Endoplasmic Reticulum Proteostasis Influences the Oligomeric State of an Amyloidogenic Protein Secreted from Mammalian Cells

Article abstract of DOI:10.1016/j.chembiol.2016.09.001

Transthyretin (TTR) is a tetrameric serum protein associated with multiple systemic amyloid diseases. In these disorders, TTR aggregates in extracellular environments through a mechanism involving rate-limiting dissociation of the tetramer to monomers, which then misfold and aggregate into soluble oligomers and amyloid fibrils that induce toxicity in distal tissues. Using an assay established herein, we show that highly destabilized, aggregation-prone TTR variants are secreted as both native tetramers and non-native conformations that accumulate as high-molecular-weight oligomers. Pharmacologic chaperones that promote endoplasmic reticulum (ER) proteostasis of destabilized TTR variants increase their fraction secreted as a tetramer and reduce extracellular aggregate populations. In contrast, disrupting ER proteostasis reduces the fraction of destabilized TTR secreted as a tetramer and increases extracellular aggregates. These results identify ER proteostasis as a factor that can affect conformational integrity and thus toxic aggregation of secreted amyloidogenic proteins associated with the pathology of protein aggregation diseases.

Full text of DOI:10.1016/j.chembiol.2016.09.001

Article
Endoplasmic Reticulum Proteostasis Influences the
Oligomeric State of an Amyloidogenic Protein
Secreted from Mammalian Cells
Highlights
Authors
d
d
d
d
Developed assay to measure transthyretin tetramers
secreted from mammalian cells
John J. Chen, Joseph C. Genereux,
Eul Hyun Suh, ..., Maria T.A. Dendle,
Jeffery W. Kelly, R. Luke Wiseman
Highly destabilized TTR tetramers are secreted in non-native
conformations
Correspondence
wiseman@scripps.edu
Pharmacologic chaperoning promotes secretion of
destabilized TTR tetramers
In Brief
Thapsigargin-induced ER stress reduces the quality of
secreted TTR
Using a new assay to measure the
oligomeric state of secreted
transthyretin, Chen et al. show that
chemical or biologic alterations in
endoplasmic reticulum protein
homeostasis can influence the
extracellular aggregation of destabilized
transthyretin variants associated with
human amyloid diseases.
Chen et al., 2016, Cell Chemical Biology 23, 1–12
October 20, 2016 ª 2016 Elsevier Ltd.
http://dx.doi.org/10.1016/j.chembiol.2016.09.001

Please cite this article in press as: Chen et al., Endoplasmic Reticulum Proteostasis Influences the Oligomeric State of an Amyloidogenic Protein
Secreted from Mammalian Cells, Cell Chemical Biology (2016), http://dx.doi.org/10.1016/j.chembiol.2016.09.001
Cell Chemical Biology
Article
Endoplasmic Reticulum Proteostasis Influences
the Oligomeric State of an Amyloidogenic
Protein Secreted from Mammalian Cells
John J. Chen,1,5 Joseph C. Genereux,1,2,5,6 Eul Hyun Suh,2 Vincent F. Vartabedian,1 Bibiana Rius,1 Song Qu,1
Maria T.A. Dendle,2 Jeffery W. Kelly,1,2,3 and R. Luke Wiseman1,4,7,
*
1Department of Molecular & Experimental Medicine, The Scripps Research Institute, 10550 North, Torrey Pines Road, MEM 220, La Jolla,
CA 92037, USA
2Department of Chemistry
3The Skaggs Institute for Chemical Biology
4Department of Chemical Physiology
The Scripps Research Institute, La Jolla, CA 92037, USA
5Co-first author
6Present address: Department of Chemistry, University of California, Riverside, Riverside, CA 92521, USA
7Lead Contact
*Correspondence: wiseman@scripps.edu
http://dx.doi.org/10.1016/j.chembiol.2016.09.001
SUMMARY
sues. Genetic and pharmacologic evidence suggests a causal
relationship between extracellular toxic aggregation of amyloi-
Transthyretin (TTR) is a tetrameric serum protein dogenic proteins and tissue degeneration, demonstrating that
protein aggregation is a predominant driving force in amyloid
disease pathogenesis (Eisele et al., 2015). Many risk factors
associated with multiple systemic amyloid diseases.
In these disorders, TTR aggregates in extracellular
environments through a mechanism involving rate-
limiting dissociation of the tetramer to monomers,
which then misfold and aggregate into soluble oligo-
mers and amyloid fibrils that induce toxicity in distal
tissues. Using an assay established herein, we show
that highly destabilized, aggregation-prone TTR
have been identified that predispose individuals to toxic aggre-
gation of amyloidogenic proteins including destabilizing muta-
tions, aging, and the composition and activity of extracellular
chaperoning pathways (Wyatt et al., 2013). However, the bio-
logic factors that influence the onset and progression of toxic
protein aggregation in amyloid disease pathogenesis remain
poorly defined.
One biologic factor that can influence the toxic aggregation of
amyloidogenic proteins is the activity of endoplasmic reticulum
variants are secreted as both native tetramers and
non-native conformations that accumulate as high-
molecular-weight oligomers. Pharmacologic chaper- (ER) pathways involved in folding, trafficking, and degradation
of the secretory proteome, collectively referred to as ER proteo-
stasis pathways (Chen et al., 2015; Powers et al., 2009). These
ones that promote endoplasmic reticulum (ER)
proteostasis of destabilized TTR variants increase
their fraction secreted as a tetramer and reduce
extracellular aggregate populations. In contrast, dis-
rupting ER proteostasis reduces the fraction of
destabilized TTR secreted as a tetramer and in-
creases extracellular aggregates. These results
identify ER proteostasis as a factor that can affect
pathways can facilitate secretion of destabilized, albeit folded,
amyloidogenic proteins from mammalian cells, leading to
increased concentrations of amyloidogenic proteins in extracel-
lular environments such as serum. This promotes concentration-
dependent aggregation of amyloidogenic proteins into toxic
oligomers and amyloid fibrils. Remodeling ER proteostasis re-
duces the secretion and extracellular aggregation of destabi-
conformational integrity and thus toxic aggregation
of secreted amyloidogenic proteins associated with
the pathology of protein aggregation diseases.
lized, aggregation-prone variants of amyloidogenic proteins
such as transthyretin (TTR) and immunoglobulin light chain
(Chen et al., 2014; Cooley et al., 2014; Plate et al., 2016; Shoul-
ders et al., 2013). This highlights the importance of ER proteosta-
sis in dictating the extracellular concentration of amyloidogenic
protein available for proteotoxic aggregation. The importance
of ER proteostasis in amyloid disease pathology is also evident
INTRODUCTION
The extracellular aggregation of destabilized, misfolding-prone from the correlation between secretion, serum levels, and dis-
proteins is linked to degenerative phenotypes in over 30 protein ease severity in patients expressing destabilizing, amyloidogenic
aggregation diseases commonly referred to as amyloid diseases variants of TTR (Sekijima et al., 2005).
(Eisele et al., 2015). These maladies include Alzheimer’s disease,
ER proteostasis could also influence extracellular protein ag-
Creutzfeldt-Jakob disease, and systemic amyloid diseases. In gregation by altering the quality of amyloidogenic proteins
these disorders, secreted amyloidogenic proteins aggregate secreted to the extracellular space. Protein misfolding is an obli-
into oligomers and amyloid fibrils that are toxic to postmitotic tis- gate step in amyloid formation (Knowles et al., 2014). Disruption
Cell Chemical Biology 23, 1–12, October 20, 2016 ª 2016 Elsevier Ltd.
