© 2006 Nature Publishing Group
S-Nitrosylated protein-disulphide isomerase links
protein misfolding to neurodegeneration
Takashi Uehara1,4, Tomohiro Nakamura1, Dongdong Yao1, Zhong-Qing Shi1, Zezong Gu1, Yuliang Ma2,
Eliezer Masliah3, Yasuyuki Nomura4& Stuart A. Lipton1,3
Stress proteins located in the cytosol or endoplasmic reticulum
(ER) maintain cell homeostasis and afford tolerance to severe
insults1–3. In neurodegenerative diseases, several chaperones
ameliorate the accumulation of misfolded proteins triggered by
oxidative or nitrosative stress, or of mutated gene products4,5.
Although severe ER stress can induce apoptosis2,6, the ER with-
stands relatively mild insults through the expression of stress
proteins or chaperones such as glucose-regulated protein (GRP)
and protein-disulphide isomerase (PDI), which assist in the
maturation and transport of unfolded secretory proteins. PDI
catalyses thiol–disulphide exchange, thus facilitating disulphide
bond formation and rearrangement reactions7–10. PDI has two
domains that function as independent active sites with homology
to the small, redox-active protein thioredoxin7,8. During neuro-
degenerative disorders and cerebral ischaemia, the accumulation
of immature and denatured proteins results in ER dysfunction11,
but the upregulation of PDI represents an adaptive response to
protect neuronal cells12–14. Here we show, in brains manifesting
sporadic Parkinson’s or Alzheimer’s disease, that PDI is S-nitro-
sylated, a reaction transferring a nitric oxide (NO) group to a
critical cysteine thiol to affect protein function15–18. NO-induced
S-nitrosylation of PDI inhibits its enzymatic activity, leads to the
accumulation of polyubiquitinated proteins, and activates the
unfolded protein response. S-Nitrosylation also abrogates
PDI-mediated attenuation of neuronal cell death triggered by
ER stress, misfolded proteins or proteasome inhibition. Thus,
PDI prevents neurotoxicity associated with ER stress and
protein misfolding, but NO blocks this protective effect in
neurodegenerative disorders through the S-nitrosylation of PDI.
Initially, we gathered two independent lines of chemical evidence
to show that PDI was S-nitrosylated in vitro and in vivo to form an
S-nitrosylatedprotein(SNO-P). First, inaspecific fluorescenceassay
for SNO-P, we demonstrated the reaction of recombinant PDI with
the physiological NO donor S-nitrosocysteine (SNOC; Fig. 1a).
This assay detects the formation of SNO-P by the conversion
of 2,3-diaminonaphthalene (DAN) to the fluorescent compound
2,3-naphthyltriazole (NAT)17,19,20. SNOC-treated PDI resulted in
significant SNO-P formation in a concentration-dependent manner.
Mammalian PDI has six cysteine residues in all, with four of them
representing two thioredoxin-like domains (one near the amino
terminus and the other near the carboxy terminus) that contain
the Cys-Gly-His-Cys sequence at the active site. To determine the
target site(s) of S-nitrosylation, we performed the DAN assay on
immunoprecipitates from HEK-293T cell lysates transfected with
active-site sequences). The fluorescence intensity in this assay of the
N-terminal (C36S, C39S) and C-terminal (C383S, C386S) mutants
was decreased by about 50% compared with the wild type. The
double mutant (N-terminal and C-terminal) was completely devoid
of fluorescence, indicating that both thioredoxin-like domains are
targets of S-nitrosylation (Fig. 1b). Next we showedthat PDIin293T
cells was S-nitrosylated by the biotin-switch method. In this assay, a
stable biotin group by chemical reduction with ascorbate, as
described previously15,20,21. SNOC markedly enhanced the level of
S-nitrosylated PDI (SNO-PDI) in cell lysates or intact cells (Fig. 1c),
whereas under the same conditions the NO†donor diethylamine-
NO (DEA-NO) or hydrogen peroxide did not (Supplementary
Fig. 1a). Additionally, in HEK-293 cells stably expressing neuronal
ous NO, and this reaction was inhibited by a NOS inhibitor (Fig. 1d,
e). Furthermore, by the biotin-switch assayweidentified the cysteine
residues that were S-nitrosylated. We found that endogenous nNOS
activity led to the S-nitrosylation of cysteine residues in both
thioredoxin-like domains of PDI (Fig. 1f and Supplementary
Fig. 1b). Moreover, with mass spectrometry we found that one of
the cysteine residues in the C-terminal thioredoxin-like domain was
possibly further oxidized to sulphinic acid (2SO2H) after exposure
to NO (Supplementary Fig. 2). These data are consistent with our
previous observation that reversible S-nitrosylation may facilitate
further oxidation of the same cysteine thiol19.
