© 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016) 129, 1892-1901 doi:10.1242/jcs.176479
RESEARCH ARTICLE
Vulnerability of newly synthesized proteins to proteostasis stress
ABSTRACT
The capacity of the cell to produce, fold and degrade proteins relies on
components of the proteostasis network. Multiple types of insults can
impose a burden on this network, causing protein misfolding. Using
thermal stress, a classic example of acute proteostatic stress, we
demonstrate that ∼5–10% of the soluble cytosolic and nuclear
proteome in human HEK293 cells is vulnerable to misfolding when
proteostatic function is overwhelmed. Inhibiting new protein synthesis
for 30 min prior to heat-shock dramatically reduced the amount of
heat-stress induced polyubiquitylation, and reduced the misfolding of
proteins identified as vulnerable to thermal stress. Following prior
studies in C. elegans in which mutant huntingtin (Q103) expression
was shown to cause the secondary misfolding of cytosolic proteins,
we also demonstrate that mutant huntingtin causes similar
‘secondary’ misfolding in human cells. Similar to thermal stress,
inhibiting new protein synthesis reduced the impact of mutant
huntingtin on proteostatic function. These findings suggest that
newly made proteins are vulnerable to misfolding when proteostasis
is disrupted by insults such as thermal stress and mutant protein
aggregation.
KEY WORDS: Neurodegenerative disease, Heat-shock,
Proteostasis, Proteomics, Protein aggregation, Ubiquitin
INTRODUCTION
A key pathologic feature of neurodegenerative diseases including
Alzheimer’s disease, fronto-temporal dementia (FTD), Parkinson’s
disease, amyotrophic lateral sclerosis and Huntington’s disease is
the presence of intracellular and/or extracellular accumulations of
insoluble protein aggregates (for a review, see Muchowski and
Wacker, 2005). Because of the similarities in the pathologies of
these diseases, it has been proposed that they share common
pathogenic mechanisms. However, no single common mechanism
has yet emerged; the literature contains reports that describe
inhibition of proteasome function (Bence et al., 2001), damage to
mitochondria (reviewed by Cho et al., 2010), damage to membrane
organelles (reviewed by Butterfield and Lashuel, 2010),
compromised chaperone function (reviewed by Kikis et al., 2010;
Voisine et al., 2010) and autophagic failure (reviewed by Wong and
Cuervo, 2010). Thus, an emerging concept is one of multisystem
failure as a component of neurodegeneration.
One hypothesis that could underpin the multi-system failure
aspects of neurodegenerative disease is that the production and/or
accumulation of these misfolded proteins might lead to an
imbalance in the cellular protein quality control network or
1
Department of Neuroscience, SantaFe HealthCare Alzheimer’s Disease Research
Center, Center for Translational Research in Neurodegenerative Disease, McKnight
2
Brain Institute, University of Florida, Gainesville, FL 32610, USA. Interdisciplinary
3
Graduate Program, University of Florida, Gainesville, FL 32610, USA. College of
Arts and Sciences, University of Florida, Gainesville, FL 32610, USA.
*Author for correspondence ([email protected])
Received 25 June 2015; Accepted 4 March 2016
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proteostasis network. This network functions in the folding, posttranslational modification and degradation of proteins involved in
diverse functional pathways (reviewed by Balch et al., 2008; Kikis
et al., 2010). A common pathologic feature of neurodegenerative
disease is the accumulation of misfolded proteins within vulnerable
neuronal or glial cell populations. In each disease, the pathologic
protein aggregates are composed of a specific protein, or in some
cases multiple proteins, that pathologically define the disease. The
accumulation of such proteins is in-and-of-itself an indication that
protein homeostasis is compromised, but recent studies have
introduced the concept of collateral misfolding in which the
accumulation of a specific misfolded mutant protein disrupts protein
homeostasis in a manner that compromises the ability of other
bystander proteins to correctly fold (Gidalevitz et al., 2006, 2009).
The concept of bystander misfolding was first uncovered in
studies involving C. elegans that express the first exon of the human
huntingtin (HTT) gene, which encodes a CAG repeat sequence that
codes for glutamine (Gidalevitz et al., 2006). Expansion of the CAG
repeat is causative for Huntington’s disease (The Huntington’s
Disease Collaborative Research Group, 1993) and produces a
protein that is prone to aggregate (Scherzinger et al., 1999). The
expression of mutant Htt in the muscle wall of C. elegans produces
inclusion pathology with muscle dysfunction (Satyal et al., 2000).
Gidalevitz and colleagues have demonstrated that expression of
mutant Htt exon-1 in C. elegans produces a burden on the
proteostatic network such that co-expressed proteins harboring
temperature-sensitive folding mutations are unable to achieve
functional structure at temperatures that such mutants are normally
functional (Gidalevitz et al., 2006). The authors hypothesized that,
in settings of proteostatic stress, proteins harboring rare sequence
variations might be vulnerable to misfolding due to an inherently
greater dependence on proteostatic machinery to achieve functional
conformations (Gidalevitz et al., 2006).