1

Please cite this article in press as: Chen et al., Endoplasmic Reticulum Proteostasis Influences the Oligomeric State of an Amyloidogenic Protein
Secreted from Mammalian Cells, Cell Chemical Biology (2016), http://dx.doi.org/10.1016/j.chembiol.2016.09.001
of ER proteostasis (i.e., ER stress) increases the population of both of the two thyroxine binding sites within the native TTR
misfolded proteins within the ER and overwhelms ER proteosta- tetramer (Choi et al., 2010). Two key advantages of these probes
sis pathways (Chen et al., 2015; Powers et al., 2009). This chal- are that they only bind to the native TTR tetramer, and they
lenges the ability of destabilized, amyloidogenic proteins to remain ‘‘dark’’ until formation of the covalent adduct within the
attain a natively folded conformation. Thus, ER stress could pro- TTR thyroxine binding pocket. This allows for selective moni-
foundly influence the conformational integrity, and thus the toring of native TTR tetramer levels independent of monomers
aggregation propensity, of amyloidogenic proteins secreted to and/or soluble oligomers. Compound 1 has been used to quan-
the extracellular space. Consistent with this prediction, ER stress tify TTR tetramer levels in human serum where TTR concentra-
promotes the trafficking of misfolded glycosylphosphatidylinosi- tions range from 3 to 8 mM (Rappley et al., 2014). We evaluated
tol-anchored proteins such as the prion protein to the cell whether this molecule could also be employed to quantify TTR
surface where they are then directed to the lysosome for degra- tetramers in media conditioned on mammalian cells where TTR
dation (Satpute-Krishnan et al., 2014). Furthermore, ER stress concentrations are significantly lower than those observed in
increases secretion of the HSP40 co-chaperone ERdj3 in com- serum (100–300 nM). We chose HEK293 cells for these experi-
plex with destabilized, amyloidogenic proteins in a mechanism ments because these cells do not synthesize endogenous
proposed to protect the extracellular environment from mis- TTR, show similar secretion efficiencies measured for trans-
folded aggregation-prone protein conformations that can be fected FTTTRs compared with liver-derived HepG2 cells, and
secreted during ER stress (Genereux et al., 2015). However, have been previously used to define how alterations in ER
the impact of ER stress, or broadly ER proteostasis, on the proteostasis influence secretion of destabilized, amyloidogenic
conformation and stability of a secreted, amyloidogenic protein proteins including TTR (Chen et al., 2014; Plate et al., 2016;
has not been demonstrated.
One challenge in investigating a role for ER proteostasis in the
Shoulders et al., 2013).
We added compound 1 (10 mM) to conditioned media
conformational integrity of secreted amyloidogenic proteins is collected from HEK293 cells overexpressing wild-type TTR
the difficulty of determining protein conformation in complex (TTRWT) or mock-transfected cells. This media was separated
biologic environments such as the extracellular space, where by anion-exchange chromatography using a UPLC System,
secreted protein concentrations are generally low. We address and TTR-compound 1 conjugate fluorescence was monitored
this challenge by developing an assay to monitor the oligomeric as described previously (Rappley et al., 2014). The addition of
state of amyloidogenic TTR variants secreted from mammalian compound 1 to media containing TTRWT revealed a set of peaks
cells. TTR is a serum protein that functions as a native tetramer showing compound 1-TTR conjugate fluorescence that was not
to transport thyroxine and retinol binding protein (Eisele et al., present in mock-transfected controls (Figure 1B). The retention
2015). However, TTR tetramer dissociation and subsequent ag- time of these peaks is comparable with that of recombinant
gregation into soluble oligomers and amyloid fibrils is implicated TTRWT added to cell media, indicating that these peaks are
in the onset and pathology of diverse diseases including familial due to the TTR tetramer (Figure S1A).
amyloid polyneuropathy and familial amyloid cardiomyopathy
Our ability to quantify the low levels of TTRWT tetramer conju-
(Eisele et al., 2015). Here, we establish an assay that utilizes a gate fluorescence in conditioned media with this UPLC assay is
non-cell-permeable kinetic stabilizer of the TTR tetramer in com- compromised by a large background signal observed near the
bination with a fluorogenic assay to quantify secreted TTR tetra- elution time of the tetramer (Figure 1B). This background band
mers in conditioned media prepared on mammalian cells. Using has been observed previously and results from the high concen-
this approach, we show that highly destabilized, aggregation- trations of bovine serum proteins (e.g., albumin) present in cell
prone TTR variants can be secreted in non-tetrameric con- culture media (Rappley et al., 2014). To address this issue, we
formations that accumulate as high-molecular-weight soluble incorporated a tandem FLAG tag sequence between the ER
aggregates. Pharmacologic chaperonesincrease secretionofde- signal sequence and the N terminus of mature TTR (FTTTRWT
;
stabilized TTR variants as tetramersandreduceextracellular pop- Figure 1C). The negatively charged FLAG tags increase the
ulations of TTR aggregates by stabilizing TTR tetramers in the ER. retention time of TTR tetramers on anion-exchange columns,
In contrast, ER stress reduces the fraction of TTR secreted as the without influencing the stability of the native tetramer (Rappley
native tetramer and increases extracellular populations of soluble et al., 2014; Schneider et al., 2001). We observed a new set of
TTR aggregates. These results show that ER proteostasis compound 1-TTR conjugate fluorescent peaks at a later elution
influences the oligomeric state of amyloidogenic TTR secreted time in media conditioned for 16 hr on cells expressing FTTTRWT
from mammalian cells. This indicates that imbalances in ER pro- (Figure 1D). This later elution time corresponds with that
teostasis can directly promote extracellular protein aggregation observed for recombinant FTTTRWT (Figure S1B) and allows for
associated with amyloid disease pathology by influencing the more effective quantification of TTR tetramers in conditioned
conformational integrity of secreted amyloidogenic proteins.
media. We then confirmed that the addition of 10 mM of com-
pound 1 to conditioned media containing FTTTRWT saturated
the 1-TTR conjugate fluorescence signal (Figure S1C). Further-
more, we used immunopurification (IP)/mass spectrometry
(MS) to show that the majority of secreted FTTTRWT is cysteiny-
lated and/or sulfated (Figure S1D). These modified subunits inte-
grate into the homo- and hetero-tetramers that comprise the 8
RESULTS
Small-Molecule Fluorogenic TTR Kinetic Stabilizers Can
Be Employed to Measure TTR Tetramers Secreted from
Mammalian Cells
Small-molecule fluorogenic TTR ligands such as compound 1 tetramer peaks observed by our UPLC assay. These modifica-
(Figure 1A) fluoresce after they covalently react with one or tions are in agreement with what has been seen before for TTR
2
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Please cite this article in press as: Chen et al., Endoplasmic Reticulum Proteostasis Influences the Oligomeric State of an Amyloidogenic Protein
Secreted from Mammalian Cells, Cell Chemical Biology (2016), http://dx.doi.org/10.1016/j.chembiol.2016.09.001
Figure 1. Secreted TTR Tetramers Can Be
Detected in Conditioned Media by Com-
pound 1 Conjugate Fluorescence
(A) Structure of the fluorogenic TTR kinetic stabi-
lizer compound 1 (Choi et al., 2010). This molecule
covalently reacts with TTR through amide forma-
tion to Lys15, liberating the p-fluorothiophenol
moiety and resulting in compound 1-TTR conju-
gate fluorescence.
(B) Plot showing the compound 1-TTR conjugate
fluorescence of media conditioned for 16 hr on
mock-transfected HEK293 cells (black) or HEK293
cells expressing TTRWT (red) separated by UPLC
anion-exchange chromatography. Compound 1
(10 mM) was incubated with conditioned media
for 16 hr prior to analysis. The inset shows com-
pound 1-TTR conjugate fluorescence for the
peaks corresponding to TTRWT. The experimental
protocol is described above.
