Redox control of 20S proteasome gating.
ABSTRACT The proteasome is the primary contributor in intracellular proteolysis. Oxidized or unstructured proteins can be degraded via a ubiquitin- and ATP-independent process by the free 20S proteasome (20SPT). The mechanism by which these proteins enter the catalytic chamber is not understood thus far, although the 20SPT gating conformation is considered to be an important barrier to allowing proteins free entrance. We have previously shown that S-glutathiolation of the 20SPT is a post-translational modification affecting the proteasomal activities.
The goal of this work was to investigate the mechanism that regulates 20SPT activity, which includes the identification of the Cys residues prone to S-glutathiolation.
Modulation of 20SPT activity by proteasome gating is at least partially due to the S-glutathiolation of specific Cys residues. The gate was open when the 20SPT was S-glutathiolated, whereas following treatment with high concentrations of dithiothreitol, the gate was closed. S-glutathiolated 20SPT was more effective at degrading both oxidized and partially unfolded proteins than its reduced form. Only 2 out of 28 Cys were observed to be S-glutathiolated in the proteasomal α5 subunit of yeast cells grown to the stationary phase in glucose-containing medium.
We demonstrate a redox post-translational regulatory mechanism controlling 20SPT activity.
S-glutathiolation is a post-translational modification that triggers gate opening and thereby activates the proteolytic activities of free 20SPT. This process appears to be an important regulatory mechanism to intensify the removal of oxidized or unstructured proteins in stressful situations by a process independent of ubiquitination and ATP consumption. Antioxid. Redox Signal. 16, 1183-1194.
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ABSTRACT: For many years, the ubiquitin-26S proteasome degradation pathway was considered the primary route for proteasomal degradation. However, it is now becoming clear that proteins can also be targeted for degradation by the core 20S proteasome itself. Degradation by the 20S proteasome does not require ubiquitin tagging or the presence of the 19S regulatory particle; rather, it relies on the inherent structural disorder of the protein being degraded. Thus, proteins that contain unstructured regions due to oxidation, mutation, or aging, as well as naturally, intrinsically unfolded proteins, are susceptible to 20S degradation. Unlike the extensive knowledge acquired over the years concerning degradation by the 26S proteasome, relatively little is known about the means by which 20S-mediated proteolysis is controlled. Here, we describe our current understanding of the regulatory mechanisms that coordinate 20S proteasome-mediated degradation, and highlight the gaps in knowledge that remain to be bridged.Biomolecules. 09/2014; 4(3):862-884.
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ABSTRACT: In eukaryotic cells, proteasomes exist primarily as 26S holoenzymes, the most efficient configuration for ubiquitinated protein degradation. Here, we show that acute oxidative stress caused by environmental insults or mitochondrial defects results in rapid disassembly of 26S proteasomes into intact 20S core and 19S regulatory particles. Consequently, polyubiquitinated substrates accumulate, mitochondrial networks fragment, and cellular reactive oxygen species (ROS) levels increase. Oxidation of cysteine residues is sufficient to induce proteasome disassembly, and spontaneous reassembly from existing components is observed both in vivo and in vitro upon reduction. Ubiquitin-dependent substrate turnover also resumes after treatment with antioxidants. Reversible attenuation of 26S proteasome activity induced by acute mitochondrial or oxidative stress may be a short-term response distinct from adaptation to long-term ROS exposure or changes during aging.Cell reports. 05/2014;
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ABSTRACT: Redox signaling is a fundamental regulation of cell fate upon differentiation.•UPS plays important role in the maintenance of pluripotency and the triggering of differentiation.•UPS regulates through degradation the redox-mediated effectors of differentiation.•Interactome network of cardiomyocytes differentiated from ESC highlighted UPS role in pluripotency.•UPS likewise genome stability were found essential in the maintenance of pluripotency state.Biochimica et Biophysica Acta (BBA) - General Subjects 11/2014; · 3.83 Impact Factor
ORIGINAL RESEARCH COMMUNICATION
Redox Control of 20S Proteasome Gating
Gustavo M. Silva,1,2Luis E.S. Netto,2Vanessa Simo ˜es,1Luiz F.A. Santos,3Fabio C. Gozzo,3
Marcos A.A. Demasi,4Cristiano L.P. Oliveira,5Renata N. Bicev,5Cle ´cio F. Klitzke,6
Mari C. Sogayar,4and Marilene Demasi1
The proteasome is the primary contributor in intracellular proteolysis. Oxidized or unstructured proteins can be
degraded via a ubiquitin- and ATP-independent process by the free 20S proteasome (20SPT). The mechanism by
which these proteins enter the catalytic chamber is not understood thus far, although the 20SPT gating con-
formation is considered to be an important barrier to allowing proteins free entrance. We have previously shown
that S-glutathiolation of the 20SPT is a post-translational modification affecting the proteasomal activities. Aims:
The goal of this work was to investigate the mechanism that regulates 20SPT activity, which includes the
identification of the Cys residues prone to S-glutathiolation. Results: Modulation of 20SPT activity by protea-
some gating is at least partially due to the S-glutathiolation of specific Cys residues. The gate was open when the
20SPT was S-glutathiolated, whereas following treatment with high concentrations of dithiothreitol, the gate was
closed. S-glutathiolated 20SPT was more effective at degrading both oxidized and partially unfolded proteins
than its reduced form. Only 2 out of 28 Cys were observed to be S-glutathiolated in the proteasomal a5 subunit
of yeast cells grown to the stationary phase in glucose-containing medium. Innovation: We demonstrate a redox
post-translational regulatory mechanism controlling 20SPT activity. Conclusion: S-glutathiolation is a post-
translational modification that triggers gate opening and thereby activates the proteolytic activities of free 20SPT.
This process appears to be an important regulatory mechanism to intensify the removal of oxidized or un-
structured proteins in stressful situations by a process independent of ubiquitination and ATP consumption.
Antioxid. Redox Signal. 16, 1183–1194.
cells (10, 26). Although only the 20S proteasome core (20SPT)
capped with the 19S regulatory particle (namely the 26S
to 30% of the total proteasome in mammalian and yeast cells
lack regulatory particles (2, 48). Alternatively, free 20SPT
operates in a ubiquitin- and ATP-independent manner to
degrade unstructured substrates, including oxidized proteins
(1, 25, 45). Recent work indicated that the 20SPT can cleave
>20% of intracellular proteins, initiating the polypeptide
processing in disordered regions, including internal domains
Because few repair systems for protein damage are known
(e.g., methionine sulfoxide reductase), it is widely accepted
that proteolysis is the cellular protective mechanism against
he 26S proteasomal complex is responsible for the
degradation of ubiquitin-tagged proteins in eukaryotic
The20SPTis responsible forthedegradation ofoxidized
and unstructured proteins. In the present work, we show
that 20SPT S-glutathiolation increases the degradation of
oxidatively modified proteins by promoting gate opening.