In a previously published proteomic analysis of two human
neural cell lines, we embarked on an effort to identify human
proteins that might be vulnerable to proteostatic stress (Xu et al.,
2012). Using a thermal stress paradigm in two independently
derived human cell lines, we identified ∼30 cytosolic and nuclear
proteins that lose solubility upon heat shock (Xu et al., 2012),
indicating that vulnerability to proteostatic stress is not restricted to
rare-variant proteins but applicable to a larger population of normal
proteins. Among the proteins identified as sensitive to thermal
proteostatic stress were several that emerged as possible biomarkers
that consistently lose solubility, including ubiquitin, CDK1, FEN1
and TDP-43 (also known as TARDBP). In cells held at 37°C, these
proteins are predominantly, sometimes exclusively, found in PBS
soluble fractions (Xu et al., 2012), whereas in the heat-stressed cells
these proteins are detected in detergent-insoluble fractions. In the
present study, we used human HEK293 cell models to compare the
expression of mutant huntingtin (Q103) as a proteostasis stressor to
thermal stress. The HEK293 cell model has been routinely used as
an expression system to study protein aggregation related to
neurodegenerative disease, including mutant Htt (Bence et al.,
Journal of Cell Science
Guilian Xu1, Amrutha Pattamatta2, Ryan Hildago3, Michael C. Pace2, Hilda Brown1 and David R. Borchelt1, *
2001; Schilling et al., 2007; Waelter et al., 2001). We demonstrate
that ∼5–10% of the soluble cytosolic and nuclear proteome in
HEK293 cells is vulnerable to misfolding when proteostatic
function is overwhelmed by thermal stress. Using this paradigm
further in HEK293 cells, we demonstrate that inhibiting new protein
synthesis 30 min prior to heat-shock prevented the appearance of
insoluble cytosolic proteins. Focusing on a subset of thermalsensitive biomarker proteins, we show that the overexpression of
mutant Htt (Q103) induces the misfolding of the same proteins that
are sensitive to thermal stress. As was the case in the thermal stress
paradigm, inhibiting new protein synthesis reduced the impact of
mutant Htt on proteostatic function. These findings suggest that
stressors that burden the proteostatic network produce the greatest
impact on newly made proteins, with a substantial portion of the
proteome being vulnerable to such stress.
RESULTS
Identification of proteostasis stress biomarkers in HEK293
cells
HEK293 cells and their derivatives, such as 293T and 293FT cells,
have been extensively used as model cells for the expression of
mutant proteins implicated in neurodegenerative disease, including
expression of mutant Htt (Bence et al., 2001; Schilling et al., 2007;
Waelter et al., 2001), mutant superoxide dismutase 1, for modeling
familial amyotrophic lateral sclerosis (Karch and Borchelt, 2008;
Prudencio et al., 2009; Wang et al., 2003), α-synuclein, for
modeling Parkinson’s disease (Paxinou et al., 2001), and tau to
model various tauopathies (Mirbaha et al., 2015). In each of these
cases, expression of a mutant protein, or in some cases a wild-type
(WT) protein, produces aggregate inclusions that resemble
structures seen in tissues of humans with the disease and relevant
animal models. Given that the HEK293 cell is a workhorse cell line
in these types of studies, we sought to identify proteins in HEK293
cells that might be vulnerable to proteostatic stress. Following
protocols established in our previous study of human SH-SY5Y and
CCF-STTG1 cells (Xu et al., 2012), we conducted a liquid
chromatography tandem mass spectrometry (LC-MS/MS) analysis
of proteins in HEK293 cells that lose solubility after thermal stress.
For comparison, as an alternative means to produce proteostatic
stress, cells were also treated with MG132 to inhibit the proteasome,
causing an accumulation of misfolded proteins that would normally
have been degraded (for a review, see Balch et al., 2008). Sequential
detergent extraction was used to separate proteins by solubility in
phosphate-buffered saline (PBS), then Nonidet P-40 (NP40), then
sodium deoxycholate (DOC), with a final extraction of the DOCinsoluble fraction in SDS (Xu et al., 2012). This sequential approach
of fractionation was used to identify the detergent that provided the
best separation of soluble and insoluble proteins. The goal was to
identify an insoluble fraction that had relatively low complexity,
enabling greater efficacy in protein identification (Xu et al., 2012).
The SDS-insoluble fractions (SDS-P) contained very little protein
and were not analyzed further. Portions of each fraction were
separated by SDS-PAGE and immunoblotted for ubiquitin to
identify which fractions contained insoluble polyubiquitin
(Fig. 1A). Interestingly, most of the ubiquitylated proteins in cells
treated with MG132 were soluble in PBS (Fig. 1A, PBS-S,
condition 2). Notably, although ubiquitin immunoblotting revealed
a smear of immunoreactivity in the insoluble fractions, CoomassieBlue-stained gels demonstrated well-defined protein bands present
in all fractions (Fig. 1B). In comparing the effect of MG132 to
thermal stress, in Coomassie-Blue-stained gels, we clearly observed
a larger number of proteins in each of the insoluble fractions from
Journal of Cell Science (2016) 129, 1892-1901 doi:10.1242/jcs.176479
heat-shocked cells (Fig. 1B; compare condition 2 to 3). Thus, by
comparison, thermal stress seemed to be a more severe insult than
proteasome inhibition.
To identify the normally soluble proteins that lose solubility upon
heat-shock, we separated PBS-soluble (PBS-S) fractions from cells
held at 37°C and DOC pellet (DOC-P) fractions of heat-shocked
and control cells by SDS-PAGE before cutting each lane into six to
eight pieces for trypsin digestion and LC-MS/MS. Compilation of
the proteins identified from these fractions resulted in the
identification of 970 proteins from the HEK293 cells with a
relatively low false-positive rate (FPR) of less than 0.1% for proteins
and less than 0.4% for peptides from 18,559 spectra. Among these
970 proteins (Table S1; a Scaffold file of the complete list of 970
proteins can be provided upon request), 315 proteins were found in
both the PBS-S and DOC-P fractions, and 321 proteins were found
only in insoluble DOC-P fractions. Regardless of cell treatment,
there were 334 proteins found only in the soluble PBS-S fractions.
The most abundant peptides for this latter class of proteins were
Fig. 1. Heat-shock treatment causes protein ubiquitylation and
aggregation. (A) Equivalent volumes of soluble and insoluble fractions from
HEK293 cells treated as noted in the figure were analyzed by immunoblotting
with anti-ubiquitin antibody (1:5000). Conditions (shown above blot): 1, 37°C
control; 2, cells treated with 10 nM MG-132 for 12 h; 3, cells placed in a 42°C
incubator for 1 h before harvesting. The image shown is representative of three
independent repetitions of the experiment. (B) Coomassie Blue staining of a
10–20% Tris-Glycine gel from experiments performed in parallel with those
shown in A in which 10 µl of each fraction generated by our protocol was loaded
in each lane. The origin of the various fractions is identified at the bottom of the
figure. BenchMark™ molecular mass markers were used for molecular mass
estimations. The image shown is the representative of three distinct
experiments.