(C) Image showing the FTTTR sequence containing
the TTR ER signal sequence (SS), two FLAG tag
sequences, and the TTR coding sequence. The
amino acid sequence for the signal sequence and
tandem FLAG tag of FTTTR is shown above.
(D) Plot showing the compound 1-TTR conjugate
fluorescence of media conditioned for 16 hr on
mock-transfected HEK293 cells (black) or HEK293
cells expressing FTTTRWT (red) separated by UPLC
anion-exchange chromatography. Compound 1
(10 mM) was incubated with conditioned media
for 16 hr prior to analysis. The inset shows com-
pound 1-TTR conjugate fluorescence for the
peaks corresponding to FTTTRWT
. The experi-
mental protocol is described above.
ants: TTRA25T, TTRL55P, TTRV30M, and
TTRV122I. These variants were selected
based on the decreased kinetic and/or
thermodynamic stability of the TTR
and other FLAG-tagged proteins (Kingsbury et al., 2008; tetramer, reflected by the t1/2 of tetramer dissociation, as a mea-
Schmidt et al., 2012; Zhang and Kelly, 2003). While these modi- sure of kinetic stability, and the Cm of urea denaturation, as a
fications could have modest effects on TTR stability (Kingsbury measure of thermodynamic stability (Sekijima et al., 2005). We
et al., 2008; Schneider et al., 2001; Zhang and Kelly, 2003), these employed the above-mentioned compound 1-TTR conjugate
modifications are not predicted to globally impact the stability of fluorescence UPLC assay to measure TTR tetramer levels in
secreted TTR tetramers.
media conditioned for 16 hr on HEK293 cells expressing
FTTTRL55P FTTTRV30M, or FTTTRV122I (Figure 2A).
These results show that the TTR-compound 1 conjugate FTTTRA25T
,
,
fluorescence UPLC assay is a useful strategy for quantifying We observed similar tetramer levels for the stable FTTTRWT and
secreted FTTTR tetramers in conditioned media prepared on the FTTTRV122I and FTTTRV30M variants. Gel-filtration chroma-
mammalian cells. Although this approach is currently limited to tography shows that secreted FTTTRWT in conditioned media
cells that do not express endogenous TTR (owing to the large migrates at a molecular weight consistent with the native
background signal observed in our UPLC assay), this model tetramer and identical to that observed for recombinant FTTTRWT
system provides an opportunity to define how alterations (Figure S2A and Chen et al., 2014). This suggests that these
in ER proteostasis influence secretion of TTR and resolve FTTTR variants primarily exist as native tetramers in conditioned
fundamental cell biology questions related to the relationship be- media. However, highly destabilized FTTTRA25T and FTTTRL55P
tween ER proteostasis and extracellular protein misfolding and displayed lower tetramer levels than the more stable variants
aggregation.
(Figure 2B), despite being present in similar total concentrations
(Figure S2B). To further demonstrate this point, we normalized
the conjugate fluorescence derived from 1 (a measure of TTR tet-
ramers) to the relative amounts of total TTR (measured by SDS-
Destabilized, Amyloidogenic TTR Tetramers Are
Unstable in Conditioned Media
To examine the effects of disease-associated TTR mutations PAGE/immunoblotting) for each FTTTR variant. This showed that
on secreted TTR tetramer levels, we focused on four TTR vari- the fraction of total secreted TTR existing as a tetramer for
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Figure 2. TTR Tetramers Composed of De-
stabilized Variants Are Unstable in Condi-
tioned Media
(A) Table showing the t1/2 of tetramer dissociation
(a measure fof kinetic stability) and Cm of urea
denaturation (a measure of thermodynamic sta-
bility) at 25ꢀC for TTRWT and the disease-associ-
ated TTR variants TTRV122I, TTRV30M, TTRL55P, and
TTRA25T. These values were reported previously
(Sekijima et al., 2005).
(B) Representative plot showing the compound
1-TTR conjugate fluorescence of media condi-
tioned for 16 hr on HEK293 cells expressing
FTTTRWT (black), FTTTRV122I (orange), FTTTRV30M
(green), FTTTRL55P (blue), or FTTTRA25T (red).
Compound 1 (10 mM) was added to conditioned
media for 16 hr prior to analysis by UPLC anion-
exchange chromatography.
(C) Graph showing the normalized compound
1-TTR conjugate fluorescence for plots such as
those shown in Figure 2B. Normalization of com-
pound 1-TTR conjugate fluorescence was per-
formed by integrating the fluorescence signal from
all peaks corresponding to the TTR tetramer and
then normalizing to the relative amounts of TTR
in each conditioned media determined by SDS-
PAGE/immunoblotting (see Figure S2B). Error bars
show SEM for n = 3.
(D) Clear native (CN)-PAGE and SDS-PAGE im-
munoblots of media conditioned for 16 hr on
HEK293 cells expressing FTTTRWT
, ,
FTTTRV122I
FTTTRV30M FTTTRL55P, or FTTTRA25T
,
.
FTTTRA25T and FTTTRL55P was reduced by >80% and 50% rela- This is consistent with the 50% lower levels of FTTTRL55P tetra-
tive to FTTTRWT, respectively (Figure 2C). Importantly, we also mers observed compared with FTTTRWT (Figure 2C). In contrast,
showed that the highly destabilized FTTTRA25T is stable in condi- no significant accumulation of TTR-soluble oligomers was
tioned media for >40 hr, indicating that reductions in tetramer observed in media conditioned on cells expressing FTTTRV30M
,
levels cannot be explained by aberrant proteolysis during the FTTTRV122I, or FTTTRWT (Figure 2D). These results suggest that
incubation with compound 1 (Figure S2C). Taken together, the highly destabilized TTR variants are either secreted as tetramers
above results indicate that significant populations of FTTTRA25T that rapidly dissociate in media during the conditioning or in non-
and FTTTRL55P in conditioned media are in non-tetrameric tetrameric conformations that can accumulate as aggregates in
conformations.
Dissociation of the native TTR tetramer is the rate-limiting step
conditioned media.
for TTR aggregation into toxic soluble oligomers and amyloid fi- Pharmacologic Chaperoning of Destabilized TTR
brils (Eisele et al., 2015). Thus, the high non-tetrameric popula- Variants in the ER Increases Tetramers and Reduces
tion of destabilized TTR variants in conditioned media could Aggregates in Conditioned Media
assemble into aggregates. We measured soluble TTR aggre- Cell-permeable TTR kinetic stabilizers, such as tafamidis (Taf),
gates in media conditioned for 16 hr on HEK293 cells expressing bind and stabilize TTR tetramers to prevent dissociation and
, ,
FTTTRA25T FTTTRL55P FTTTRV30M, or FTTTRV122I using clear subsequent aggregation (Razavi et al., 2003). These molecules
native (CN)-PAGE/immunoblotting (Chen et al., 2014). This also increase secretion of highly destabilized TTR variants,
showed high levels of TTR aggregates in conditioned media such as TTRA25T (Chen et al., 2014; Sekijima et al., 2005). This
containing FTTTRA25T (Figure 2D). These high levels of aggre- increased secretion is proposed to result from a pharmacologic
gates are consistent with low levels of FTTTRA25T tetramers chaperoning mechanism wherein intracellular binding of Taf to
observed by our UPLC assay (Figure 2C). Interestingly, gel-filtra- one or both of the two thyroxine binding sites of the TTR tetramer
tion chromatography demonstrated that nearly all FTTTRA25T in stabilizes the tetrameric structure in the ER and promotes traf-
conditioned media migrates as high-molecular-weight soluble ficking through the secretory pathway (Sekijima et al., 2005).
oligomers, with no significant amount of FTTTRA25T identified in We examined this mechanism by monitoring the populations
the fractions containing the native tetramer (Chen et al., 2014) of total TTR, TTR aggregates, and TTR tetramers in media
(Figure S2A). Combined with our UPLC results (Figures 2B and conditioned on HEK293 cells expressing FTTTRWT
2C), this indicates that the majority (>80%) of the FTTTRA25T or FTTTRA25T in the presence or absence of Taf.