Next, we sought to determine whether SNO-PDIwas produced in
we incubated dopaminergic SH-SY5Y cells with the mitochondrial
complex I inhibitor rotenone, which is known to induce a parkinso-
nian phenotype, at least in part, in a NO-dependent fashion22.
Exposure to rotenone led to the generation of SNO-PDI in these
cells (Fig. 1g). To extend this finding to humans, we examined PD
brains obtained shortly afterdeath. We found evidencefor SNO-PDI
formation in each of four PD brains but not in controls obtained
from patients who had died of disorders that were not of central
nervous system origin (Fig. 1h and Supplementary Table 1).
Additionally, brains from another major neurodegenerative disorder
associated with protein aggregation and nitrosative stress, Alzheimer’s
disease (AD), also showed evidence of SNO-PDI (Fig. 1i, Sup-
plementary Fig. 3 and Supplementary Table 1), consistent with the
notion that this finding could represent a common denominator
linking free-radical stress and protein misfolding.
To determine whether S-nitrosylation affects PDI function, we
of rhodanese induced by guanidinium, as previously described9.
Rhodanese aggregation occurred in a time-dependent manner, and
1Center for Neuroscience and Aging, and2Proteomic Facility, Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, California 92037, USA.
3Department of Neurosciences, University of California at San Diego, 9500 Gilman Drive, La Jolla, California 92039, USA.4Department of Pharmacology, Graduate School of
Pharmaceutical Sciences, Hokkaido University, Sapporo, 060-0812, Japan.
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© 2006 Nature Publishing Group
incubation with recombinant wild-type PDI, but not dominant-
negative PDI (produced by the N-terminal and C-terminal
mutant)13, suppressed this aggregation by about 80%. S-Nitro-
sylation of PDI also significantly inhibited this chaperone activity
(Fig. 2a). Next we measured isomerase activity in a standard assay
that uses as a substrate an inactive form of RNase A containing
scrambled disulphide bonds10. PDI catalyses the renaturation
(refolding) of this inactive RNase A. Recovery of RNase A activity
by wild-type PDI was attenuated about 50% by S-nitrosylation
(Fig. 2b). Thus, S-nitrosylation inhibited the functional activities
of PDI. In addition, direct oxidation of PDI by hydrogen peroxide
could also decrease its activity (Supplementary Fig. 4), indicating
that the sulphinated PDI derivative observed by mass spectrometry
after exposure to NO (Supplementary Fig. 2) might also be patho-
physiologically relevant to the inhibition of PDI activity.
From these results we reasoned that PDI might function in
attenuating protein misfolding and consequent aggregation in
Figure 1 | S-Nitrosylation of PDI in vitro and in vivo. a, Recombinant PDI
218C. SNO-PDI thus generated was assessed by DAN assay (n ¼ 4
experiments). Open symbols, with SNOC; filled symbol, without SNOC.
Values are mean ^ s.e.m. b, Cell lysates transduced with wild-type (WT) or
mutant (N-terminal (Nmut), C-terminal (Cmut), or N-terminal and
C-terminal (N&C)) PDI were immunoprecipitated with anti-PDI antibody.
Immunoprecipitates were then incubated in the presence or absence of
SNOC and subjected to DAN assay. Values are mean ^ s.e.m., n ¼ 5;
asterisks, P , 0.01 by analysis of variance. c, Top: cell lysates from human
293T cells were incubated with SNOC at room temperature to assay for
SNO-PDI. Control samples were subjected to decayed (old) SNOC.
SNO-PDIwas detected by biotin-switch assay 30min after SNOC exposure.
Bottom: total PDI in cell lysates by western analysis. d–f, Top panels:
HEK-293 cells stably expressing nNOS were assayed for endogenous
SNO-PDI. nNOS was activated by Ca2þionophore A23187 (5mM) in the
presence or absence of NOS inhibitor (N-nitro-L-arginine; NNA). Bottom
panels: Total PDI. Activation of nNOS increased endogenous SNO-PDI (d).