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RESEARCH ARTICLE
Journal of Cell Science (2016) 129, 1892-1901 doi:10.1242/jcs.176479
from DYNC1H1 (dynein), TUBB3B (tubulin), HUWE1 (E3
ubiquitin ligase), PKM2 ( pyruvate kinase), FLNA (filamin),
PLS3 ( plastin) and TPR (nucleoprotein). Ultimately, there were
44 proteins identified in HEK293 cells as proteins that seemed to
specifically lose solubility in thermal stress (Table S2); the most
abundant of these was ubiquitin. Given the much larger number of
proteins for which the only peptides identified were in soluble
fractions, we conclude that ∼10–20% of the soluble proteome might
be vulnerable to thermal stress.
To determine which types of proteins lose solubility in these
HEK293 cells, we analyzed the LC-MS/MS data using a statistical
method as previously described (Xu et al., 2012, 2013). Apart from
ubiquitin, we identified 13 additional proteins in which the
frequency of peptide identification in the DOC-insoluble fraction
of heat-shocked cells was significantly (P<0.05) more abundant
than predicted in two experiments (Table 1). There were 33
additional proteins in which we identified no peptides in the DOCinsoluble fractions from 37°C cells in both LC-MS/MS
experiments, but the frequency of peptide identification in the
DOC-insoluble fraction of one experiment was less than five and
thus did not achieve statistical significance in both experiments
(Table S2). If we include the larger dataset in analyses of protein
classification, most of the proteins identified as showing shifts in
solubility were nuclear proteins (Fig. S1), which is consistent with
our previous findings in thermal stressed SH-SY5Y and CCFSTTG1 cells (Xu et al., 2012). In comparing the list of proteins
identified by LC-MS/MS as sensitive to thermal stress in HEK293
cells to those identified in the two neural cell lines, we note that six
proteins that lost solubility were common to all three cell lines
(Fig. 2). An additional seven proteins were identified by LC-MS/
MS as losing solubility in both the HEK293 and SH-SY5Y cells.
Based, in part, on the commercial availability of specific antibodies,
we identified five proteins whose losses of solubility could be used
as biomarkers of proteostatic stress, these being ubiquitin, MATR3,
FEN1, TDP-43 and CDK1.
Mutant Htt overexpression causes bystander misfolding
To determine whether proteostatic stress caused by misfolded
protein pathology produces bystander misfolding in mammalian
cells, we examined the effect of expressing fragments of mutant Htt
(Htt-103Q) on the solubility of the biomarkers proteins identified
above (ubiquitin, MATR3, FEN1, TDP-43 and CDK1). HEK293FT
cells were transfected with N-terminal fragments of Htt fused to
cyan fluorescent protein (CFP) to allow us to flow sort the
transfected cells and isolate them from untransfected cells (Fig. 3A).
We compared the effects of expressing Htt–CFP encoding a 25Q
repeat, to the effects of expressing Htt–CFP encoding a 103Q repeat.
Immunoblots of sorted cells demonstrated the expression of both the
Htt25Q–CFP and Htt103Q–CFP protein fragments (Fig. 3B). In
cells expressing the Htt103Q–CFP fragment, the expressed protein
formed inclusions, whereas the Htt25Q–CFP fragment showed a
diffuse distribution in the cytoplasm (Fig. 3C). Cells transfected
with either Htt25Q–CFP or Htt103Q–CFP showed increased levels
of polyubiquitin, but the intensity of immunoreactivity was
consistently higher in the cells expressing the Htt103Q–CFP
protein (Fig. 4A). These cell lysates were then subjected to
detergent extraction and centrifugation to separate soluble and
detergent-insoluble proteins, and then these fractions were
examined by SDS-PAGE and immunoblotting. In cells expressing
the Htt103Q–CFP protein, we observed increased levels of
Table 1. Detergent-insoluble proteins in lysates of heat-shocked HEK293 cells
Gene Symbol
Protein name
UBC*
Ubiquitin
GTF2I
Isoform 1 of general
transcription factor II-I
Matrin-3
MATR3*
RRP12
KPNA2
TTLL12
MYBBP1A
PRPF8
NSUN2
MCM7
PMVK
CDK1*
FEN1*
TARDBP*
Isoform 1 of RRP12-like
protein
Importin subunit alpha-2
Tubulin-tyrosine ligase-like
protein 12
Isoform 1 of Myb-binding
protein 1A
Pre-mRNA-processingsplicing factor 8
tRNA (cytosine-5-)methyltransferase
NSUN2
Isoform 1 of DNA
replication licensing
factor MCM7
Phosphomevalonate
kinase
Cyclin-dependent kinase 1
Flap endonuclease 1
Isoform 2 of TAR DNAbinding protein 43
Accession
number
Experiment 1
Experiment 2
37°C
42°C
37°C
42°C
Detected as
soluble?
Range of P-value
by G-test
8
38
1
15
Y
6.9×10−6–2.3×10−4
0
29
0
8
N
5.1×10−9–0.007
IPI00179330
(+16)
IPI00054042
(+3)
IPI00017297
(+1)
IPI00101186
6
52
6
20
N
2.6×10−10–0.007
3
23
1
10
N
6.9×10−5–0.009
IPI00002214
IPI00029048
0
0
26
10
0
0
6
6
Y
Y
3.9×10−8–0.024
0.002–0.024
IPI00005024
(+1)
IPI00007928
29
48
12
28
N
0.012–0.032
6
16
4
13
Y
0.035–0.038
IPI00306369
0
20
0
5
Y
0.023–0.047
IPI00299904
0
12
0
5
Y
4.8×10−4–0.047
IPI00220648
0
5
0
5
Y
0.047
IPI00026689
IPI00026215
IPI00025815
0
0
0
4
3
2
0
0
0
14
14
10
Y
Y
Y
1.3×10−4–0.088
1.3×10−4–0.165
0.002–0.306
*Proteins validated by western blot assay. The number in parentheses under the accession number indicates that some of the peptides attributed to this specific
gene can also be attributed to other genes; the number of other possibilities is given.