20SPT S-glutathiolation would take place via the oxidation
of Cys residues to sulfenic acid species followed by glu-
responsible for both an increased protein oxidation and a
modification of the redox status of the proteasome con-
tributing to the removal of oxidized proteins before their
aggregation without ATP consumption because the
mechanism proposed precludes the protein ubiquitylation
process. The present results show an important mecha-
nism for coping with stressful conditions to avoid protein
1Laborato ´rio de Bioquı ´mica e Biofı ´sica, Instituto Butantan, Sa ˜o Paulo, Brasil.
2Departamento de Gene ´tica e Biologia Evolutiva, Instituto de Biocie ˆncias, Universidade de Sa ˜o Paulo, Brasil.
3Instituto de Quı ´mica, Universidade Estadual de Campinas, Brasil.
4Departamento de Bioquı ´mica, Instituto de Quı ´mica, Universidade de Sa ˜o Paulo, Brasil.
5Instituto de Fı ´sica, Universidade de Sa ˜o Paulo, Brasil.
6Laborato ´rio Especial de Toxinologia Aplicada, Instituto Butantan, Brasil.
ANTIOXIDANTS & REDOX SIGNALING
Volume 16, Number 11, 2012
ª Mary Ann Liebert, Inc.
metabolic protein damage, and the 20SPT is the preferential
protease responsible for the removal of such proteins (24).
Although it is still under discussion, convincing evidence has
suggested that oxidized proteins are degraded in a ubiquitin-
independent manner (1, 25, 45). The mechanism by which
oxidized proteins enter the 20SPT catalytic channel is not
currently understood. Both higher hydrophobicity and loss of
secondary structure were investigated and appear to underlie
the process (5, 19, 39). Notably, many components of the
ubiquitin-proteasome system are highly sensitive to oxidative
or an uncoupling of the 26S complex (24, 52). Although the
autophagy-lysosome system can play an important role in the
prevention of protein aggregation (11), no convincing data
have yet revealed its role in the removal of mildly oxidized
proteins. Altogether, the knowledge accumulated to date is in
agreement with the hypothesis that the 20SPT is able to re-
move oxidized proteins.
Because the 20SPT lacks regulatory units, it is unclear how
its proteolytic activity is regulated. Most likely, gating regu-
lation and substrate interaction with the 20S core particle
would underlie the entrance of substrates into the 20SPT.
However, the mechanisms that regulate the gating of the free
20SPT pool are still elusive. Furthermore, there has been no
systematic study of the conformational state of the free 20SPT
pool in any cellular model. The 20SPT is composed of four
heptameric rings (a7b7b7a7) arranged in a barrel-like config-
uration, and the a-rings control substrate entrance via a dy-
namic gating process (42). As previously demonstrated, the
closed conformation of the 20SPT is maintained by a lattice
formed by interactions among the N-terminal tails of the a
subunits (4, 22). Deletion of the a3N-terminal domain in-
creased the proteasomal peptidase activity and promoted the
and a7N-terminal domains was necessary to increase the
20SPT proteolytic activity (4). The opening of the eukaryotic
20SPT gate can occur concomitant with its coupling to the 19S
regulatory particle (28, 49) in a process dependent on specific
activators (e.g., yeast Blm10) (12) and on the presence of poly-
ubiquitylated substrates (40).
Our hypothesis is that post-translational modifications,
including S-glutathiolation, could also control the activity of
the free 20SPT pool by regulating the gating process in a
manner that is independent of the 19S regulatory particle. The
residues primarily during oxidative challenges has been de-
scribed in diverse eukaryotic organisms from yeast to plants
and mammals (14–16, 35, 46). This post-translational modifi-
cation of the 20SPT affects its peptidase activities (15, 46) and
is reversed by thiol-disulfide oxido-reductases (46). Although
S-glutathiolation appears to be a widespread metabolic
modification of the 20SPT, neither the identification of the
subunits and Cys residues susceptible to S-glutathiolation nor
its structural and functional meaning have been elucidated
thus far, which is due, in part, to the large number of Cys
residues present in this protein complex. Here, we report that
20SPT within cells is under redox regulation by glutathione
that affects gate opening and the degradation of oxidized or
from yeast cells grown to the stationary phase contains 2 out
of the 28 analyzed Cys residues modified by a glutathione
Proteolysis rates are increased when the 20SPT
In a previous work, we showed that the 20SPT isolated
from yeast cells grown to stationary phase in a glucose-
containing medium was S-glutathiolated (46). This post-
translational modification alters the proteasomal site-specific
activities (peptidase activity) (14, 15, 46). In the present work,
we tested the proteolytic ability of 20SPT in different redox
forms: the S-glutathiolated form was obtained from cells
grown in YPD medium (referred to as nPT-SG), and the re-
with 20mM dithiothreitol (DTT) (referred to as PT-SH). We
conducted a set of experiments in vitro with the nPT-SG as a
model of physiologically S-glutathiolated 20SPT, and we also
used similar preparations of PT-SH. Both cores were incu-
bated with proteins known to be degraded bythe 20SPT, such
as oxidized bovine serum albumin (BSAox), casein, and glu-
taredoxin 2 (Grx2). Grx2 was selected because it is either de-
graded by the 20SPT or poly-ubiquitylated inside yeast cells
(46). Moreover, the ability of Grx2 to deglutathiolate the
20SPT concomitant with its degradation has been previously
All proteins tested were degraded more extensively by the
nPT-SG core than by the PT-SH core (Fig. 1A–C). To quantify
the peptide fragments generated by both redox forms, the
20SPT preparations were incubated with either BSAoxderiva-
tized with dinitrophenylhydrazine (BSAox-DNPH) or fluores-
cein isothiocyanate (FITC)-modified casein (casein-FITC). The
peptides derived from BSAox-DNPH refer exclusively to the
oxidized fragments generated by hydrolysis. The nPT-SG
species produced at least twice as many peptides from each
substrate (Fig. 2A and B), confirming the proteolysis rate ob-
served by sodium dodecyl sulfate polyacrylamide gel electro-
20SPT (Supplementary Fig. S1; Supplementary Data are
available online at www.liebertonline.com/ars).
Here, we show that the proteolytic rate for the degradation
of these proteins (oxidized, unstructured, and oxidoreduc-
tases) increases when acted upon bythe S-glutathiolated form
of 20SPT (nPT-SG). Because both processes are dependent on
the loss of intracellular reductive ability, it is likely that the
intracellular pool of oxidized proteins increases concomi-
tantly with proteasomal S-glutathiolation (15). This conclu-
sion is in agreement with the observation that the S-
glutathiolated 20SPT more efficiently degraded oxidized
proteins (Figs. 1 and 2). We hypothesized here that the redox
control of gating is the mechanism that underlies proteolysis
by glutathiolated 20SPT. The nPT-SG would prevail on its
open-gate conformation and would facilitate the access of
protein substrates into the inner catalytic chamber, thereby
increasing proteolytic rates.