, ,
FTTTRL55P
in conditioned media exists as soluble aggregates. Similarly,
The presence of Taf did not increase total TTR levels of
we observe a significant amount of soluble TTR aggregates in FTTTRWT or FTTTRL55P in conditioned media (Figures 3A
media conditioned on cells expressing FTTTRL55P (Figure 2D). and 3B). This is consistent with previous results showing that
4
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Please cite this article in press as: Chen et al., Endoplasmic Reticulum Proteostasis Influences the Oligomeric State of an Amyloidogenic Protein
Secreted from Mammalian Cells, Cell Chemical Biology (2016), http://dx.doi.org/10.1016/j.chembiol.2016.09.001
Figure 3. Pharmacologic Chaperoning of
TTR Tetramers in the ER Reduces Aggre-
gates and Increases Tetramers of Destabi-
lized TTR Variants in Conditioned Media
(A) Representative CN-PAGE and SDS-PAGE im-
munoblots of media conditioned for 16 hr on
HEK293 cells expressing FTTTRWT FTTTRL55P, or
,
FTTTRA25T in the presence or absence of Taf
(10 mM).
(B) Graph showing the normalized total amount of
FTTTRWT (black), FTTTRL55P (blue), or FTTTRA25T
(red) in media conditioned for 16 hr on HEK293
cells expressing these different TTR variants in
the presence (solid bars) or absence (open bars)
of Taf (10 mM). Total amounts of TTR were
measured by SDS-PAGE/immunoblotting as
shown in (A). TTR levels were normalized to
vehicle-treated cells. Error bars show SEM for
n = 3. *p < 0.05.
(C) Representative plot showing compound 1-TTR
conjugate fluorescence of media conditioned
for 16 hr on HEK293 cells expressing FTTTRWT
incubated in the presence (red) or absence
(black) of Taf (10 mM). Compound 1 (10 mM) was
added to conditioned media for 16 hr prior to
analysis by UPLC anion-exchange chromatog-
raphy.
(D) Graph showing the integrated compound
1-TTR conjugate fluorescence (including all
peaks) for FTTTRWT (gray), FTTTRL55P (blue), and
FTTTRA25T (red) in media conditioned for 16 hr on
HEK293 cells expressing the indicated FTTTR
variant in the presence (solid bars) or absence
(open bars) of Taf (10 mM). Representative plots
are shown in (C), (E), and (F). Error bars show SEM
for n = 3. *p < 0.05.
(E) Representative plot showing compound 1-TTR
conjugate fluorescence of media conditioned for
16 hr on HEK293 cells expressing FTTTRL55P
incubated in the presence (red) or absence
(black) of Taf (10 mM). Compound 1 (10 mM) was
added to conditioned media for 16 hr prior to
analysis by UPLC anion-exchange chromatog-
raphy.
(F) Representative plot showing compound 1-TTR conjugate fluorescence of media conditioned for 16 hr on HEK293 cells expressing FTTTRA25T incubated in the
presence (red) or absence (black) of Taf (10 mM). Compound 1 (10 mM) was added to conditioned media for 16 hr prior to analysis by UPLC anion-exchange
chromatography.
cell-permeable kinetic stabilizers do not increase secretion of tetramer, allowing accurate quantification of secreted TTR tetra-
FTTTRWT (Sekijima et al., 2005). However, we observe a 2-fold in- mers by compound 1-TTR conjugate fluorescence. This effect
crease in total FTTTRA25T in conditioned media, reflecting phar- has been demonstrated previously in human serum (Rappley
macologic chaperoning of this highly destabilized TTR variant et al., 2014). Consistent with this, Taf did not influence
(Chen et al., 2014; Sekijima et al., 2005). As expected, because 1-FTTTRWT conjugate fluorescence in conditioned media (Fig-
of the report by Chen et al. (2014), Taf also reduced aggregate ures 3C and 3D), demonstrating the efficacy of compound 1
populations of FTTTRL55P and FTTTRA25T in conditioned media displacement of Taf in our system and showing that the presence
(Figure 3A). This suggests that the binding of TTR tetramers by of Taf does not influence our ability to accurately measure
Taf in the ER stabilize tetramers throughout the secretion pro- secreted TTR tetramers using the covalent TTR kinetic stabilizer
cess, as well as in conditioned media, preventing aggregation compound 1.
into soluble oligomers.
Interestingly, the addition of Taf during conditioning increased
We next quantified TTR tetramers in conditioned media sam- FTTTRL55P and FTTTRA25T tetramers in media (Figures 3D–3F). Taf
ples containing secreted FTTTR variants using our compound 1 increased FTTTRL55P tetramers 2-fold relative to vehicle (Figures
conjugate fluorescence UPLC assay. Although Taf and com- 3D and 3E). This increase is consistent with the Taf-dependent
pound 1 bind the same thyroxine binding sites on the TTR reduction in FTTTRL55P-soluble oligomers (Figure 3A). The addi-
tetramer, the covalent nature of compound 1 binding allows tion of Taf during the conditioning also increased the media con-
this fluorogenic ligand to efficiently displace Taf within the centration of FTTTRA25T tetramers (Figures 3D and 3F). This is
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Figure 4. Tafamidis-Sulfonate (Taf-SO3–) Is
a Non-Cell-Permeable Kinetic Stabilizer of
the TTR Tetramer
(A) Synthetic scheme used to prepare Taf-SO3
À
.
(B) Graph showing the acid-induced aggregation
of TTRWT (3.6 mM tetramer) at pH 4.4 measured by
turbidityÀat 400 nm (gray) or 330 nm (white). Taf or
Taf-SO3 was added at the indicated molar
equivalents to the TTR tetramer (3.6 mM). Error bars
show SEM for n = 3.
(C) Immunoblot showing TTR in media conditioned
on HEK293 cells expressing FTTTRA25T in the
presence of TTR tetramer kinetic stabilizers Taf,
Taf-SO3À, 1, thyroxine (T4), or flufenamic acid (FA)
(10 mM). The lysate samples are shown as
a
control.
Inhibition of pH-induced TTR aggrega-
tion is an established assay to measure
the capacity for small molecules like Taf
to bind and stabilize TTR tetramers (Ra-
zavi et al., 2003; Sekijima et al., 2005).
The addÀition of increasing equivalents of
Taf-SO3 to recombinant TTRWT shows
a
dose-dependent reduction in pH-
consistent with the significant reduction in FTTTRA25T soluble induced aggregation, with complete inhibition observed at 1.5
oligomers afforded by Taf treatment (Figure 3A). These results equivalents of Taf-SO3À relative to the TTR tetramer (Figure 4B).
show that cell-permeable, small-molecule TTR kinetic stabilizers This inhibition of aggregation is nearly identical to that observed
such as Taf can bind and stabilize TTR tetramers in the ER, as with Taf (Bulawa et al., 2012), confirming that Taf-SO3À efficiently
well as in the extracellular space, to increase the population of binds and stabilizes TTR tetramers.
destabilized TTR secreted in the native tetrameric conformation
and reduce extracellular populations of soluble aggregates.