NNA prevented this increase (e). Mutation of critical cysteine thiol groups
of PDI also prevented its S-nitrosylation (f). g, SNO-PDI increased in cells
exposed to rotenone in a NOS-dependent manner. Top: lysates from
SH-SY5Y cells exposed to rotenone for 6h in the presence or absence of
NNA. Bottom: Total PDI. h, i, Brain tissuesfrom controls, from PD patients
with diffuse Lewy body disease (h), or AD patients (i) were subjected to
Supplementary Fig. 3 and Supplementary Table 1).
Figure 2 | S-Nitrosylation of PDI regulates its enzymatic activity. a, Effect
of S-nitrosylation on PDI chaperone activity. Unfolded rhodanese was
diluted in buffer containing wild-type PDI (blue), SNO-PDI (red), SNOC
(green), dominant-negative PDI (N-terminal and C-terminal mutant; cyan)
or buffer alone (black). Rhodanese aggregation was monitored (n ¼ 8
experiments).Valuesaremean ^ s.e.m.b,S-NitrosylationofPDIattenuates
its isomerase activity. Scrambled RNase A from bovine pancreas was
incubated with wild-type (wt) PDI, dominant-negative (dn) PDI or
SNO-PDI. Asterisks, P , 0.01 for n ¼ 5 experiments. Values are
mean ^ s.e.m. c, PDIinhibits the aggregation of synphilin-1. SH-SY5Ycells
were transfected with green fluorescent protein (GFP)–synphilin-1
(GFP–Synp) and wild-type or dominant-negative PDI. Inclusion body
formation was monitored by deconvolution microscopy 24h after exposure
to SNOC or control solution. Images were deconvolved with SlideBook
software (Intelligent Imaging Innovations, Inc.). d, Percentage of cells with
GFP–Synp inclusions. Values are mean ^ s.e.m. for n ¼ 2,500 transfectants
counted in five experiments; asterisks, P , 0.05. e, Co-localization of
synphilin-1 and polyubiquitin (polyUb) by immunofluorescence (n ¼ 3).
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neurodegenerative diseases, and that the formation of SNO-PDI
might inhibit neuroprotective activity. Moreover, previous work had
suggested that ER stress can directly or indirectly influence the
aggregation of both ER and cytosolic proteins4,5,23. We therefore
attempted to show an inhibitory effect of PDI on the aggregation of
synphilin-1, as observed in Lewy body inclusions in the brains of PD
patients. Initially, we tested whether PDI could prevent the ubiqui-
tinated, Lewy-body-like inclusions that are formed in the cytosol
after synphilin-1 overexpression in cultured SH-SY5Y cells (Fig. 2c).
When wild-type PDI was co-expressed with synphilin-1, discrete
inclusions were greatly decreased, and instead ubiquitin-negative
synphilin-1 was localized diffusely in the cytosol (Fig. 2c–e). As a
control, immunofluorescent staining of transfected SH-SY5Y cells
revealed that overexpression of PDI did not alter its predominant
intracellular distribution in the ER (Supplementary Fig. 5). NO
attenuated the protective effect of PDI on synphilin-1 inclusions
(Fig. 2d). These findings suggest that PDI is involved in protein
folding linked to PD in a NO-sensitive manner.
Excitotoxic damage is also thought to have a function in neuro-
degenerative disorders such as PD by triggering the production of
freeradicals, including NO,inpartthrough theexcessivestimulation
of N-methyl-D-aspartate (NMDA)-type glutamate receptors; mild
that exposure of cerebrocortical neurons to NMDA induced
SNO-PDI in a NOS-sensitive fashion (Fig. 3a). We surmised that
might contribute to the accumulation of unfolded and consequently
polyubiquitinated proteins marked for degradation by the protea-
some. We therefore next examined whether the accumulation of
polyubiquitinated proteins occurred in response to NMDA by using
a polyubiquitin-specific antibody. Within 12h of exposure to
NMDA, we detected polyubiquitin immunoreactivity in neurons,
but the cells remained viable at this time point (Fig. 3b, c). By 24h,
many of the polyubiquitinated neurons had undergone apoptosis.