S-glutathiolation modifies proteasomal gating
To test the hypothesis raised above, we used transmission
electron microscopy (TEM) to investigate whether protea-
somal gating is modified by thiolation. A high frequency
(75%–5%) of open structures was observed in the nPT-SG
1184 SILVA ET AL.
samples (Fig. 3A and Supplementary Fig. S2A), whereas
after treatment with DTT (20mM), the frequency of closed
particles predominated and represented approximately
90%–10% of the total complexes (Fig. 3B and Supplemen-
tary Fig. S2B). Our results are in agreement with reports in
the literature that demonstrate the dynamic state of the
proteasomal gate (36, 41, 47).
As a complementary and independent assessment of the
overall dimensions of the proteasome, small-angle X-ray
scattering (SAXS) experiments were performed. According
to the results obtained (Table 1 and Supplementary Data),
there is a significant three-dimensional (3D) structural shift
between both redox forms of the 20SPT, which is in
agreement with the TEM data already discussed. For both
20SPT redox forms, the SAXS data indicated a cylindrical
shape of the particles with an internal hole. The outer di-
ameter decreased from 106 A˚
identical preparations were treated with DTT (PT-SH).
(nPT-SG) to 72 A˚when
redox-modified 20S catalytic unit
of the proteasome (20SPT) prepa-
dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) of (A)
oxidized bovine serum albumin
(20lg; BSAox), (B) casein (20lg),
and (C) glutaredoxin 2 (15lg;
Grx2) after incubation for 120, 15,
and 60min, respectively, with na-
tively S-glutathiolated 20S protea-
teasome (5lg; PT-SH). After this
incubation, the samples were fil-
tered through YM-100 microfilters
(Millipore) to remove the 20SPT,
and the filtrates were used to load
the gels. To test the integrity of
the preparations, 0.0125 % SDS-
containing buffer (+SDS) was uti-
lized as a positive control. BSA was
oxidized in the presence of 5mM
H2O2and 100lM diethylene tria-
mine pentaacetic acid (DTPA) for
30 minutes at room temperature,
and the remaining H2O2 was re-
moved by cycles of filtration and
redilution through YM-10 micro-
filters (Millipore). All incubations
were performed at 37?C. St, stan-
dard proteins not incubated with
Protein degradation by
PROTEASOME GATING CONTROL1185
Remarkably, the inner diameter was almost completely
closed in the PT-SH samples (Table 1), confirming the data
visualized by TEM (Fig. 3B). The shift from the open to the
closed conformation was previously demonstrated to be
accompanied by slight changes in the outer diameter and
length (37). The proteasomal lengths and diameters ob-
tained by SAXS are in agreement with data in the literature
(Supplementary Table S1). However, the inner diameter
measured in the present work (80 A˚; nPT-SG) is higher than
that determined by the few crystallographic studies that
have been performed or evaluated by TEM (Supplementary
Table S1). A comparative revision of the 20SPT dimensions
is presented in the Supplementary Data section (Supple-
mentary Table S1).
Only Cys residues of the a subunits were observed
to be modified by glutathione
the 20SPT to degrade oxidized/unstructured proteins and its
gate conformation, it was necessary to identify the Cys resi-
dues that were post-translationally modified in both nPT-SG
and PT-SH. Therefore, we initially isolated and characterized
the 20SPT subunits by two-dimensional electrophoresis (2-
DE) coupled to MALDI-TOF fingerprinting (Supplementary
Fig. S3, Supplementary Table S2). Next, the Cys-containing
20SPT subunits were digested with trypsin and prepared for
liquid chromatography–tandem mass spectrometry (LC-MS/
MS) analysis. Two S-glutathiolated Cys residues (Cys76
and Cys221) in the a5 subunit were identified in the nPT-SG
by tandem mass spectrometry analysis. To better characterize
all of the Cys residues that are potentially prone to S-
glutathiolation, the purified nPT-SG preparations were trea-
ted in vitro with 10mM glutathione (GSH). In this series of
experiments, we identified two other subunits (a6 and a7) in
addition to the a5 subunit and a total of seven GSH-modified
Cys residues (+305.1 Da) among the 35 Cys residues present
in mature yeast 20SPT (Table 2; Fig. 4A). The samples that
were reduced by DTT (PT-SH) were subjected to LC-MS/MS
analysis as a control (Fig. 4B). The latter preparations did not
present any glutathione-modified Cys residues. We cannot
discard the possibility that the b subunits were also modified
seven of the Cys-containing fragments in the b subunits
among the 20 predicted. Nevertheless, all of the Cys-
containing fragments in the a subunits were identified (Sup-
plementary Table S3). This 2-DE/mass spectrometry analysis
was employed at least five times with reproducible results.
(nPT-SG preparations), the Cys residues were observed in
different oxidative states as follows: reduced (-SH, which were
modified by iodoacetamide), modified via S-glutathiolation or
hyper-oxidized to sulfinic acid (Cys-SO2H) (Supplementary
Table S3). Remarkably, Cys-SO2H was detected in all of the
Cys residues (except Cys66 from the a6 subunit) prone to
S-glutathiolation (Supplementary Table S3), indicating that
the formation of Cys sulfenic acid (Cys-SOH) is a common
intermediate in both processes (hyper-oxidation and S-
glutathiolation). In fact, we have previously shown that 20SPT
S-glutathiolation occurs via a Cys-SOH intermediate (15). It is
A low pKaof the thiol group and solvent accessibility are im-
portant factors for increasing the thiol protein reactivity.
Given the location of the S-glutathiolated Cys residues in
the 3D structure of the 20SPT, Cys221 from the a5 subunit is
the only modified residue whose thiol group is highly acces-
sible to the solvent (Fig. 5A; Supplementary Fig. S 4A and B).
Furthermore, the environment around Cys221 allows for the
docking of a GSH molecule (Fig. 4B and Supplementary
Fig. S4C) that fits very well into the proteasomal bulk where
GSH-charged groups (both N- and C-terminal carboxyl
groups and the N-terminal amine) can establish important
fied 20SPT preparations. (A) BSAoxthat had reacted with di-
nitrophenylhydrazine (DNPH), a carbonyl protein reactant
(31), was incubated with the 20SPT preparations for 60min
followed by the addition of 20% trichloroacetic acid. The su-
pernatant was retained for spectrometric measurement at
370nm. (B) Fluorescein isothiocyanate (FITC)-modified casein
(casein-FITC) was incubated with the proteasomal prepara-
tions for15min followed by theaddition of20% trichloroacetic
acid. The supernatant was sampled for fluorometric determi-
nation (excitation, 492nm; emission, 515nm). Both the casein-
FITC and DNPH-treated BSAoxsamples were processed using
the same conditions in the absence of the proteasome as con-
trols. The results shown represent the mean–SD and are ex-
pressed as arbitrary units of absorbance (hydrazone adducts)
or fluorescence (FITC). *p<0.000021; **p<0.000003.