Cell-permeable TTR kinetic stabilizers such as Taf, com-
pound 1, thyroxine (T4), and flufenamic acid increase the secre-
tion of highly destabilized TTR variants such as TTRA25T from
mammalian cells through pharmacologic chaperoning of TTR
tetramers in the ER (Figures 3A and 3B and Chen et al., 2014;
Sekijima et al., 2005). This is shown by the increased levels of
Tafamidis-Sulfonate Is a Non-Cell-Permeable TTR
Kinetic Stabilizer that Selectively Binds Secreted TTR
Tetramers
The next question we wanted to investigate is the exact nature of FTTTRA25T in media conditioned on HEK293 cells treated with
the oligomeric state of TTR secreted from mammalian cells. Taf these different cell-permeable kinetic stabilizers (Figure 4C).
À
stabilizes TTR tetramers in the ER and maintains this stabilized However, the addition of Taf-SO3 did not increase FTTTRA25T
À
tetramer throughout the secretion process and in the extracel- levels in conditioned media. This shows that Taf-SO3 is
lular space. However, the ER stabilization of TTR tetramers unable to pharmacologically chaperone this destabilized TTR
afforded by Taf prevents us from defining the oligomeric state variant in the ER. Collectively, these results indicate that Taf-
of TTR secreted from mammalian cells. For this type of analysis, SO3À retains its ability to bind and stabilize extracellular TTR tet-
we require a TTR kinetic stabilizer that selectively and rapidly ramers, but is not able to acÀt as a pharmacologic chaperone
stabilizes TTR tetramers only upon secretion to the extracellular within the ER. Thus, Taf-SO3 can be employed to selectively
environment. The crystal structure of Taf bound to the two stabilize TTR tetramers only upon secretion to conditioned
thyroxine binding sites of TTR tetramer shows that the carbox- media and evaluate the relative populations of TTR secreted
ylate group at the 6-position of the benzoxazole ring resides at from mammalian cells as tetramers and non-tetrameric
the periphery of the thyroxine binding site near the outside of conformations.
the TTR tetramer (Bulawa et al., 2012). This suggests that we
could replace the carboxylate functionality of Taf with a sulfonate Highly Destabilized TTR Variants Can Be Secreted in
moiety at this position without disrupting the capacity to bind Non-Native Conformations
and stabilize TTR tetramers. The addition of a sulfonate at this In order to define the conformation of TTR secreted from
position, which has a lower pKa than a carboxylate, should limit mammalian cells, we initially measured total, aggregate, and
the cell permeability of Taf, preventing intracellular stabilization tetramer levels of TTR in media conditioned for 16 hr on
of TTR tetramers in the ER and allowing selective stabilization HEK293 cells expressing FTTTRL55P in theÀpresence of vehicle,
of TTR tetramers, but only once secreted to the media. Toward- Taf, or Taf-SO3À. The addition of Taf-SO3 reduced aggregate
ward that aim, we synthesized Taf-SO3À and confirmed its iden- levels of FTTTRL55P >75%, mirroring the reduction in aggregates
tity and purity (Figures 4A, S3A, and S3B).
observed in Taf-treated cells (Figures 5A and 5B). However,
6
Cell Chemical Biology 23, 1–12, October 20, 2016

Please cite this article in press as: Chen et al., Endoplasmic Reticulum Proteostasis Influences the Oligomeric State of an Amyloidogenic Protein
Secreted from Mammalian Cells, Cell Chemical Biology (2016), http://dx.doi.org/10.1016/j.chembiol.2016.09.001
Figure 5. Highly Destabilized TTR Variants
Can Be Secreted as Native Tetramers and
in Non-Tetrameric Conformations
(A) Representative CN-PAGE and SDS-PAGE
immunoblots of media conditioned for 16 hr on
HEK293 cells expressing FTTTRWT FTTTRL55P, or
,
FTTTRA25T in the presence of Taf (10 mM) or Taf-
SO3À (all at 10 mM).
(B) Graph showing the quantification of aggre-
gate (open bars; measured by CN-PAGE) or total
(solid bars; measured by SDS-PAGE) FTTTRL55P
in media conditioned on HEK293 cells in the
presence of vehicle (gray), Taf (10 mM; red), or
À
Taf-SO3 (10 mM; blue). Representative immu-
noblots are shown in (A). Error bars show SEM
for n = 3.
(C) Graph showing tetramer levels in media con-
ditioned on HEK293 cells expressing FTTTRL55P in
the presÀence of vehicle (gray), Taf (10 mM; red), or
Taf-SO3 (10 mM; blue). Tetramer levels were
quantified by integrating compound 1-TTR con-
jugate fluorescence measured by UPLC and are
shown relative to Taf-treated cells. A representa-
tive UPLC trace is shown in Figure S4A. Error bars
show SEM for n = 4. **p < 0.01.
(D) Graph showing the quantification of aggregate
(open bars; measured by CN-PAGE) or total (solid
bars; measured by SDS-PAGE) FTTTRA25T in me-
dia conditioned for 16 hr on HEK293 cells in the
presÀence of vehicle (gray), Taf (10 mM; red), or Taf-
SO3 (10 mM; blue). Representative immunoblots
are shown in (A). Error bars show SEM for n = 8.
***p < 0.005, **p < 0.01.
(E) Graph showing tetramer levels in media
conditioned for 16 hr on HEK293 cells expressing
FTTTRA25T in the presence of vehicle (gray), Taf
(10 mM; red), or Taf-SO3À (10 mM; blue). Tetramer
levels were quantified by integrating compound
1-TTR conjugate fluorescence measured by UPLC
and are shown relative to Taf-treated cells. A
representative UPLC trace is shown in Figure S4B.
Error bars show SEM for n = 4. ***p < 0.005.
(F) Graph showing the normalized compound 1-TTR conjugate fluorescence of TTR tetramers in media conditioned for 16 hr on HEK293 cells expressing
FTTTRA25T in the presence of vehicle (gray), Taf (10 mM; red), or Taf-SO3À (10 mM; blue). Compound 1-TTR conjugate fluorescence was normalized to the average
À
relative amounts of total FTTTRA25T (measured by SDS-PAGE; A) to account for differences in total protein in media prepared in the presence of Taf or Taf-SO3
.
Error bars show SEM for n = 4. *p < 0.05.
À
neither Taf nor Taf-SO3À significantly influenced total FTTTRL55P tion of Taf-SO3 did not significantly increase total FTTTRA25T
levels in conditioned media (Figures 5A and 5B). These results levels in conditioned media, further highlighting the inability of
indicate that Taf-SO3À kinetically stabilizes FTTTRL55P tetramers this molecule to pharmacologically chaperone destabilized
in the extracellular space, preventing TTR aggregation into solu- TTR tetramers in the ER (FigÀures 5A and 5D). However, unlike
ble oligomers. Consistent with this prediction, the addition of Taf, the addition of Taf-SO3 reduced extracellular FTTTRA25T
Taf-SO3 increased FTTTRL55P tetramer levels in conditioned soluble aggregates by only 50% when compared with untreated
À
media 2-fold relative to vehicle-treated controls, as measured controls (Figures 5A and 5D). This suggests that a population of
by our UPLC assay (Figures 5C and S4A). This increase is nearly FTTTRA25T is secreted from mammalian cells in a non-tetrameric
À
identical to that observed in Taf-treated cells. These results indi- conformation that is refractory to stabilization by Taf-SO3
cate that FTTTRL55P is predominantly secreted as the native
.