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Total spectra
RESEARCH ARTICLE
Journal of Cell Science (2016) 129, 1892-1901 doi:10.1242/jcs.176479
Fig. 2. Venn diagram illustrating the overlap in protein identification by
LC-MS/MS in detergent-insoluble fractions from heat-shocked cells.
The LC-MS/MS data from the present study of HEK293 cells (see Table S2;
proteins meeting statistical significance in one or both LC-MS/MS
experiments) was compared to previously published LC-MS/MS data from
SH-SY5Y and CCF-STTG1 cells (see Tables 1 and 2 in Xu et al., 2012). Six
proteins were found to lose solubility in all three cell lines: ubiquitin (UBQ),
CDK1, FEN1, MATR3, GTF2I and SND1. Seven additional proteins were
found in both SH-SY5Y cells and HEK293 cells: KPNA2, ANP32E, PDCD11,
MYBBP1A, NSUN2, TYMS and RRP12. Five proteins were found in both CCFSTTG1 and HEK293 cells: BYSL, TARDBP, STAT1, TTLL12 and RBM12B.
insoluble MATR3, FEN1, TDP-43 and CDK1 (Fig. 4B–D). In
unstressed cells, these proteins were either exclusively or largely,
found in PBS-soluble fractions. These findings indicate that the
aggregation of mutant Htt fragments produces a sufficient level of
proteostasis stress to cause other cellular proteins to lose solubility.
Newly made proteins are vulnerable to proteostatic stress
Fig. 3. Isolation of HEK293FT cells expressing Htt fragments fused to
CFP. (A) Workflow diagram of fluorescence activated cell sorting (FACS).
(B) Immunoblot using an antibody against Htt protein (1:1000) confirmed the
expression of Htt fragments in transfected cells. (C) Representative images of
the transfected cells in fluorescent and bright field, demonstrating the high
frequency of inclusions in the cells transfected with Htt103Q–CFP. Scale bars:
200 µm.
waited 24 h, and then treated some of the cells with cycloheximide
for 6 h before detergent extraction and immunoblotting for ubiquitin
(Fig. 6). Similar to the heat-shock paradigm, cells expressing mutant
huntingtin that were treated with cycloheximide showed lower
levels of insoluble polyubiquitin immunoreactivity (Fig. 6A,
compare lanes 7 and 8). Quantification and statistical analysis of
replicate experiments demonstrated that cycloheximide treatment
significantly reduced the levels of insoluble ubiquitin (Fig. 6A,
graph). Immunoblots of the insoluble fractions from these cells
demonstrated that these fractions contained mutant Htt fragments as
expected for this aggregation prone protein (Fig. 6B). Immunoblots
of the PBS soluble fractions demonstrated high levels of Htt-N17118Q fragments along with soluble forms of Htt-N171-82Q
fragments (Fig. 6B). Additionally, these blots demonstrated that
the cycloheximide treatment did not dramatically reduce the overall
accumulation of Htt in these transfected cells. Immunofluorescence
stains of transfected cells demonstrated that in both cultures most of
the expressed Htt was diffusely distributed in the cells (Fig. 6C), a
pattern consistent with soluble protein as indicated by the
immunoblots. However, in the cells expressing Htt-N171-82Q we
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Previous studies in yeast have demonstrated that inhibiting protein
synthesis for 30 min prior to thermal stress prevents the
accumulation of polyubiquitin after heat-shock (Medicherla and
Goldberg, 2008), and thus we turned back to the thermal stress
paradigm to determine whether the proteins identified in HEK293
cells as vulnerable to thermal stress were newly-made versions of
these proteins. To accomplish this goal, we treated cells with
cycloheximide (CHX), a protein synthesis inhibitor, 30 min prior to
heat-shock. As predicted from the yeast work (Medicherla and
Goldberg, 2008), treatment of cells with cycloheximide before heatshock prevented the accumulation of insoluble polyubiquitylated
proteins (Fig. 5A, last 2 lanes; Fig. 5B,C). Similarly, treatment of
cells with CHX prevented the appearance of CDK1, FEN1, TDP-43
and MATR3 in insoluble fractions (Fig. 5B, lanes 4–6; Fig. 5C).
Adding CHX to the cells just prior to harvest did not prevent the loss
of solubility (Fig. S2, lanes 7–9), indicating the effects of CHX on
protein solubility required at least 30 min of prior incubation.
Immunoblots of the soluble fractions from these cells demonstrated
that levels of CDK1, FEN1, TDP-43 and MATR3 in the soluble
fractions of cells treated with CHX were similar to that of the
untreated cells (Fig. S3). Thus, the absence of these proteins in
insoluble fractions of cells treated with CHX was not due to the
absence of the protein. Rather, the data show that pre-existing forms
of these proteins (older than 30 min) retain solubility after heatshock.
To determine whether proteostatic stress caused by mutant
huntingtin expression shows a similar pattern of vulnerability, we
transfected HEK293FT cells with the huntingtin vectors encoding
the N171 fragment of Htt (with 18 and 82Q) (Schilling et al., 2007),
RESEARCH ARTICLE
Journal of Cell Science (2016) 129, 1892-1901 doi:10.1242/jcs.176479
Fig. 4. Expression of mutant Htt fragments in HEK293FT cells causes
collateral misfolding of cytosolic and nuclear proteins. At 48 h after
transfection with expression vectors for Htt25Q–CFP or Htt103Q–CFP, cells
were separated by FACS and then subjected to sequential detergent extraction
and sedimentation (see Materials and Methods). Immunoblots of insoluble
fractions showing that cells expressing Htt103Q–CFP specifically accumulated
insoluble ubiquitin (A), MATR3 (B), FEN1 (C), TDP-43 and CDK1 (D). The
images shown are representative of three independent transfection
experiments.