Quantitative protein degradation by redox-modi-
1186SILVA ET AL.
saline interactions with charged groups of the side chains of
proteasomal residues (E197, Q225, K229, and K235; Fig. 5B).
In the case of Cys76, the other natively S-glutathiolated resi-
due, no docking was obtained, likely because the thiol group
is not located on the protein surface. It is probable that the S-
glutathiolation of Cys76 is dependent on structural changes
that allow the reaction of GSH with the thiol group to occur.
Remarkably, Cys76 is fully conserved among the 20SPT a5
subunits from yeast, plants and mammals (Supplementary
S-glutathiolation promotes the allosteric modification
of site-specific proteasomal activity
The increased degradation of proteins by nPT-SG may
appear contradictory to our previous finding that S-
like (ChT-L) and post-acidic proteasomal activities, which
were measured using fluorogenic peptides (15, 46) (see Sup-
plementary Table S4). Our hypothesis is that, in addition to
affecting gating, S-glutathiolation also promotes an allosteric
modification of the proteasomal catalytic sites. In fact, ac-
cording to SAXS analysis (Table 1), the 20SPT conformation
experienced changes not only in its outer and inner diameters
but also in its longitudinal length depending on its redox
state. To address this hypothesis, we performed experiments
with the mutated 20SPT lacking the N-terminal sequences of
its a3 and a7 subunits. As reported (3), this mutated form of
the 20SPT is permanently in its open conformation and is
highly active compared with the wild-type 20SPT. These re-
sults were reproduced in our lab (not shown). We then per-
formed a series of experiments with purified preparations of
the mutated (DNa3a7) 20SPT to evaluate whether thiol reac-
tants would modify its activity, as had been observed in the
reactant), 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD; sulf-
hydryl and sulfenic acid reactant), or oxidized glutathione
(GSSG; sulfhydryl reactant) promoted the inhibition of the
ChT-L DNa3a7 20SPT activity (Table 3), which could not be
explained by changes in the diameter of the 20SPT gate but
could be related to changes in the length of this core particle
(Supplementary Table S1). In the context of this work, em-
in the 20SPT-Cys-SOH form that is equivalent to that formed
by the oxidation of the DNa3a7 20SPT with 5mM H2O2in the
presence of diethylene triamine pentaacetic acid (DTPA; to
incubation at increasing GSH concentrations. The ChT-L
DNa3a7 20SPT activity was inhibited in a dose-dependent
manner by GSH (Table 3). Next, we performed SAXS analyses
of the DNa3a7 20SPT preparations to evaluate whether treat-
ment with 1mM GSH modifies the proteasome conformation.
No alteration in either the external or internal diameters was
detected by comparing samples incubated with GSH or DTT,
trol is dependent on the cysteine (Cys)
redox state. (A) Representative images
obtained by transmission electron mi-
croscopy of nPT-SG in the open con-
analyzed immediately after treatment
with 20mM DTT for 30min followed
by a washing procedure to eliminate
DTT, as described in the Materials and
Methods section. The squares were
amplified as shown on the right. The
combined conformations (open and
closed) were observed in both 20SPT
preparations (nPT-SG and PT-SH), as
shown in Supplementary Fig. S1A and
B (Supplementary Data).
20S proteasomal gating con-
PROTEASOME GATING CONTROL1187
although the maximum length of the DNa3a7 20SPT was
210nm in the sample incubated with GSH and 230nm in the
absence of GSH (Supplementary Table S1). These results indi-
cated that partial inhibition of the ChT-L peptidase activity is
most likely related to an allosteric phenomenon triggered by S-
glutathiolation, which involves changes in the length of 20SPT
that are distinct from the gating.
Collectively, the present results demonstrate that the
S-glutathiolated 20SPT exhibits higher degradation rates
of oxidized and partially unstructured proteins, most likely
because of its gate opening. Based on our results, we hy-
pothesize that the opening of the catalytic chamber, which is
triggered by the thiolation of the proteasomal a5 subunit,
would facilitate the ability of cells to cope with misfolded
proteins without requiring ATP. It is likely that the 20SPT
plays a prominent role in the response to oxidative stress
because various reports have demonstrated that ATP has no
stimulating effect on the degradation of oxidized proteins in
cell lysates (44). Accordingly, Davies and colleagues (45)
concluded that oxidized proteins are degraded by the 20S
proteasome in a manner independent of both the 19S regu-
lator and, consequently, ubiquitin by demonstrating that the
disruption of the ubiquitylation system did not impair the
degradation of oxidized proteins.
According to some reports (10, 22, 26), the so-called latent
form of the free 20SPT is closed. However, in reports de-
scribing the gating of the 20SPT (yeast and human 20SPT), a
mixed pool of open and closed conformations is observed (2,
21, 29, 36–38). Notably, in the protocols described in the lit-
erature, DTT is present in the purification procedure at low
concentrations (0.1–2mM), which would not remove the
glutathionyl moiety from the 20SPT. Among the DTT con-
centrations used in our experiments (not shown), a minimum
of 20mM was necessary for alteration of the gating (Fig. 3B,
Table 1) to occur concomitantly with the disappearance of the
glutathione-modified Cys residues (Fig 4B). As previously
demonstrated, the reduction of the 20SPT inside cells may be
accomplished by oxidoreductases (46).
Glutathiolation is emerging as a relevant post-translational
modification that is involved in redox regulation. In several
proteins under redox control by glutathiolation, only one or
very few Cys residues are involved (34). In the case of the
redox process mediated by peroxides, it is also expected that
few Cys residues exhibit high reactivity toward this oxidant
(53). Once reactive protein thiols are oxidized to sulfenic acid,
oxidation to sulfinic or sulfonic acids and S-glutathiolation
and the formation of intra- and interprotein disulfides or
sulfenylamide derivatives (53). Notably, depending on the
thiol location in the protein (pKa; solvent accessibility) and
the cellular compartment of a given protein, the peroxide-
sensitive thiol targets may be directly oxidized without
disruption to the overall redox homeostasis. Therefore, the
finding that only 2 ofthe 28 Cys residues detected in the20SPT
were S-glutathiolated indicates that this post-translational
modification may represent a redox signaling process. In fact,
it appears that there is a microenvironment appropriate for
GSH binding in the vicinity of Cys221 from the a5 subunit
(Fig. 5B). The other glutathiolated Cys residue (Cys76) is fully
conserved among all of the a5 subunits analyzed (Supple-
mentary Fig. S5), suggesting a prominent role for this amino
acid. Additionally, Cys42 from the a7 subunit (S-glutathiolated
in vitro) is highly conserved (Supplementary Fig. S5).