To examine this further, we measured FTTTRA25T tetramer
tetramer, which can be stabilized by Taf-SO3À in the extracellular levels in conditioned media and observed that addition of Taf-
space. In addition, these results show that FTTTRL55P aggregateÀs SO3 increased FTTTRA25T tetramer levels as compared with
À
observed in media conditioned in the absence of Taf or Taf-SO3
vehicle-treated cells (Figures 5E and S4B). Importantly, we
primarily result from the dissociation of secreted FTTTRL55P tetra- observe no change in the FTTTRA25T tetramer levels in condi-
À
mers and subsequent aggregation during the media conditioning tioned media that was prepared in the presence of Taf-SO3
and incubation with 1.
and then incubated for 0–12Àhr prior to addition of compound 1,
We also performed an identical analysis using media condi- demonstrating that Taf-SO3 stabilizes these tetramers in the
tioned for 16 hr on HEK293 cells expressing FTTTRA25T. The addi- extracellular space (Figure S4C). In order to compare the relative
Cell Chemical Biology 23, 1–12, October 20, 2016
7

Please cite this article in press as: Chen et al., Endoplasmic Reticulum Proteostasis Influences the Oligomeric State of an Amyloidogenic Protein
Secreted from Mammalian Cells, Cell Chemical Biology (2016), http://dx.doi.org/10.1016/j.chembiol.2016.09.001
FTTTRA25T tetramer levels in conditioned media prepared in in HEK293 cells attenuates, but does not ablate, the Tg-depen-
the presence of Taf (where we observe >90% reduction in dent reduction in tetramer secretion (Figure S5F). This suggests
À
aggregates) or Taf-SO3 (where we observe a 50% reduction that this effect is sensitive to ER proteostasis load, indicating
in aggregates), we normalized the observed FTTTRA25T tetramer that tissues with high secretory load are likely to be most suscep-
levels to the amounts of total FTTTRA25T determined by SDS- tible to ER-stress-dependent alterations in the quality of
PAGE. This normalization is necessary to account for the secreted TTR. Interestingly, Tg similarly reduces the amount of
2-fold increase in total FTTTRA25T afforded by Taf-dependent FTTTRWT secreted as the native tetramer by 15% (Figures 6F,
pharmacologic chaperoning of TTR tetramers in the ER (Figures 6G, and S5G).
5A and 5D). This approach shows that Taf-SO3À stabilizes only
The addition of Tg alters the distribution of peaks observed for
65% of the FTTTRA25T tetramer population in the native FTTTRA25T (Figure S5B) and FTTTRWT (Figure 6F) in our UPLC
tetramer, as compared with Taf (Figure 5F). This supports the assay. IP/MS analysis reveals that this results from a modest
notion that this highly destabilized TTR variant is secreted from reduction in secretion of posttranslationally modified FTTTRWT
À
cells as both a tetramer that can be stabilized by Taf-SO3
accompanied by a corresponding increase in the population of
and in a non-tetrameric conformation (e.g., aggregate or mis- unmodified protein (Figure S5H). However, these modest alter-
folded monomer) that is insensitive to Taf-SO3À-dependent ations in TTR posttranslational modifications induced by Tg do
stabilization.
not appear sufficient to explain the 2-fold decrease in FTTTRA25T
tetramer secretion we observed.
Thapsigargin-Induced ER Stress Reduces the Fraction
of FTTTRA25T Secreted as a Tetramer
DISCUSSION
Imbalances in ER proteostasis (i.e., ER stress) could influence
the conformation of FTTTRA25T secreted as a native tetramer. Here, we address the question of how ER proteostasis influ-
ER stress induced by thapsigargin (Tg) reduces the fraction of ences the oligomeric state of secreted TTR, an amyloidogenic
total FTTTRA25T secreted from mammalian cell culture models protein involved in multiple amyloid diseases. We establish an
(Shoulders et al., 2013). However, the impact of Tg-induced ER assay to quantify TTR tetramers secreted from mammalian cells.
stress on the oligomeric state of secreted FTTTRA25T has not This assay takes advantage of two small molecules: (1) the non-
À
been determined. We utilized SDS-PAGE, CN-PAGE, and cell-permeable small-molecule TTR kinetic stabilizer Taf-SO3
UPLC assays to define the impact of Tg-induced ER stress on that stabilizes TTR tetramers upon secretion to the extracellular
the oligomeric state of this destabilized TTR variant in condi- space and (2) the fluorogenic TTR small-molecule kinetic stabi-
tioned media.
lizer compound 1 that fluoresces upon binding and covalent
À
We conditioned media containing Taf-SO3 for 16 hr on attachment to TTR tetramers. Using these molecules, we show
HEK293 cells expressing FTTTRA25T in the presence or absence that highly destabilized TTR variants such as TTRA25T can
of Tg. We confirmed Tg-induced ER stress in these cells by be secreted as both the native tetramer and in non-native
showing increased expression of the ER-stress-responsive conformations that accumulate as soluble aggregates in the
chaperone BiP in cell lysates (Figure S5A). As reported previ- extracellular environment. Pharmacologic chaperoning of these
ously (Shoulders et al., 2013), Tg reduces total FTTTRA25T levels destabilized TTRs in the ER increases their secretion as native
in conditioned media by 25% (Figures 6A and 6B). Despite this tetramers and reduces the accumulation of extracellular soluble
decrease in total FTTTRA25T, we observed an increase in TTR ag- oligomers. In contrast, disrupting ER proteostasis reduces the
gregates in media conditioned in the presence of Tg (Figures 6A population of TTR secreted as the native tetramer and increases
and 6B). To better define the impact of Tg on extracellular extracellular concentrations of soluble aggregates. These results
FTTTRA25T aggregates, we normalized the observed populations show that alterations in ER proteostasis can directly influence
of soluble aggregates to the total amount of TTR measured by the conformational integrity, and thus aggregation propensity,
SDS-PAGE. This normalization takes into account the reduced of secreted TTR in the extracellular space. The experimental
total FTTTRA25T in conditioned media prepared in the presence approach described herein provides a new opportunity to further
of Tg (Figure 6B). This analysis indicates that Tg increases the examine the mechanism of TTR aggregation and identify new
normalized TTR aggregate levels 2-fold (Figure 6C).
biologic factors that influence the conformational integrity and
Next, we used our compound 1-TTR conjugate fluorescence aggregation propensity of secreted TTR.
UPLC assay to show that Tg treatment reduces secreted
A major implication of our findings is that disruptions of ER
FTTTRA25T tetramer levels in conditioned media by 60% (Figures proteostasis in tissues secreting amyloidogenic proteins such
6D and S5B). Normalizing these tetramer signals to account for as TTR could influence their disease-associated extracellular
the Tg-dependent reduction in total FTTTRA25T demonstrates aggregation at distal sites. In this model, imbalances in hepatic
that Tg reduces normalized tetramer levels by 50% (Figure 6E). ER proteostasis could reduce the conformational integrity of
Lower doses of Tg similarly reduce the relative fraction of secreted TTR, facilitating extracellular formation of toxic aggre-
FTTTRA25T secreted as the native tetramer (Figures S5C–S5E). gates. These types of imbalances could be induced by hepatic
This Tg-dependent reduction in tetramer secretion is consistent ER stress or organismal aging, both of which can disrupt ER pro-
with the Tg-dependent 2-fold increase in FTTTRA25T aggregate teostasis in the liver (Brown and Naidoo, 2012). Clinical evidence
levels (Figure 6C). This suggests that treatment with Tg shifts a supports the potential involvement of this type of mechanism
population of FTTTRA25T that is normally secreted as the native in TTR amyloid disease. For example, domino liver transplant re-
tetramer to a population that accumulates as extracellular solu- cipients receiving livers expressing destabilized TTRs develop
ble aggregates. Interestingly, reducing expression of FTTTRA25T TTR amyloid deposition at a considerably faster rate as
8
Cell Chemical Biology 23, 1–12, October 20, 2016

Please cite this article in press as: Chen et al., Endoplasmic Reticulum Proteostasis Influences the Oligomeric State of an Amyloidogenic Protein
Secreted from Mammalian Cells, Cell Chemical Biology (2016), http://dx.doi.org/10.1016/j.chembiol.2016.09.001
Figure 6. Thapsigargin-Induced ER Stress
Reduces Secretion of FTTTRA25T as Tetra-
mers and Increases Extracellular TTR Ag-
gregates
(A) Representative CN-PAGE and SDS-PAGE im-
munoblots of media conditioned for 16 hr on
HEK293 cells expressing FTTTRA25T in the pres-
ence of vehicle or thapsigargin (Tg) (0.5 mM). All
media were conditioned in the presence of Taf-
SO3À (10 mM).