DISCUSSION
In the present study, using a heat-shock paradigm in HEK293 cells,
we identified multiple proteins that rapidly lose solubility upon
thermal stress. Many of the same proteins were previously identified
as losing solubility upon thermal stress in human SH-SY5Y and
CCF-STTG1 cells (Xu et al., 2012). On the basis of consistency
among the cell lines examined and on the availability of commercial
antibodies, we identified a set of 5 proteins whose losses of
solubility could be used as biomarkers of proteostatic stress, these
being ubiquitin, MATR3, FEN1, TDP-43 and CDK1. Here we
demonstrate that these proteins lose solubility when N-terminal
fragments of mutant huntingtin are expressed at a level that induces
intracellular inclusion formation. Thus, these biomarker proteins for
thermal stress are also vulnerable to bystander misfolding caused by
pathologic protein aggregation. Using protein synthesis inhibitors,
we found that it is the newly synthesized versions of these biomarker
proteins that are selectively sensitive to proteostatic stress. Apart
from CDK1, FEN1, TDP-43 and MATR3, our LC-MS/MS data
from thermal stress experiments suggests that 10–20% of the soluble
proteome (defined as proteins that are normally soluble in PBS)
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Fig. 5. Inhibiting protein synthesis for 30 min before heat-shock prevents the
accumulation of insoluble polyubiquitin, CDK1, FEN1, TDP43 and MATR3.
HEK293 cells were heat-shocked with and without CHX (treated cells were
exposed to CHX for 30 min before heat-shock), and then proteins insoluble in
DOC were separated by the small-scale protocol for detergent extraction and
sedimentation (see Materials and Methods). (A) Immunoblot analysis with
antibodies to ubiquitin demonstrated that CHX treatment nearly completely
blocked the accumulation of detergent-insoluble polyubiquitin. SOD1 was
detected by immunoblotting of the PBS soluble fractions to demonstrate that
volumetric equalization of the samples provided a reliable means of maintaining
similar protein content in the various soluble fractions. The images shown are
representative of more than three independent experiments. (B) Using the smallscale method, DOC insoluble fractions were separated for immunoblot analysis
with antibodies to ubiquitin (1:1000), CDK1 (1:500), FEN1 (1:500), TDP-43
(1:5000) and MATR3 (1:10,000). The treatment the cells received (37°C, 42°C
heat shock, or CHX treatment), and the antibody used for immunoblotting are
shown on the figure. (C) Immunoblots of DOC-insoluble fractions from three
separate experiments were quantified. The data for each immunoblot were
normalized to the value for the protein band visible in the lysates of heat-shocked
cells, which was uniformly the most intense signal. The relative intensity of the
protein band from cells at 37°C and cells treated with CHX is graphed as the mean
relative percentage intensity (±s.d.). Statistical analyses of the data demonstrated
that pre-treatment with CHX significantly reduced the level of insoluble CDK1,
FEN1, TDP43 and MATR3 (P<0.05, Student’s t-test, two-tailed, unequal variance).
Journal of Cell Science
observed that a subset of cells harbored inclusions (Fig. 6C, arrows).
Overall, these findings are consistent with our data from the heatstress paradigms and suggest that newly synthesized proteins
represent the major component of the proteome that is vulnerable to
misfolding during proteostatic stress caused by mutant huntingtin
expression.
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Journal of Cell Science (2016) 129, 1892-1901 doi:10.1242/jcs.176479
might be transiently vulnerable to proteostatic stress. Although it
comes as no surprise that newly made proteins are heavily
dependent upon the proteostatic network, our data indicate that it
is these proteins that represent the population that is most vulnerable
to collateral misfolding when an aggregating proteotoxin such as
mutant Htt accumulates.
The proteins we identify as being sensitive to thermal stress are
involved in multiple cellular functions and although the modest loss
of function of any one of these proteins might be inconsequential, it
is difficult to know what the consequences would be of a concerted
partial loss of function of multiple proteins in a particular functional
pathway. For example, CDK1 is a master regulator of mitosis and is
responsible for the phosphorylation of a large number of substrates
(for a review, see Domingo-Sananes et al., 2011), FEN1 is essential
for excision DNA repair (for a review, see Tsutakawa et al., 2011),
and MATR3 and TDP-43 are RNA-binding proteins that regulate
mRNA splicing and possibly other RNA metabolic processes
(Coelho et al., 2015; Polymenidou et al., 2011). Partial loss of
function of these activities and potentially many others could
compromise cellular function or viability. In acute proteostatic stress
settings, if only newly made proteins are vulnerable to the stressor,
then one would not expect sustained loss of function to occur as the
system would reset once the source of stress was removed. However,
in settings of chronic proteostasis stress, one could envision that the
proteins we identified as vulnerable could have a substantial loss of
functional molecules that could produce biological effects. Cultured
cells are not well suited for modeling chronic insults and future
effort to understand the impact of chronic insults, such as Htt
aggregation, on proteostatic function and bystander misfolding will
require applying these techniques to in vivo models.
We recognize that our LC-MS/MS sampling of the proteome is
not all inclusive. There is sampling error (Xu et al., 2012), and
proteins of low abundance are inherently difficult to quantify by the
spectral counting approach we have used here because spectral
numbers below five generally do not achieve statistical significance.
Additionally, our method focuses on identifying cytosolic and
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Fig. 6. CHX treatment of cells transfected
with mutant Htt prevents the accumulation
of insoluble polyubiquitin. HEK293FT cells
were transiently transfected with expression
vectors for HttN171-18Q and HttN171-82Q, or
left untransfected, and then incubated for 24 h
at 37°C. A set of the cells transfected with
HttN171-82Q were treated with CHX for 6 h,
and then harvested for small-scale
detergent extraction and sedimentation.