Interestingly, the activity and gating of the vascular KATP
Table 1. Proteasomal Surface Dimensions Obtained
From Small-Angle X-Ray Scattering Measurements
Outer diameter (A˚)
Pore diameter (A˚)
Length L (A˚)
0 to 10
Table 2. 20S Proteasome S-Glutathiolated Cysteine Residues
The 20SPT preparations were treated with 10mM GSH (PT-SG) or were natively S-glutathiolated (nPT-SG). All subunits containing Cys
residues identified on the two-dimensional electrophoresis gel coupled to MS-fingerprinting were digested with trypsin followed by an
GSH, glutathione; PT-SG, in vitro S-glutathiolated 20S proteasome.
1188SILVA ET AL.
this case, glutathiolation stabilizes the gate in its closed con-
modification that modifies the gate opening of the 20SPT,
thereby regulating the ability of the 20SPT to degrade target
proteins. Our results suggest a need to reevaluate the funda-
mental aspects of the currently favored models of the regu-
lation of proteasomal gating based on the destabilization of
the N-termini of the a3 and a7 subunits (3, 4, 22). The latter
model summarizes the important dynamics of the a subunits
that contribute to gating control upon assembly of the 26S
proteasome, but it does not explain the mechanism that trig-
gers such dynamics of the free 20SPT pool. The mechanism
described here was shown to be closely associated with the
intracellular redox status because cells growing in a less oxi-
dative environment possess proteasomes with low levels of S-
glutathiolation (46), which corresponds to a population of
core particles in the reduced state and with closed gates. The
present model agrees with the idea that during the oxidant-
mediated damage of proteins, degradation may be facilitated
inside cells to avoid energy consumption. The mechanism by
which oxidized proteins interact with the 20SPT particle for
degradation is unknown. A loss of secondary structure and
the consequent increase in the exposure of hydrophobic pat-
ches has been claimed as the phenomena underlying this
process (5, 19, 39, 44). We propose that the redox modification
of the 20SPT under oxidative conditions would facilitate the
proteolytic processing of unstructured proteins, including
oxidized proteins. The critical function of the proteolytic
systems in maintaining a continuous turnover of all intracel-
lular proteins, including those that are still functional, is
necessary to prevent the accumulation of intracellular dam-
age and its associated consequences. This function represents
an important aspect of protein quality control. Limiting the
half-life of the cellular proteins reduces their chances of
damage and avoids their risk for aggregation.
of the majority of proteins implicated in neurodegenerative
processes (43). Because intracellular inclusions that are
the thiol-modified proteasomal Cys
residues obtained by LC-ESI-MS/
MS. LC-ESI-Q-TOF (Waters Synapt
HDMS) analysis of the tryptic
peptide from the 20SPT a5 subunit
spectrum of a triply charged ion
with an m/z ratio of
moiety (+305.1) attached to the Cys
residue. The monoisotopic mass of
the deprotonated peptide (LDEN-
NAQLSCITK) is equal to 1752.82
Da. (B) MS/MS spectrum corre-
shown in A from DTT-treated sam-
[M+2H]2+possesses an m/z ratio of
724.84 and a monoisotopic mass of
1447.67 Da, indicating the reduced
form of the Cys residue. The re-
shown above each spectrum.
Representative spectra of
PROTEASOME GATING CONTROL1189
derived from protein aggregation and associated with neu-
rodegenerative diseases arerich inubiquitylated proteins,it is
assumed that ubiquitin-proteasome system (UPS) impair-
43). Thus, UPS has been investigated extensively in many
neurodegenerative diseases. In fact, there are data suggesting
that the mono-ubiquitylation of protein aggregates is an im-
portant signal to remove aggregates via autophagy (18, 27).
However, processes favoring the ubiquitin-independent re-
moval of oxidized or unstructured proteins that are prone to
aggregation appear to also be an immediate and important
mechanism to avoid neurotoxicity. In fact, the 20SPT directly
interacts with the prion protein (8), soluble oligomers, and
insoluble filaments of a-synuclein or amyloid-Ab peptide
aggregates (32, 55). Additionally, monomeric a-synuclein
is easily degraded by the 20S proteasome in a ubiquitin-
independent manner (33, 50). Therefore, the redox regulation
process described here represents an important aspect of
protein quality control.
Proteasomal S-glutathiolation is a reversible and protective
mechanism that allows for the removal of unstructured
and oxidatively damaged proteins. Because either hyper-
oxidation or complexation to metals of proteasomal Cys resi-
one would predict deleterious consequences from the accu-
mulation ofproteinsprone to aggregationina highlyoxidative
environment. Accordingly, irreversible proteasome oxidation
(9) and metal accumulation (17) are described in the brain of
Alzheimer’s patients together to proteasome impairment.
According to the present results, the increased proteolysis
rates due to proteasomal S-glutathiolation are primarily de-
pendent on the 20SPT gate opening despite the partial inhi-
bition of site-specific activity. According to another line of
investigation in our laboratory, the peptide fragments origi-
nating from degradation of identical proteins differ in their
cleavage sites depending on the proteasomal redox status,
including the generation of immune-competent fragments
(unpublished). Similar to the case of the immuno- and thymo-
proteasomes in their specific contexts, the redox modification
of the standard proteasome might play an important role in
the regulation of cell redox signaling.
Materials and Methods
Bovine serum albumin, casein, casein-FITC, cytochrome c,
DTPA, dimedone, DTT, GSH, and NBD were purchased
from Sigma-Aldrich (St. Louis, MO). The fluorogenic sub-
strate succinyl-Leu-Leu-Val-Tyr-MCA was purchased from
proteasomal structure. (A) Surface structure of the a-ring
highlighting the solvent-accessible sulfur atom (yellow) of
Cys221 from the a5 subunit and the surface oxygen (red)
from the Cys66 residue of the a6 subunit. (B) Modeling of
glutathione docking onto Cys221 of the a5 subunit was
performed by Gold 4.1–Protein-Ligand Docking (Cambridge
Crystallographic Data Centre). The proteasome is shown by
a surface representation, and the glutathione is represented
by sticks. The proteasomal residues interacting with the
GSH-charged groups are highlighted in white in the surface
representation and are depicted as blue sticks underneath.
The sulfur atoms are depicted in yellow. The distances (A˚)
between the GSH-charged groups and the lateral chains of
the proteasomal amino acids are shown. The graphical im-
ages were generatedusing
Location of Cys221 in the a5 subunit in the 3D
Table 3. ChT-L Activity of Purified Preparations
of the DNa3a7 20SPT After Treatment
With Sulfhydryl Reactants
The DNa3a7 20SPT was purified from cells grown to stationary
phase in YPD medium. The assays were performed with 10lg 20SPT
pre-incubated with the reactants for 10min followed by the addition
of 65lM succinyl-Leu-Leu-Val-Tyr-MCA. The results are expressed
as a percentage of the control samples, which are set at 100.
aThe purified DNa3a7 20SPT was oxidized with 5mM H2O2in the
presence of 100lM DTPA. After H2O2 washing, the oxidized
samples were reacted with GSH at increasing concentrations, as
shown. In all experiments, the reactants were removed by cycles of
filtration and re-dilution prior to the next step.