(B) Graph showing total TTR (open bars; measured
by SDS-PAGE) and aggregate TTR (closed bars;
measured by CN-PAGE) in media conditioned for
16 hr on HEK293 cells expressing FTTTRA25T in the
presence of vehicle (gray) or Tg (red; 0.5 mM). All
medÀia were conditioned in the presence of Taf-
SO3 (10 mM). Representative immunoblots are
shown in (A). Data are shown relative to vehicle-
treated cells. Error bars show SEM for n = 12.
***p < 0.005.
(C) Graph showing normalized TTR aggregates in
the conditioned media described in (A) and (B).
Normalized aggregates were calculated by
dividing the relative amounts of aggregate by the
total amount of TTR in each sample measured by
SDS-PAGE. This normalization is necessary to
account for the reduced total FTTTRA25T levels
observed in Tg-treated cells. Error bars show SEM
for n = 12. ***p < 0.005.
(D) Graph showing total TTR (open bars; mea-
sured by SDS-PAGE) and tetrameric TTR (closed
bars; measured by integrated compound 1-TTR
conjugate fluorescence from UPLC traces) in
media conditioned for 16 hr on HEK293 cells ex-
pressing FTTTRA25T in the presence of vehicle
(gray) or Tg (red; 0.5 mM). All media were condi-
À
tioned in the presence of Taf-SO3 (10 mM).
A representative UPLC trace is shown in Fig-
ure S5B. Data are shown relative to vehicle-
treated cells. Error bars show SEM for n = 15.
***p < 0.005.
(E) Graph showing normalized TTR tetramers in
the conditioned media described in (D). Normal-
ized tetramers were calculated by dividing
the relative tetramer levels by the total amount
of TTR in each sample measured by SDS-
PAGE. This normalization is necessary to account for the reduced total FTTTRA25T levels observed in Tg-treated cells. Error bars show SEM for n = 15.
***p < 0.005.
(F) Representative plot showing compound 1-TTR conjugate fluorescence of media conditioned for 16 hr on HEK293 cells expressing FTTTRWT in the presence
À
(red) or absence (black) of Tg (0.5 mM). Media were conditioned in the presence of Taf-SO3 (10 mM). Compound 1 (10 mM) was added to conditioned media
for 16 hr prior to analysis by UPLC anion-exchange chromatography.
(G) Graph showing normalized FTTTRWT tetramers in the conditioned media described in (F). Normalized tetramers were calculated by dividing the relative
tetramer levels by the total amount of TTR in each sample measured by SDS-PAGE. This normalization is necessary to account for the reduced total FTTTRWT
levels observed in Tg-treated cells. Independent quantifications of total FTTTRWT (measured by SDS-PAGE) and tetrameric FTTTRWT (measured by integrated
compound 1-TTR conjugate fluorescence from UPLC traces) in these conditioned media are shown in Figure S5G. Error bars show SEM for n = 6. *p < 0.05.
compared to the donor. (Llado et al., 2010; Stangou et al., 2005). gates, revealing a potential mechanism to explain the relation-
Similarly, transcriptional profiling of livers from mice overex- ship between ER proteostasis and toxic aggregation of secreted
pressing TTRWT suggests that the expression of stress- TTRs that could contribute to the distal deposition of TTR oligo-
responsive regulatory factors inversely correlate with the distal mers in human disease.
deposition of TTR amyloid fibrils on the heart (Buxbaum et al.,
Disruptions in ER proteostasis could also promote the secre-
2012). These results indicate that alterations in liver ER proteo- tion and subsequent extracellular aggregation of other misfolded
stasis promote the extracellular aggregation and distal deposi- proteins. ER stress is implicated in the onset and pathology
tion of toxic TTR oligomers on the heart. Our results show that of many protein aggregation diseases including Alzheimer’s
imbalances in ER proteostasis can increase secretion of TTR in disease, Creutzfeldt-Jakob disease, and retinitis pigmentosa
non-native conformations that accumulate as soluble aggre- (Hetz and Mollereau, 2014; Hiramatsu et al., 2015; Roussel
Cell Chemical Biology 23, 1–12, October 20, 2016
9

Please cite this article in press as: Chen et al., Endoplasmic Reticulum Proteostasis Influences the Oligomeric State of an Amyloidogenic Protein
Secreted from Mammalian Cells, Cell Chemical Biology (2016), http://dx.doi.org/10.1016/j.chembiol.2016.09.001
et al., 2013). While it is difficult to discern cause-and-effect rela- favorable as these types of pharmacologic chaperones have
tionships for ER stress in the pathogenesis of these disorders, the most potential to stabilize the entire population of protein in
our results suggest that alterations in ER proteostasis (e.g., ER extracellular environments.
stress) could promote extracellular aggregation through the
In addition, targeting ER proteostasis pathways could provide
secretion of aggregation-prone proteins in non-native conforma- a unique opportunity to broadly attenuate the toxic extracellular
tions. Consistent with this prediction, ER stress increases the protein aggregation associated with diverse protein aggregation
trafficking of misfolded PrP proteins to the cell surface (Sat- diseases. One such strategy is to activate unfolded protein
pute-Krishnan et al., 2014). Furthermore, misfolded conforma- response (UPR)-associated transcription factors that endoge-
tions of amyloidogenic proteins such as the amyloid precursor nously regulate ER protein folding, trafficking, and degradation
protein can be secreted to extracellular environments in complex pathways (Chen et al., 2015). Activation of these transcription
with the ER-stress-induced co-chaperone ERdj3 (Genereux factors has previously been shown to reduce the secretion and
et al., 2015). However, the impact of ER stress on the extracel- subsequent aggregation of amyloidogenic variants of TTR and
lular aggregation of these misfolded proteins remains to be immunoglobulin light chain (Chen et al., 2014; Cooley et al.,
defined. Our work establishes a new experimental strategy that 2014; Plate et al., 2016; Shoulders et al., 2013). Adapting ER pro-
can be adapted to other amyloidogenic or aggregation-prone teostasis through activation of UPR-associated transcription
proteins to evaluate how ER stress influences their proteotoxic factors could also influence the conformational integrity of amy-
aggregation in downstream secretory environments such as loidogenic proteins secreted to extracellular environments.
the extracellular space.