(A) Immunoblots for ubiquitin demonstrated
that the levels of insoluble polyubiquitin were
highest in the cells transfected with Htt-N17182Q, and that CHX treatment can decrease
the level of insoluble ubiquitin. Quantification
(mean±s.d.) and statistical analyses of
replicate experiments (n=3) demonstrated
that the levels of insoluble ubiquitin were
significantly (*P<0.05, Student’s t-test, twotailed, unequal variance) lower in
untransfected cells, cells expressing HttN171-81Q, and cells expressing Htt-N17182Q treated with CHX. For positive controls,
untransfected cells were heat-shocked for 3 h
with and without CHX pre-treatment (30 min
at 37°C) (lanes 1–3). M, marker proteins, from
the bottom up in A, 50 kDa, 60 kDa, 80 kDa
and 100 kDa marker proteins. UTf,
untransfected. (B) Immunoblots with anti-Htt
antibody 2B4 detected high levels of insoluble
Htt-N171-82Q proteins in these cells with
trace levels of insoluble Htt-N171-18Q
protein. Both Htt-N171-18Q and Htt-N17182Q were detected in the PBS-soluble
fractions of the same cell lysates. DOCinsoluble fractions were also immunoblotted
for ubiquitin (lanes identified on the figure).
(C). Immunostaining of similar transfected cell
cultures demonstrated inclusion structures in
a subset of cells expressing HttN171-82Q
(arrows). The images shown are
representative of three independent
experiments.
nuclear proteins that lose solubility and thus we have not sampled
the membrane-bound component of the proteome and can make no
estimate on their vulnerability to proteostatic stress. Overall, our
data provide the first estimation of the number and types of cytosolic
and nuclear proteins that might be vulnerable to bystander
misfolding.
Our principal means of identifying misfolded proteins relies on
the loss of protein solubility in detergent. In studies of proteins
implicated in neurodegenerative disease, there are many examples
in which loss of solubility in detergent distinguishes a misfolded
form of a protein from a more natively folded form (Ishihara et al.,
1999; Johnston et al., 2000; McKinley et al., 1991; Scherzinger
et al., 1999; Wang et al., 2003). Unlike these disease-specific
examples, we believe that the change in solubility observed in our
experimental paradigm occurs when exposed hydrophobic domains
of non-natively folded proteins drive non-specific interactions to
produce large heterogenous complexes. Because we analyzed the
cells during continuous thermal stress, we did not expect to see
inclusions containing these proteins to form. Among the four
proteins that we identified as vulnerable to proteostasis stress, one of
the most obvious candidate proteins to produce inclusion bodies is
TDP-43. This protein has been shown to localize to cytosolic stress
granules upon heat-shock (McDonald et al., 2011), which could
change its solubility (Liu-Yesucevitz et al., 2010). TDP-43 is also
a component of inclusion pathology found in individuals with
fronto-temporal dementia (FTD-U) that present with ubiquitin
immunoreactive inclusions that are tau negative (Neumann et al.,
2006). Subsequently, this protein has been found in inclusions in
sporadic ALS (Neumann et al., 2006) and mutations were then
discovered in TDP-43 in patients with familial ALS and FTD
(Kabashi et al., 2008; Rutherford et al., 2008; Sreedharan et al.,
2008). More recently, TDP-43 immunoreactive inclusion pathology
has been reported in patients with Alzheimer’s disease, Pick’s
disease and Huntington’s disease (Freeman et al., 2008; Hasegawa
et al., 2007; Uryu et al., 2008). A notable feature of TDP-43 is that it
encodes a domain with high sequence similarity to yeast prion
domains of Sup35 and Psi (King et al., 2012). In yeast,
overexpression of wild-type TDP-43 produces inclusion structures
(Couthouis et al., 2011), and TDP-43 is a major component of
inclusion body pathology of ALS and FTD. Thus, TDP-43 would
seem to be a prime candidate to form inclusions upon proteostasis
stress. However, when we immunostained our heat-shocked cells
with antibodies to TDP-43 to determine whether cells form TDP-43
immunoreactive stress granules, or inclusions, within the timeframe of our experiments none were observed (Fig. S4). Thus, we
conclude that the insoluble TDP-43 observed in our stressed-cell
models is likely to have been dispersed in small structures that
coalesce into large heterogeneous aggregates when cells are lysed.
The forms of CDK1, FEN1, TDP-43 and MATR3 that were
detected in the insoluble fractions migrated at the expected size
and were thus not ubiquitylated. Additionally, as previously
reported for the SH-SY5Y and STTG-1 cells, in heat-stressed
HEK293 cells almost all of the ubiquitin peptides possessing the
K-GlyGly motif, which identifies a site of ubiquitin conjugation,
were linked to ubiquitin itself at K48 or K63 to form polyubiquitin
chains. The K-GlyGly modification of K48 was identified from
16 mass spectrometry samples (corresponding to 16 gel pieces)
with 20 unweighted spectra. Ubiquitylation at K63 was also found
but to a much lesser extent. As expected, most of the ubiquitinconjugated peptides (both K48 and K63) identified by LC-MS/
MS were found in insoluble fractions after 42°C treatment. At
present, we do not know the identity of the ubiquitylated proteins
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Journal of Cell Science (2016) 129, 1892-1901 doi:10.1242/jcs.176479
that lose solubility. Apart from ubiquitin itself, only histone H3.1
was identified definitively to contain K-GlyGly peptide
conjugates that are signatures of ubiquitinylation. Given that
inhibiting new protein synthesis also inhibited the accumulation
of polyubiquitin, we assume that ubiquitin must be conjugated to
misfolded protein substrates, but if 10–20% of the soluble
proteome is misfolded by thermal stress to become a potential
substrate for ubiquitination, then it might be that no particular
protein is ubiquitylated a sufficient number of times to allow
detection. It is also important to note that it is clear that most
ubiquitin is conjugated to itself. It is possible that sequestering the
ubiquitin from possible conjugation to misfolded protein
substrates is a means of limiting the volume of proteins to be
degraded, or is a means to delay degradation, allowing induced
chaperones an opportunity to repair some of the proteins that lost
solubility as result of proteostatic stress.
In conclusion, we demonstrate here that the overexpression of
mutant Htt in human cells can induce what appears to be the
equivalent of the bystander misfolding that was first described in
C. elegans models (Gidalevitz et al., 2006). In cells in which
proteostatic stress has been induced by thermal stress or mutant
Htt expression, newly made components of the proteome appear to
be the most vulnerable to ‘secondary’ misfolding. These findings
imply that reducing the burden of newly made proteins might be a
way to allow the proteostatic network to regain balance. Inhibitors
of protein synthesis would surely have short-term toxicity issues,
but a treatment that substantially reduced protein synthesis for
some period might be one means to push the system back in
balance.