polyubiquitinated cells, indicating that PDI has a function in
preventing the accumulation of unfolded, polyubiquitinated pro-
teins in response to NMDA insult, and subsequent neuronal cell
death. Next we evaluated the involvement of the unfolded protein
response (UPR) signalling pathway that is activated by the accumu-
lation of misfolded proteins or ER dysfunction. Representing this
pathway we detected CHOP mRNA induction and XBP-1 mRNA
processing by activated IRE1-a after exposure of cortical cultures to
Figure 3 | NMDA stimulates the accumulation of polyubiquitinated
proteins and UPR pathway. a, SNO-PDI was detected in a NOS-sensitive
manner in primary cortical cultures exposed to NMDA. b, Primary cortical
and immunostained for polyubiquitinated protein (green) and neuron-
specific MAP2 (red). Hoechst-stained DNA (blue) was used to assess
condensed, apoptotic nuclei (white in merged image). c, Quantification of
apoptotic and/or polyubiquitinated neurons similar to those shown in b.
Left, apoptotic cells; middle, polyubiquitinated cells; right, apoptotic and
polyubiquitinated cells. Values are ratio (£100%) of affected to total
neurons, expressed as mean ^ s.e.m. for n ¼ 7,000 neurons counted in six
experiments; asterisks, P , 0.01. Open circles, Ad-LacZ; blue circles,
Ad-wtPDI; green squares, Ad-dnPDI; red triangles, Ad-wtPDI plus N-nitro-
L-arginine. d, NMDA-stimulated processing of XBP-1 mRNA and induction
of CHOP. Processing of endogenous XBP-1 mRNA was evaluated by PstI
endonuclease digestion of XBP-1 cDNA. TG, thapsigargin; bp, base pairs.
Figure 4 | Neuroprotection by PDI against ER stress, proteasome
inhibition, or Pael receptor expression. a–d, SH-SY5Y cells were
transduced for 24h with expression vectors for control lacZ (a–c), PDI
constructs(a–d),or Pael receptor(d).Cultureswerethen incubated for15h
in the presence or absence of 100mM SNOC with 5mM thapsigargin (a),
10mgml21tunicamycin (b) or 0.1mM proteasome inhibitor MG132 (c).
Exposure to SNOC abolished the protective effect of PDI on cell death
induced by ER stress, proteasome inhibitor or Pael receptor. dn, dominant-
negative; wt, wild type. For each panel, values are mean ^ s.e.m. for
n ¼ 3,500 cells counted in five experiments; asterisks, P , 0.01. e, Possible
mechanism of SNO-PDI contributing to the accumulation of aberrant
proteins and to cell death in human neurodegenerative disorders. UPS,
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NMDA1,2. This UPR was attenuated by overexpression of wild-type
PDI but not by dominant-negative PDI (Fig. 3d). Additionally,
the NOS inhibitor N-nitro-L-arginine blocked NMDA-induced
apoptosis and the UPR, indicating that a pathophysiologically
relevant amount of NO was produced under these conditions
(Fig. 3c, d and Supplementary Fig. 6). Taken together, these findings
indicate that NMDA activates a NO-mediated UPR through ER
dysfunction, but this dysfunction can be mitigated by PDI activity.
For further clarification of the relationship between the protective
function of PDI and its S-nitrosylation, we investigated the effect of
PDI on neuronal death after ER stress or proteasome inhibition
(resulting in the accumulation of polyubiquitinated proteins that
cannot be degraded by the proteasome). For this purpose we used
(Fig. 1), allowing us to tease apart the effect of NO on cell death and
PDI S-nitrosylation. We found that cell death precipitated by
thapsigargin and tunicamycin (to induce ER stress) or MG132 (to
inhibit the proteasome) was largely abrogated by wild-type PDI;
however, this protective effect was reversed by exposure to SNOC
(Fig. 4a–c and Supplementary Fig. 6). Similarly, wild-type PDI
ameliorated cell death triggered by overexpression of the Pael
receptor, a protein that abnormally accumulates in Parkinson’s
disease and serves as a potent inducer of the UPR and substrate of
the E3 ubiquitin ligase parkin6,28; exposure to SNOC also reversed
this protective effect (Fig. 4d). These results are consistent with
the notion that NO impairs the protective role of PDI through
S-nitrosylation. From these findings we conclude that cell death in
response to proteasome inhibition or ER stress, which contributes to
ER dysfunction, UPR activation and protein misfolding, can be
attenuated by PDI.