Thus, our results reveal another mechanism by which targeting
Interestingly, the increased secretion of TTR variants in non- ER proteostasis through UPR-associated transcription factor
tetrameric conformations may reflect a mechanism to promote activation or other approaches could impact extracellular pro-
restoration of ER proteostasis during ER stress. While ER pro- teotoxic aggregation.
teostasis pathways are traditionally thought to prevent the traf-
ficking of misfolded or non-native conformations, the secretion SIGNIFICANCE
of misfolded protein conformations could provide a mechanism
to relieve the burden of misfolded ER proteins that accumulate Many human amyloid diseases are causatively associated
during ER stress. Consistent with this mechanism, misfolded with the extracellular aggregation of proteins that deposit
prion proteins trafficked to the cell surface in response to ER as amyloid fibrils on postmitotic tissues. Risk factors asso-
stress are directed to the lysosome for degradation through a ciated with this pathologic protein aggregation include de-
mechanism called rapid ER-stress-induced export (Satpute- stabilizing mutations in amyloidogenic proteins, aging, and
Krishnan et al., 2014). Similarly, ER stress increases secretion the composition and activity of extracellular chaperoning
of the HSP40 co-chaperone ERdj3 to the extracellular space, pathways. However, the biologic pathways that influence
which is predicted to protect the extracellular environment the extracellular aggregation of amyloidogenic proteins
from misfolded and/or aggregation-prone proteins secreted are largely undefined. Here, we establish a new assay to
during ER stress (Genereux et al., 2015). ERdj3 can also be define the oligomeric state of the amyloidogenic protein
secreted in complex with destabilized, aggregation-prone pro- transthyretin (TTR) secreted from mammalian cells. In vitro
teins including TTRA25T, providing a mechanism to preemptively TTR aggregation proceeds through a mechanism involving
protect the extracellular environment from misfolded protein dissociation of the native tetramer to monomers that then
conformations (Genereux et al., 2015). While these pathways misfold and aggregate into the soluble oligomers and amy-
are likely effective at preventing toxic misfolding and/or aggrega- loid fibrils implicated in TTR amyloid disease pathogenesis.
tion of non-native proteins secreted during ER stress, dysregula- Interestingly, we show that highly destabilized, aggregation-
tion of these pathways induced by genetic or aging-related prone TTR variants can be secreted from mammalian cells in
factors could compromise extracellular proteostasis mainte- non-native conformations that promote their extracellular
nance, potentially explaining the age-dependence of many pro- aggregation. Intracellular stabilization of TTR using cell-
tein aggregation diseases.
permeable small-molecule kinetic stabilizers, which bind
Surprisingly, our results show that pharmacologic chaper- to the native tetramer, increase secretion of destabilized
oning of destabilized TTR tetramers in the ER offers an advan- TTRs as tetramers and attenuate extracellular aggregation
tage for reducing extracellular aggregates of highly destabilized through a pharmacologic chaperoning mechanism. How-
TTR variants. While Taf-dependent stabilization of FTTTRA25T tet- ever, disrupting the protein-folding pathways within the
ramers in the ER nearly ablates extracellular aggregate load, endoplasmic reticulum (ER) responsible for the folding and
selective stabilization of secreted FTTTRA25T using Taf-SO3 only trafficking of TTR can increase secretion of destabilized
partially reduces extracellular aggregates. This importance of TTR tetramers in non-native conformations and increase
intracellular pharmacologic chaperoning to ameliorate extracel- extracellular aggregate loads. These results show that intra-
lular protein aggregation has implications in the establishment of cellular alterations in ER pathways involved in the folding
new small-molecule approaches to treat TTR and other amyloid and trafficking can influence the quality, and thus aggrega-
diseases. Our results suggest that non-cell-permeable mole- tion propensity, of amyloidogenic proteins such as TTR
cules will be less effective in their ability to attenuate disease secreted to extracellular environments. This reveals ER pro-
toxicity, as they will not prevent secretion of non-native confor- tein-folding pathways as a new biologic factor the altered
mations that can accumulate extracellularly as toxic aggregates. activity of which can contribute to the pathologic aggrega-
In contrast, cell-permeable molecules such as Taf would be tion of amyloidogenic proteins involved in human disease
10 Cell Chemical Biology 23, 1–12, October 20, 2016

Please cite this article in press as: Chen et al., Endoplasmic Reticulum Proteostasis Influences the Oligomeric State of an Amyloidogenic Protein
Secreted from Mammalian Cells, Cell Chemical Biology (2016), http://dx.doi.org/10.1016/j.chembiol.2016.09.001
SUPPLEMENTAL INFORMATION
and suggests that targeting these pathways provides a new
opportunity to attenuate the toxic aggregation associated
Supplemental Information includes Supplemental Experimental Procedures
with amyloid disease pathology.
and five figures and can be found with this article online at http://dx.doi.org/
10.1016/j.chembiol.2016.09.001.
EXPERIMENTAL PROCEDURES
AUTHOR CONTRIBUTIONS
Immunoblotting, CN-PAGE, and SDS-PAGE
CN-PAGE was performed on conditioned media prepared on HEK293 cells
J.J.C., J.C.G., .E.H.S., J.W.K., and R.L.W. designed the research. J.J.C.,
expressing FTTTR variants using a 4%–16% gradient native PAGE gel (Life
J.C.G., E.H.S., V.F.V., B.R., S.Q., M.T.A.D., and R.L.W. designed and per-
Technologies) as described previously (Chen et al., 2014). SDS-PAGE was
formed the experiments. J.J.C., J.C.G., V.F.V., B.R., S.Q., M.T.A.D., and
performed on conditioned media or whole-cell lysates using 15% Tris-glycine
R.L.W. interpreted and analyzed the data. J.J.C., J.C.G., E.H.S., and R.L.W.
gels. CN-PAGE and SDS-PAGE gels were transferred onto a nitrocellulose
wrote the manuscript.
membrane (Bio-Rad) for immunoblotting. Membranes were blocked and
then incubated with primary antibodies. Primary antibodies were then de-
ACKNOWLEDGMENTS
tected using appropriate IRDye-conjugated secondary antibodies and then
analyzed using the Odyssey Infrared Imaging System (Li-COR Biosciences).
We thank Ryan Paxman, Lars Plate, Angelo Solania, Dennis Wolan, and Evan
Powers (TSRI) for experimental help and critical reading of this manuscript. We
thank the NIH (DK102635 and NS092829) and the Amyloidosis Foundation
(R.L.W.) for financial support. J.C.G. was supported by the NIH (HL099245)
and an American Heart Association Postdoctoral Fellowship.
Measurement of TTR Tetramers in Conditioned Media
HEK293 cells overexpressing FTTTR variants were plated on poly-D-lysine-
coated plates. Fresh media was added to the cells and incubated in the pres-
ence of vehicle, Taf, or Taf-SO3À, as indicated. Conditioned media was
diluted 1:1 into water containing 1 to reach the indicated final concentration.
This media was stored at ambient temperature prior to UPLC analysis to
allow for modification of TTR tetramers. Media samples were separated over
a HiQ anion-exchange column in 25 mM Tris (pH 7.0) and 1 mM EDTA with
a linear gradient of 1 M NaCl using the ACQUITY UPLC H-Class Bio System
(Waters). Fluorescence of TTR conjugates was observed by excitation at
328 nM and monitoring emission at 430 nM. Peaks were integrated using
Empower 3 software according to the manufacturer instructions.
Received: April 15, 2016
Revised: July 12, 2016
Accepted: September 2, 2016
Published: October 6, 2016
REFERENCES
Brown, M.K., and Naidoo, N. (2012). The endoplasmic reticulum stress
–
response in aging and age-related diseases. Front. Physiol. 3, 263.
Synthesis of Taf-SO3
Taf-SO3À was synthesized in an analogous manner to Taf (Razavi et al., 2003).
2-(3,5-Dichlorophenyl)benzo[d]oxazole (2a). 2-Hydroxyaniline (3 g, 29 mmol)
and 3.5-dichorobenzoyl chloride (5.8 g, 29 mmol) were dissolved in pyridine
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and the mixture refluxed. The crude product was crystallized from DMSO and
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À
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LC-MS and NMR; however, previous a previous report found that modifica-
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