MATERIALS AND METHODS
Cell culture
Human embryonic kidney 293 (HEK293) cells [CRL-1573™, American
Type Culture Collection (ATCC), Rockville, MD, USA] (3–4 passages
from frozen seed stock) were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) with 10% fetal bovine serum (FBS) and 4 mM
L-glutamine at 37°C in 5% CO2. Cells used to generate samples for
LC-MS/MS were cultured in 100-mm dishes. Cells were heat-shocked at
42°C for 1 h before harvesting, or held in 37°C incubators as controls. To
inhibit the proteasome, cells at 70–80% confluency were treated with
culture medium containing 10 nM MG-132 [N-(benzyloxycarbonyl)leucinylleucinylleucinal, Z-Leu-Leu-Leu-al] (EMD Millipore, Billerica,
MA) for 12 h before harvesting.
For experiments in which cells were treated with cycloheximide {CHX;
3-[2-(3,5-Dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]glutarimide; SigmaAldrich, St Louis, MO} before heat shock, HEK293 cells were cultured in
60-mm dishes at 37°C. After the cells had reached 90% confluency, the
medium was replaced with fresh medium and then 5 µl DMSO containing
2.5 mg CHX was added into each dish and mixed well. The cells were
kept in a 37°C incubator for 30 min, and then moved to a 42°C incubator
for 3 h. Control experiments included cells in which 5 µl of DMSO was
added before the 3 h of heat shock at 42°C, and cells in which 5 µl of
DMSO+CHX was added right after heat shock and just before harvesting
(see Table S3).
For Htt transfection experiments, 293FT cells (Invitrogen, Carlsbad, CA;
3-4 passages from seed stock) were used because they transfect more
efficiency than the original HEK293 cells. The 293FT cells were plated in
poly-D-lysine-coated 60-mm dishes. When cells were 80% confluent, they
were transfected with expression vectors for Htt103Q–CFP and Htt25Q–
CFP (kind gift of Ron Kopito, Stanford University, CA), using
Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol;
100 units/ml penicillin and 100 μg/ml streptomycin were added to avoid
contamination. At 48 h post-transfection, the cells were harvested by trypsin
treatment, then diluted into complete growth medium to stop the reaction,
and then washed in PBS. The PBS-suspended cells were subjected to
Journal of Cell Science
RESEARCH ARTICLE
fluorescence-activated cell sorting (FACS) with a BD FACSARIA II sorter
(BD Biosciences, San Jose, CA) to isolate transfected cells. The same
number of fluorescent and non-fluorescent cells for each transfection was
collected for analysis.
Sequential detergent extraction
The sequential method of detergent extraction and ultracentrifugation
followed previously published protocols (Xu et al., 2012). Briefly, cells
cultured in 100-mm dishes were harvested by scraping in PBS, then
sequentially extracted in buffers containing three different detergents to
fractionate the proteins according to their solubility (Xu et al., 2012). The
sequential extraction procedure produced eight samples: total crude cell
lysate, PBS-S (PBS soluble), NP40-S (NP-40 soluble), DOC-S
(deoxycholate soluble), SDS-S (SDS soluble); PBS-P (PBS insoluble
fraction), NP40-P (PBS soluble but NP-40 insoluble fraction), and DOCP (NP-40 soluble but deoxycholate insoluble fraction). The pellet after
SDS extraction contained little protein, and this fraction was not
analyzed. All the samples used for LC-MS/MS were generated by
above protocols.
In experiments in which cells were transfected, or treated with CHX or
other drugs, smaller culture plates were used (60 mm), and thus fewer cells
were extracted. The initial homogenization in PBS for these cells was
conducted in 200 µl, then 400 µl TEN with 1% NP-40 was used to extract
the PBS insoluble pellet, followed by centrifugation at top speed in a
benchtop microcentrifuge for 10 min. The NP40 insoluble pellet was then
extracted in TEN with 2% DOC with the resuspended protein homogenate
transferred to a new 1.5 ml centrifuge tube before centrifugation at 16,000 g
for 10 min. Finally, each of the DOC insoluble pellets were resuspended in
65 µl 1× Laemmli buffer with 15 µl loaded on SDS-PAGE for immunoblot
assay.
Mass spectrometry
The methods used for mass spectrometry have been described by Xu et al.
(2012). Briefly, 45 µl of each fraction to be analyzed (e.g. PBS-S or DOCP) was loaded per lane for SDS-PAGE (4–20% Tris-Glycine gel) followed
by Coomassie Blue staining. The lanes for each sample were cut
longitudinally, and then each gel strip was cut into five to eight pieces,
from low to high molecular mass. Standard in-gel trypsin digestion was
used prior to LC-MS/MS following a protocol used by the Protein
Chemistry Core (ICBR, University of Florida, Gainesville, FL). The
digested peptides from all the samples were analyzed with a QSTAR® XL
instrument (Applied Biosystems, Foster City, CA), a hybrid quadrupole
time-of-flight mass spectrometer. A 90-min gradient was used for liquid
chromatography separation. Tandem mass spectra were extracted by
Analyst QS (version 1.1). Charge state deconvolution and deisotoping
were not performed. All MS/MS samples were analyzed using Mascot
(Matrix Science, London, UK; version Mascot) and X! Tandem (The GPM,
thegpm.org; version 2007.01.01.1). X! Tandem was set up to search a
subset of the ipi.HUMAN.v3.80 database assuming trypsin. Mascot was set
up to search the IPI_human_20070808 database (3.32, 67,524 entries)
(Kersey et al., 2004) assuming the digestion enzyme trypsin. Mascot and
X! Tandem were searched with a fragment ion mass tolerance of
0.30 Da and a parent ion tolerance of 0.30 Da. Iodoacetamide derivative
of cysteine was specified in Mascot and X! Tandem as a fixed modification.
