–27579.
31. Bagriantsev SN, Kushnirov VV, Liebman SW (2006) Analysis of amyloid
aggregates using agarose gel electrophoresis. Methods Enzymol 412: 33–48.
32. Halfmann R, Lindquist S (2008) Screening for amyloid aggregation by SemiDenaturing Detergent-Agarose Gel Electrophoresis. J Vis Exp 17: e838.
33. Khurana R, Coleman C, Ionescu-Zanetti C, Carter SA, Krishna V, et al. (2005)
Mechanism of thioflavin T binding to amyloid fibrils. J Struct Biol 151: 229–238.
34. Patton EE, Willems AR, Sa D, Kuras L, Thomas D, et al. (1998) Cdc53 is a
scaffold protein for multiple Cdc34/Skp1/F-box proteincomplexes that regulate
cell division and methionine biosynthesis in yeast. Genes Dev 12: 692–705.
35. Prendergast JA, Ptak C, Kornitzer D, Steussy CN, Hodgins R, et al. (1996)
Identification of a positive regulator of the cell cycle ubiquitin-conjugating
enzyme Cdc34 (Ubc3). Mol Cell Biol 16: 677–684.
36. Tabb MM, Tongaonkar P, Vu L, Nomura M (2000) Evidence for separable
functions of Srp1p, the yeast homolog of importin alpha (Karyopherin alpha):
role for Srp1p and Sts1p in protein degradation. Mol Cell Biol 20: 6062–6073.
37. Duden R, Hosobuchi M, Hamamoto S, Winey M, Byers B, et al. (1994) Yeast
beta- and beta’-coat proteins (COP). Two coatomer subunits essential for
endoplasmic reticulum-to-Golgi protein traffic. J Biol Chem 269: 24486–24495.
38. Kryndushkin DS, Engel A, Edskes H, Wickner RB (2011) Molecular chaperone
Hsp104 can promote yeast prion generation. Genetics 188: 339–348.
39. Arimon M, Grimminger V, Sanz F, Lashuel HA (2008) Hsp104 targets multiple
intermediates on the amyloid pathway and suppresses the seeding capacity of
Abeta fibrils and protofibrils. J Mol Biol 384: 1157–1173.
40. Shorter J, Lindquist S (2004) Hsp104 catalyzes formation and elimination of selfreplicating Sup35 prion conformers. Science 304: 1793–1797.
41. Parsell DA, Kowal AS, Singer MA, Lindquist S (1994) Protein disaggregation
mediated by heat-shock protein Hsp104. Nature 372: 475–478.
42. Shorter J, Lindquist S (2008) Hsp104, Hsp70 and Hsp40 interplay regulates
formation, growth and elimination of Sup35 prions. EMBO J 27: 2712–2724.
43. Rikhvanov EG, Romanova NV, Chernoff YO (2007) Chaperone effects on
prion and nonprion aggregates. Prion 1: 217–222.
44. Winkler J, Tyedmers J, Bukau B, Mogk A (2012) Chaperone networks in protein
disaggregation and prion propagation. J Struct Biol 179: 152–160.
45. Miller-Fleming L, Giorgini F, Outeiro TF (2008) Yeast as a model for studying
human neurodegenerative disorders. Biotechnol J 3: 325–338.
46. Eisenberg D, Jucker M (2012) The amyloid state of proteins in human diseases.
Cell 148: 1188–1203.
47. Shorter J, Lindquist S (2005) Prions as adaptive conduits of memory and
inheritance. Nat Rev Genet 6: 435–450.
48. Wolfe KJ, Cyr DM (2011) Amyloid in neurodegenerative diseases: friend or foe?
Semin Cell Dev Biol 22: 476–481.
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March 2013 | Volume 8 | Issue 3 | e58218
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