These results show that SNO-PDI forms in brains of patients with
PD and AD, neurodegenerative disorders that are characterized by
abnormal protein accumulations. Cell models of neurodegeneration
produced by exposure to the pesticide rotenone, NO or NMDA also
result in the formation of SNO-PDI. S-Nitrosylation of PDI inhibits
its activity, allows the accumulation of polyubiquitinated proteins
and contributes to neuronal cell death (Fig. 4e). To determine
whether the level of SNO-PDI in neurodegenerative human brain
is of pathophysiological significance, we calculated the ratio of
SNO-PDI (by biotin-switch assay) to total PDI (from western
blotting) and found that this ratio was similar to that encountered
in our neuronal cell models manifesting polyubiquitinated proteins
and cell death (Supplementary Fig. 3). This finding indicates that
pathophysiologically relevant amounts of SNO-PDI are present in
human brains with PD and AD.
related protein synphilin-1, and affords neuroprotection. Previous
in cellular models of PD29. In addition, there is increasing evidence
that the accumulation of aggregated or misfolded proteins links
cellular stress to the pathogenesis of PD6,20,30. Other reports have
shown that NO can be involved in neurodegeneration by a variety of
mechanisms15,19,20,24–27,30. Our data demonstrate a previously unrec-
ognized relationship between NO and protein misfolding in degen-
erative disorders, showing that PDI can be a target of NO after
mitochondrial insult in cellular models of PD and in human neuro-
degenerative diseases. Nitrosative stress resulting in PDI dysfunction
therefore provides a mechanistic link between deficits in molecular
chaperones, accumulation of misfolded proteins, and neuronal
demise in neurodegenerative disorders. The elucidation of this
SNO-PDI-mediated pathway that contributes to neuronal injury
and apoptosis might permit the development of new therapeutic
approaches for neurodegenerative diseases and other disorders
associated with abnormal protein accumulation and nitrosative
Fluorimetric detection of S-nitrosothiols. We measured S-nitrosothiols by the
conversion of DAN to fluorescent NAT, as described17,19. NAT was quantified
with a FluoroMax-2 spectrofluorometer and DataMax software (Instruments
S.A.). Serial NAT dilutions were used to construct a standard curve.
Cell injury/death assays. Cerebrocortical neurons or SH-SY5Y cells were
transduced with a wild-type or mutated PDI gene or with a Pael receptor
expression construct and incubated for 24h (Supplementary Fig. 7). The cells
were then treated for 15h with 5mM thapsigargin, 10mgml21tunicamycin,
0.1mM MG-132 or diluent in the presence or absence of 100mM SNOC.
Neurotoxicity of the Pael receptor was analysed as described6. Cortical neurons
exposed to NMDA were incubated as described previously24. Hoechst staining
was used to assess morphological changes of apoptotic nuclei. MAP2 staining
was used to assess injury or retraction of neuronal processes. Additional details
are described in Supplementary Information.
Expression and purification of recombinant PDI proteins, isolation of PDI
complementary DNA and construction of adenoviral vectors, detection of
S-nitrosylated proteins with the biotin-switch assay, liquid chromatography–
PDI enzymatic activity assays, detection of aggregated synphilin-1, immuno-
cytochemistry, XBP-1 mRNA splicing and CHOP mRNA induction, human
subjects, and statistics are all described in the Supplementary Information.
Received 27 December 2005; accepted 4 April 2006.
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Supplementary Information is linked to the online version of the paper at
Acknowledgements We thank X. Fang for preparation of cerebrocortical
cultures, T. William for technical assistance with the analysis of mass spectra,
and R. Takahashi for the Pael receptor construct. T.U. was supported in part by
the Mitsubishi Pharma Research Foundation and a Grant-in-Aid from the
Ministry of Education, Culture, Sports and Technology of Japan. S.A.L. was
supported in part by grants from the NIH, the American Parkinson’s Disease
Association, San Diego Chapter, and an Ellison Senior Scholars Award in Aging.
Author Contributions T.U. and T.N. performed most of the experiments,
contributing equally to the work, and helped to write the manuscript. D.Y.,
Z.Q.S. and Z.G. provided the biochemical data, and also contributed equally to
the work. Y.M. analyzed the mass spectrometry data. E.M. provided the human
subjects, and Y.N. provided constructs and advice. S.A.L., the senior author,
designed the project, helped to analyse the data, wrote the manuscript and
provided the financial support.
Author Information Reprints and permissions information is available at
npg.nature.com/reprintsandpermissions. The authors declare that they have no
competing financial interests. Correspondence and requests for materials should
be addressed to S.A.L. (email@example.com).
NATURE|Vol 441|25 May 2006