S-carbamoylmethylcysteine cyclization (N-terminus) of the N-terminus,
deamidation of asparagine and glutamine, oxidation of methionine and
ubiquitination residue of lysine were specified in Mascot and X! Tandem as
variable modifications.
Scaffold (version 3_0_07, Proteome Software Inc., Portland, OR) was
used to validate MS/MS-based peptide and protein identifications. Peptide
identifications were accepted if they could be established at greater than
95.0% probability as specified by the Peptide Prophet algorithm (Keller
et al., 2002). Protein identifications were accepted if they could be
established at greater than 99.0% probability and contained at least two
identified peptides. Protein probabilities were assigned by the Protein
Prophet algorithm (Nesvizhskii et al., 2003). Proteins that contained similar
peptides and could not be differentiated based on MS/MS analysis alone
were grouped to satisfy the principles of parsimony.
Journal of Cell Science (2016) 129, 1892-1901 doi:10.1242/jcs.176479
Data compilation and semi-quantification
The numbers of unweighted spectrum counts per protein were tabulated
from the unfiltered Scaffold data for comparison. The method of spectral
counting was used for relative protein quantitation to test whether the
abundance of a particular protein was significantly higher in one sample
than another sample (Prokai et al., 2009). The method for relative
quantitation followed published protocols (Higgs et al., 2005; Old et al.,
2005), in which the change in abundance was determined by the ratio of the
total number of identified MS/MS spectra (unweighted spectral count from
Scaffold) for a particular protein in the heat-shock treated and control
groups, respectively. A statistical G-test (likelihood ratio test for
independence) was then utilized to determine the statistical probability
that the abundance of a particular protein in a particular fraction was higher
or lower than expected (Old et al., 2005; Sokal and Rohlf, 1995). To increase
statistical power for G-test analysis of the initial high-confidence protein
identifications (99% protein confidence, 95% peptide confidence and
containing two unique peptides), we included in the data sets peptides with
lower Mascot scores that represent true positive identifications at 50%
probability to match. Differences in protein composition between fractions
were considered highly significant if the G-test significance was P<0.05.
The gene ontology analysis of both the heat-induced insoluble
proteins used the PANTHER (Protein ANalysis THrough Evolutionary
Relationships) system (http://www.pantherdb.org) (Thomas et al., 2006).
SDS-PAGE and immunoblotting
For the samples used for LC-MS/MS, equal volumes of each fraction (10 µl)
were loaded per well, and the proteins were separated by 10–20% TrisGlycine SDS-PAGE. One gel was stained with Coomassie Blue and a parallel
gel was used for immunoblotting using monoclonal antibodies against
ubiquitin [5-25 (1:1000), Covance, Princeton, NJ]. For SDS-PAGE of
samples for immunoblotting we used either 10–20% or 4-20% Tris-Glycine
gels. Antibodies used in immunoblotting were against ubiquitin [rabbit antiubiquitin (1:1000), cat. no. Z045801-5, DAKO, Carpinteria, CA; and mouse
monoclonal anti-ubiquitin antibody, cat. no. MCA-UBi-1(1:1000), Encor
Biotech Inc. Gainesville, FL], CDK1 (rabbit polyclonal, 1:500, cat. no.
HPA003387, Sigma-Aldrich), FEN1 antibody (rabbit polyclonal, 1:500, cat.
no. SAB4500881, Sigma-Aldrich), TDP-43 antibody (mouse monoclonal,
1:5000, cat. no. MCA-3H8, Encor Biotech. Inc. Gainesville, FL), MATR3
(rabbit monoclonal, 1:10,000, cat. no. ab151714, Abcam, Cambridge, UK)
and Htt (monoclonal, 1:1000, clone 2B4, cat. no. MAB5492, Millipore,
Boston, MA) were used according to standard protocols.
Cultured cell immunocytochemistry
The culture medium was removed from the cells in 60-mm dishes, then 2 ml
4% paraformaldehyde was added to the cells and held at 25°C for 20 min,
followed by washing three times in PBS (5–10 min each). After blocking in
3% normal goat serum in PBS with 0.05% Triton X-100, anti-TDP-43
antibody (1:1000 dilution, MCA-3H8, Encor Biotech) or anti-Htt antibody
(2B4, 1:3000 dilution, Millipore) was added and then incubated at 4°C
overnight. After washing in PBS with 0.05% Triton X-100, Alexa-Fluor586-conjugated goat anti-mouse-IgG was added and incubated at 25°C in
darkness for 1 h. After three washes in PBS, all liquid was drained and one
drop of mounting medium with DAPI was added to the dish before
coverslips were placed onto samples. An Olympus BX60 epi-fluorescence
microscope was used to capture the fluorescence images.
Acknowledgements
The authors are grateful to Interdisciplinary Center for Biotechnology Research
(ICBR) in University of Florida for assistance with LC-MS/MS analysis. We thank
Stanley Stevens (University of South Florida) for helpful suggestions regarding data
analysis. We are grateful to Dr Gerry Shaw for providing antibodies to ubiquitin and
TDP-43.
Competing interests
The authors declare no competing or financial interests.
Author contributions
All authors reviewed and edited the manuscript. G.X. performed most of the
experimental work. A.M., R.H., M.C.P. and H.B. assisted GX in culturing cells,
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Journal of Cell Science
RESEARCH ARTICLE
performing immunoblots or performing immunofluorescence. G.X. and D.R.B.
designed the experiments, analyzed the data and drafted the manuscript.
Funding
This study was supported by the National Institutes of Health [grant numbers
R21AG025426, R01NS44278 to D.R.B.], the Santa Fe HealthCare Alzheimer’s
Disease Research Center, and the Huntington’s Disease Society of America and
CHDI Foundation – Coalition for a Cure. Deposited in PMC for release after 12 months.
Data Availability
The mass spectrometry proteomics data have been deposited to the
ProteomeXchange Consortium via the PRIDE [1] partner repository with the dataset
identifier PXD003888.
Supplementary information
Supplementary information available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.176479/-/DC1
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