NBD, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole; GSSG, oxidized
1190 SILVA ET AL.
Calbiochem (Merck, Darmstadt, Germany). The molecular
GE Biosciences (GE Healthcare Europe, Glattbrugg, Switzer-
land). The Bradford protein assay reagent was purchased
from Bio-Rad (Hercules, CA).
Yeast strain and growth
The Saccharomyces cerevisiae RJD1144/JD 122 (MATa
::Ylplac211 URA3) strain, derived from the JD47-13C strain,
was kindly donated by Dr. Raymond Deshaies (Division of
Biology, Caltech, Pasadena, CA). The RJD1144 strain con-
tained the 20S proteasome PRE1 gene modified with the
FLAG peptide and a poly-histidine tail sequence (51). The
cells were cultured in YPD medium containing 4% glucose
(referred to as YPD) at 30?C with reciprocal shaking and
harvested after 60h of incubation. The SUB556 strain, de-
rived from SUB62, was kindly donated by Dr. Michael H.
Glickman (Department of Biology, Technion-Israel Institute
of Technology, Haifa, Israel). The 20SPT from the SUB556
strain contains N-terminal deletions in both the a3 and a7
Extraction and purification of the 20S proteasome
The 20SPT from the RJD1144 strain was purified by nickel
affinity chromatography with a continuous gradient of im-
Purifier, GE Healthcare). Neither DTT nor any other thiol
reductant was utilized in any step of the entire purification
procedure, which differs from nearly all other protocols de-
scribed in the literature to date. The final preparations were
passed through a PD10-desalting column according to the
manufacturer’s protocol (GE Biosciences). The untagged
20SPT from the RJD1144 strain and the DNa3a7 20SPT from
the SUB556 strain were purified by conventional chroma-
tography (14). The untagged 20SPT was utilized as a control
relative to the tagged sample.
Reduction and S-glutathiolation of the 20S proteasome
When specified, the preparations of the purified 20SPT
(1mg) extracted from cells grown in YPD-rich medium were
incubated overnight at 4?C with 300mM DTT. Following this
incubation, the proteasome
through a PD10-desalting column according to the manufac-
turer’s protocol (GE Biosciences) to remove the DTT, imid-
azole, and NaCl. The eluted protein fractions were tested for
Only protein fractions in which no DTT was detected were
selected for further procedures. These preparations are herein
referred to as the DTT-reduced 20SPT (PT-SH). To obtain the
in vitro S-glutathiolated protein (PT-SG), aliquots of the native
20SPT (nPT-SG) were incubated at room temperature for
20min in the presence of 10mM GSH in 50mM Tris buffer,
pH 7.5. Following the incubation, GSH was removed by cy-
cles of centrifugation and rediluted through YM-100 micro-
filters. After the determination of the protein concentration,
aliquots of the PT-SH and PT-SG preparations were selected
for additional assays.
PAGE analysis of proteins
SDS-PAGE was performed as previously described (7).
After incubation at the indicated conditions (Fig. 1), the pro-
Tris-HCl [pH 6.8], 10% glycerol, 2% SDS, and 0.02% bromo-
phenol blue) and applied to the gel.
Liquid chromatography-quadrupole-time of flight mass-
spectrometry identification of S-glutathiolated
The trypsin-digested products were analyzed by LC-MS/
MS in a Synapt HDMS instrument (Waters, Millford, MA)
coupled online to a nanoAcquity ultra performance liquid
chromatography system. The digests were loaded and de-
salted using a 180-lm·20-mm Waters Symmetry C18 col-
umn. After the desalting step, the samples were directed to a
100-lm·100-mm Waters BEH130 C18 column at a flow rate
of 1.0lL/min. Mobile phases A and B consisted of 0.1% for-
mic acid/water and 0.1% formic acid/acetonitrile, respec-
tively. The gradient conditions used were as follows: at 0min
the concentration of B started at 3% and increased linearly to
30 % in 20min; the concentration of B then increased up to
70% in 40min and remained at this level until 50min; finally,
in the next minute, the concentration decreased to 3%. The
typical operating conditions of the mass spectrometer in the
data-dependent analysis experiments were as follows: capil-
lary voltage, 3.0kV; cone voltage, 40V; power supply tem-
perature, 100?C; and collision energy, 6 and 4eV in the Trap
and Transfer in the MS mode. The collision energy was se-
lected as a function of the precursor charge and the m/z value.
The instrument was externally calibrated using phosphoric
acid oligomers over an m/z range of 100 to 3000.
Negative staining of the 20SPT particles by TEM
Drops (12lL) of the purified 20SPT preparations (0.5lg/
lL) were applied onto carbon-coated 400 mesh copper grids.
After 1min the excess liquid was blotted with a tissue paper,
leaving a small amount of residual fluid. Negative staining
was performed with 12lL of 2% phosphotungstic acid, pH
7.2, for 10s and then the samples were blotted dry. The grids
were examined in an LEO 906E transmission electron micro-
scope (Zeiss, Germany) at an acceleration voltage of 100kV.
The images were acquired using a CCD camera MegaView III
in conjunction with the iTEM - Universal TEM Imaging Plat-
form software (Olympus Soft Imaging Solutions GmbH,
Germany). Our protocol did not consider the side-on view of
the 20SPT; thus, we did not utilize a vacuum to prepare the
grids (Dr. Edward Morris, personal communication). A
quantitative analysis was manually performed by counting
the frequency of open or closed structures from identical
proteasomal populations. The possibility of saturated images
was excluded because the microscope was operated under
similar light conditions and also because many of the images
obtained showed both closed and open conformations to-
gether, as shown in Supplementary Fig. S2A and B.
Small-angle X-ray scattering
The SAXS experiments were performed using Bruker
NanostarTMequipment (Karlsruhe, Germany). The data
were collected at room temperature using samples of the
PROTEASOME GATING CONTROL1191
nPT-SG, PT-SH (both at 2.2mg/mL), and DNa3a7 20SPT
(0.7–1mg/mL) resuspended in 20mM Tris/HCl, pH 7.5,
buffer. The measurements were obtained using samples
placed in reusable quartz capillaries glued on stainless steel
and the background under the same conditions. Several 1-h
frames were taken to enable the monitoring of the sample
stability. The data treatment, background subtraction and
frame average were performed using the SUPERSAXS pro-
gram package (Oliveira and Pedersen, unpublished). The
complete methodology and data analysis are described in
The GRID methodology (20) was utilized for the docking
analysis of the glutathione (GS-) that was covalently attached
to Cys residues of the proteasome. The 3D structure of the
yeast 20SPT was obtained from the PDB file 1RYP (resolution:
residues were placed in their lowest energy positions, and
their energies were minimized using the Tripos force field
with Pullman charges and conjugate gradient minimization,
keeping all other protein residues rigid. The modeling of
glutathione docking was performed on Gold–Protein-Ligand
Docking (Cambridge Crystallographic Data Center), and all
graphical images were generated using Pymol software (De-
their critical reading of the present work. We are grateful to
Dr. Alberto Malvezzi and Dr. Sylvia Carneiro who helped
with the structural modeling and the electron microscopy
studies, respectively. We thank Adrian Hand for technical
support. This work was supported by Fundac ¸a ˜o de Amparo a
and 07/58147-6) and Instituto Nacional de Cie ˆncia, Tecnolo-
gia e Inovac ¸a ˜o de Processos Redox em Biomedicina (Re-
doxome, CNPq, FAPESP, CAPES).
Author Disclosure Statement
The authors declare no conflict of interest.
1. Asher G, Reuven N, and Shaul Y. 20S proteasomes and
protein degradation ‘‘by default’’. Bioessays 28: 844–849,
2. Babbitt SE, Kiss A, Deffenbaugh AE, Chang YH, Bailly E,
Erdjument-Bromage H, Tempst P, Buranda T, Sklar LA,
Baumler J, Gogol E, and Skowyra D. ATP hydrolysis-
dependent disassembly of the 26S proteasome is part of the
catalytic cycle. Cell 121: 553–565, 2005.
3. Bajorek M, Finley D, and Glickman MH. Proteasome disas-
sembly and downregulation is correlated with viability
during stationary phase. Curr Biol 13: 1140–1144, 2003.
4. Bajorek M, and Glickman MH. Keepers at the final gates:
regulatory complexes and gating of the proteasome channel.
Cell Mol Life Sci 61: 1579–1588, 2004.
5. Baugh JM, Viktorova EG, and Pilipenko EV. Proteasomes
can degrade a significant proportion of cellular proteins in-
dependent of ubiquitination. J Mol Biol 386: 814–827, 2009.
6. Bingol B, and Sheng M. Deconstruction for reconstruction:
the role of proteolysis in neural plasticity and disease. Neu-
ron 69: 22–32, 2011.
7. Bollag DM, and Edelstein SJ. Protein Methods. New York:
8. Cecarini V, Bonfili L, Cuccioloni M, Mozzicafreddo M, An-
geletti M, and Eleuteri AM. The relationship between the 20S
proteasomes and prion-mediated neurodegenerations: po-
tential therapeutic opportunities. Apoptosis 15: 1322–1335,
9. Cecarini V, Ding Q, and Keller JN. Oxidative inactivation of
the proteasome in Alzheimer’s disease. Free Radic Res 41:
10. Coux O, Tanaka K, and Goldberg AL. Structure and func-
tions of the 20S and 26S proteasomes. Annu Rev Biochem 65:
11. Cuervo AM, Wong ES, and Martinez-Vicente M. Protein
degradation, aggregation, and misfolding. Mov Disord 25
Suppl 1: S49–54, 2010.
12. Cunha FM, Demasi M, and Kowaltowski AJ. Aging and
calorie restriction modulate yeast redox state, oxidized
protein removal, and the ubiquitin-proteasome system. Free
Radic Biol Med 51:664–670, 2011.
13. Dange T, Smith D, Noy T, Rommel PC, Jurzitza L, Legendre
A, Finley D, Goldberg AL, and Schmidt M. Blm10 promotes
proteasomal substrate turnover by an active gating mecha-
nism. J Biol Chem, 2011 [Epub ahead of print].
14. Demasi M, Shringarpure R, and Davies KJ. Glutathiolation
of the proteasome is enhanced by proteolytic inhibitors. Arch
Biochem Biophys 389: 254–263, 2001.
15. Demasi M, Silva GM, and Netto LE. 20 S proteasome from
Saccharomyces cerevisiae is responsive to redox modifications
and is S-glutathionylated. J Biol Chem 278: 679–685, 2003.
16. Dixon DP, Skipsey M, Grundy NM, and Edwards R. Stress-
induced protein S-glutathionylation in Arabidopsis. Plant
Physiol 138: 2233–2244, 2005.
17. Duce JA, and Bush AI. Biological metals and Alzheimer’s
disease: implications for therapeutics and diagnostics. Prog
Neurobiol 92: 1–18, 2010.
18. Engelender S. Ubiquitination of alpha-synuclein and au-
tophagy in Parkinson’s disease. Autophagy 4: 372–374, 2008.
19. Ferrington DA, Sun H, Murray KK, Costa J, Williams TD,
Bigelow DJ, and Squier TC. Selective degradation of oxi-
dized calmodulin by the 20 S proteasome. J Biol Chem 276:
20. Goodford PJ. A computational procedure for determining
energetically favorable binding sites on biologically impor-
tant macromolecules. J Med Chem 28: 849–857, 1985.
21. Gregori L, Hainfeld JF, Simon MN, and Goldgaber D.
Binding of amyloid beta protein to the 20 S proteasome. J
Biol Chem 272: 58–62, 1997.
22. Groll M, Bajorek M, Kohler A, Moroder L, Rubin DM, Huber
R, Glickman MH, and Finley D. A gated channel into the
proteasome core particle. Nat Struct Biol 7: 1062–1067, 2000.
23. Groll M, Ditzel L, Lowe J, Stock D, Bochtler M, Bartunik HD,
and Huber R. Structure of 20S proteasome from yeast at 2.4
A resolution. Nature 386: 463–471, 1997.
24. Grune T, Catalgol B, Licht A, Ermak G, Pickering AM, Ngo
JK, and Davies KJA. HSP70 mediates dissociation and re-
association of the 26S proteasome during adaptation to ox-
idative stress. Free Rad Biol Med 51:1355–1364, 2011.
25. Inai Y and Nishikimi M. Increased degradation of oxidized
proteins in yeast defective in 26 S proteasome assembly.
Arch Biochem Biophys 404: 279–284, 2002.
1192SILVA ET AL.
26. Jung T, Catalgol B, and Grune T. The proteasomal system.
Mol Aspects Med 30: 191–296, 2009.
27. Kirkin V, McEwan DG, Novak I, and Dikic I. A role for
ubiquitin in selective autophagy. Mol Cell 34: 259–269,
28. Ko ¨hler A, Cascio P, Leggett DS, Woo KM, Goldberg AL, and
Finley D. The axial channel of the proteasome core particle is
gated by the Rpt2 ATPase and controls both substrate entry
and product release. Mol Cell 7:1143–1152, 2001.
29. Kopp F, Hendil KB, Dahlmann B, Kristensen P, Sobek A,
and Uerkvitz W. Subunit arrangement in the human
20S proteasome. Proc Natl Acad Sci U S A 94: 2939–2944,
30. Kriegenburg F, Poulsen EG, Koch A, Kruger E, and
Hartmann-Petersen R. Redox control of the ubiquitin-
proteasome system: from molecular mechanisms to func-
tional significance. Antiox Redox Signal 15: 2265–2299, 2011.
31. Levine RL, Williams JA, Stadtman ER, and Shacter E. Car-
bonyl assays for determination of oxidatively modified
proteins. Methods Enzymol 233: 346–357, 1994.
32. Lindersson E, Beedholm R, Hojrup P, Moos T, Gai W, Hendil
KB, and Jensen PH. Proteasomal inhibitionby alpha-synuclein
filaments and oligomers. J Biol Chem 279: 12924–12934, 2004.
33. Liu CW, Corboy MJ, DeMartino GN, and Thomas PJ. En-
doproteolytic activity of the proteasome. Science 299: 408–
34. Mieyal JJ, Gallogly MM, Qanungo S, Sabens EA, and Shelton
MD. Molecular mechanisms and clinical implications of re-
versible protein S-glutathionylation. Antioxid Redox Signal
10: 1941–1988, 2008.
35. Niture SK, Velu CS, Bailey NI, and Srivenugopal KS. S-
thiolation mimicry: quantitative and kinetic analysis of re-
dox status of protein cysteines by glutathione-affinity chro-
matography. Arch Biochem Biophys 444: 174–184, 2005.
36. Osmulski PA and Gaczynska M. Atomic force microscopy
reveals two conformations of the 20 S proteasome from fis-
sion yeast. J Biol Chem 275: 13171–13174, 2000.
37. Osmulski PA and Gaczynska M. Nanoenzymology of the
20S proteasome: proteasomal actions are controlled by the
allosteric transition. Biochemistry 41: 7047–7053, 2002.
38. Osmulski PA, Hochstrasser M, and Gaczynska M. A tetra-
hedral transition state at the active sites of the 20S protea-
some is coupled to opening of the alpha-ring channel.
Structure 17: 1137–1147, 2009.
39. Pacifici RE, Kono Y, and Davies KJ. Hydrophobicity as the
signal for selective degradation of hydroxyl radical-modified
hemoglobin by the multicatalytic proteinase complex, pro-
teasome. J Biol Chem 268: 15405–15411, 1993.
40. Peth A, Besche HC, and Goldberg AL. Ubiquitinated pro-
teins activate the proteasome by binding to Usp14/Ubp6,
which causes 20S gate opening. Mol Cell 36:794–804, 2009.
41. Rabl J, Smith DM, Yu Y, Chang SC, Goldberg AL, and
Cheng Y. Mechanism of gate opening in the 20S proteasome
by the proteasomal ATPases. Mol Cell 30: 360–368, 2008.
42. Religa TL, Sprangers R, and Kay LE. Dynamic regulation of
archaeal proteasome gate opening as studied by TROSY
NMR. Science 328: 98–102, 2010.
43. Rogers N, Paine S, Bedford L, and Layfield R. Review: the
ubiquitin-proteasome system: contributions to cell death or
survival in neurodegeneration. Neuropathol Appl Neurobiol
36: 113–124, 2010.
44. Shang F and Taylor A. Ubiquitin-proteasome pathway and
cellular responses to oxidative stress. Free Radic Biol Med 51:
45. Shringarpure R, Grune T, Mehlhase J, and Davies KJ. Ubi-
quitin conjugation is not required for the degradation of
oxidized proteins by proteasome. J Biol Chem 278: 311–318,
46. Silva GM, Netto LE, Discola KF, Piassa-Filho GM, Pimenta
DC, Barcena JA, and Demasi M. Role of glutaredoxin 2 and
cytosolic thioredoxins in cysteinyl-based redox modification
of the 20S proteasome. FEBS J 275: 2942–2955, 2008.
47. Smith DM, Chang SC, Park S, Finley D, Cheng Y, and
Goldberg AL. Docking of the proteasomal ATPases’ car-
boxyl termini in the 20S proteasome’s alpha ring opens the
gate for substrate entry. Mol Cell 27: 731–744, 2007.
48. Tanahashi N, Murakami Y, Minami Y, Shimbara N, Hendil
KB, and Tanaka K. Hybrid proteasomes. Induction by
interferon-gamma and contribution to ATP-dependent pro-
teolysis. J Biol Chem 275: 14336–14345, 2000.
49. Tian G, Park S, Lee MJ, Huck B, McAllister F, Hill CP, Gygi
SP, and Finley D. An asymmetric interface between the
regulatory and core particles of the proteasome. Nat Struct
Mol Biol 18: 1259–1267, 2011.
50. Tofaris GK, Layfield R, and Spillantini MG. alpha-synuclein
metabolism and aggregation
independent degradation by the proteasome. FEBS Lett 509:
51. Verma R, Chen S, Feldman R, Schieltz D, Yates J, Dohmen J,
and Deshaies RJ. Proteasomal proteomics: identification of
mass spectrometric analysis of affinity-purified protea-
somes. Mol Biol Cell 11: 3425–3439, 2000.
52. Wang X, Yen J, Kaiser P, and Huang L. Regulation of the 26S
proteasome complex during oxidative stress. Sci Signal 3:
53. Winterbourn CC and Hampton MB. Thiol chemistry and speci-
ficity in redox signaling. Free Radic Biol Med 45: 549–561, 2008.
54. Yang Y, Shi W, Chen X, Cui N, Konduru AS, Shi Y, Trower
TC, Zhang S, and Jiang C. Molecular basis and struc-
tural insight of vascular K(ATP) channel gating by
S-glutathionylation. J Biol Chem 286: 9298–9307, 2011.
55. Zhao X and Yang J. Amyloid-beta peptide is a substrate of the
human 20S proteasome. ACS Chem Neurosci 1: 655–660, 2010.
Address correspondence to:
Dr. Marilene Demasi
Laborato ´rio de Bioquı ´mica e Biofı ´sica
Avenida Vital Brasil, 1500
Sa ˜o Paulo, SP, CEP: 05503-001
Dr. Luis E.S. Netto
Instituto de Biociencias
Universidade de Sa ˜o Paulo
Rua do Matao, 277
Date of first submission to ARS Central, August 2, 2011; date
of final revised submission, January 4, 2012; date of accep-
tance, January 4, 2012.
PROTEASOME GATING CONTROL1193
20SPT¼20S catalytic unit of the proteasome
DNa3a7 20SPT¼20SPT purified from the SUB556 strain
BSAox¼oxidized bovine serum albumin
Cys-SOH¼cysteine sulfenic acid
Cys-SO2H¼cysteine sulfinic acid
DTPA¼diethylene triamine pentaacetic acid
of flight-mass spectrometry
nPT-SG¼natively S-glutathiolated 20S proteasome
purified from yeast grown in YPD
medium to stationary phase
PAGE¼polyacrylamide gel electrophoresis
PT-SH¼DTT-reduced 20S proteasome
PT-SG¼in vitro S-glutathiolated 20S proteasome
SAXS¼small-angle X-ray scattering
SDS¼sodium dodecyl sulfate
TEM¼transmission electron microscopy
1194SILVA ET AL.