Detection of protein S-sulfhydration by a tag-switch technique

Article abstract of DOI:10.1002/anie.201305876

Protein S-sulfhydration (forming -S-SH adducts from cysteine residues) is a newly defined oxidative posttranslational modification and plays an important role in H₂S-mediated signaling pathways. In this study we report the first selective, tag-switch method which can directly label protein S-sulfhydrated residues by forming stable thioether conjugates. Furthermore we demonstrate that H₂S alone cannot lead to S-sulfhydration and that the two possible physiological mechanisms include reaction with protein sulfenic acids (P-SOH) or the involvement of metal centers which would facilitate the oxidation of H₂S to HS^.. Selective detection: The first selective tag-switch method can be used to directly label protein persulfide units (sites of S-sulfhydration) in the form of stable thioether conjugates. It is thought that H₂S alone cannot lead to S-sulfhydration and that the two possible physiological mechanisms include reaction with protein sulfenic acids (P-SOH) and the involvement of metal centers, which would facilitate the oxidation of H₂S to HS^..

Full text of DOI:10.1002/anie.201305876

Angewandte
Chemie
DOI: 10.1002/anie.201305876
Protein S-Sulfhydration
Detection of Protein S-Sulfhydration by a Tag-Switch Technique**
Dehui Zhang, Igor Macinkovic, Nelmi O. Devarie-Baez, Jia Pan, Chung-Min Park,
Kate S. Carroll, Milos R. Filipovic,* and Ming Xian*
Abstract: Protein S-sulfhydration (forming -S-SH adducts
from cysteine residues) is a newly defined oxidative posttrans-
lational modification and plays an important role in H2S-
mediated signaling pathways. In this study we report the first
selective, “tag-switch” method which can directly label protein
S-sulfhydrated residues by forming stable thioether
conjugates. Furthermore we demonstrate that H2S
alone cannot lead to S-sulfhydration and that the
two possible physiological mechanisms include
reaction with protein sulfenic acids (P-SOH) or
the involvement of metal centers which would
facilitate the oxidation of H2S to HSC.
difficulties in developing selective detection methods for S-
sulfhydration.[4]
So far two methods have been utilized in the detection of
S-sulfhydration (Scheme 1). The first method is a modified
biotin switch technique.[5a] It employs an alkylating agent S-
H
ydrogen sulfide (H2S) has been recently
classified as a critical cell-signaling molecule.[1]
Literature published in the past few years increas-
ingly suggests that H2S is a mediator of many
physiological and/or pathological processes.[2]
Some of these effects are ascribed to the forma-
tion of protein persulfides, or protein S-sulfhydra-
tion (i.e. conversion of cysteine residues -SH to
Scheme 1. Current strategies for profiling protein S-sulfhydration.
persulfides -S-SH). This has been defined as a new oxidative
posttranslational modification (oxPTM).[3,4] Formation of
persulfides is potentially significant because it provides
a possible mechanism by which H2S alters the functions of
a wide range of cellular proteins and enzymes.[5] To date, the
underlying mechanisms of S-sulfhydration mediated by H2S
are still unclear.[3,4] A significant challenge is that the
persulfide group (-S-SH) shows reactivity akin to that of
other sulfur species, especially thiols (-SH), which causes
methyl methanethiosulfonate (MMTS) to differentiate thiols
and persulfides. Thiols (-SH) in proteins are first blocked by
MMTS. Persulfides (-S-SH) are believed to remain unreacted
and be available for subsequent conjugation to N-[6-(bio-
tinamido)hexyl]-3ꢀ-(2ꢀ-pyridyldithio)propionamide (biotin-
HPDP). Using this method, a large number of proteins
were identified as targets for S-sulfhydration and the basal
sulfhydration level of some proteins was estimated to be as
high as 25%. In the second method,[5c] it suggested that both
-SH and -SSH units can be blocked by alkylating reagents like
iodoacetic acid (IAA). Then the persulfide adducts can be
reduced by dithiothreitol (DTT) to form free -SH groups, and
subsequently labeled with iodoacetamide-linked biotin
(IAP).
[*] Dr. D. Zhang,[+] Dr. N. O. Devarie-Baez, Dr. C.-M. Park, Prof. M. Xian
Department of Chemistry, Washington State University
Pullman, WA 99164 (USA)
From a chemistry perspective, both methods are problem-
atic. In Method 1, the underlying mechanism of selectivity of
MMTS for thiol versus persulfide is unclear. Studies have
demonstrated that persulfides and thiols should have similar
reactivity towards electrophiles such as MMTS.[4] In
Method 2, it is unclear how DTT reduction would distinguish
persulfide modifications from other DTT-reducible residues,
such as disulfides and S-nitrosothiols.
E-mail: mxian@wsu.edu
I. Macinkovic,[+] Dr. M. R. Filipovic
Department of Chemistry and Pharmacy
University of Erlangen-Nuremberg
Erlangen (Germany)
E-mail: milos.filipovic@fau.de
Dr. J. Pan, Prof. K. S. Carroll
Department of Chemistry, The Scripps Research Institute
Jupiter, FL 33458 (USA)
Therefore, the chemical foundations of current methods
are questionable, which may lead to erroneous results.
Apparently more reliable methods for the detection of
protein S-sulfhydration are needed. Having realized the
very similar reactivity of both thiols and persulfides, we
proposed a tag-switch technique to detect S-sulfhydration.
Herein we report the development and application of this
method.
[+] These authors contributed equally to this work.
[**] M.X. thanks the NSF-CAREER Program (0844931) and the American
Chemical Society (Teva USA Scholar Award). M.R.F. and I.M. thank
the University of Erlangen-Nuremberg within Emerging Field
Initiative (EFI-MRIC) for support. K.S.C. thanks NIH R01
GM102187.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201305876.
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from persulfide 2. This should not be a concern in
the case of protein persulfides because polysulfide
formation is not expected to occur easily on
protein persulfides.
We next used a cysteine substrate 4 to screen
the appropriate nucleophile for the tag-switch step
(Table 1). The preparation of 4 is shown in
Scheme S2 in the Supporting Information. It
should be noted that R-S-S-BT products like 4
Scheme 2. Proposed tag-switch technique for detecting S-sulfhydration.
As illustrated in Scheme 2, we proposed that S-sulfhydra-
tion can be selectively detected by the tag-switch method (i.e.
using two reagents to label protein persulfides in two steps).
In the first step a SH-blocking reagent will be introduced and
it should tag both -SH and -SSH to form intermediate T. If an
appropriate tag is employed, the disulfide bonds in persulfide
adducts may show much enhanced reactivity to certain
nucleophiles relative to the reactivity of common disulfides
in proteins. Therefore we could introduce a tag-switching
reagent (containing both the nucleophile and a reporting
molecule such as biotin) to label only the persulfide adducts.
It should be noted that thiol adducts from the first step are
thioethers, which are not expected to react with the nucleo-
phile.
Table 1: Screening nucleophiles for the tag-switch step.
A major challenge in this technology is whether the newly
generated disulfide linkage from persulfide moieties can
display a unique reactivity for a suitable nucleophile to an
extent that distinguishes them from common disulfides. SH-
blocking reagents are well known.[6] However, those fulfilling
the criteria for this assay are limited. For example, irreversible
thiol-blocking reagents such as maleimides and iodoaceta-
mides displayed good selectivity and fast reactivity for
thiols.[6] If such reagents react with persulfides, alkyl disulfide
adducts are produced and their reactivity should not differ
from that of cysteine or glutathionylated protein disulfides.
Therefore, these reagents are not suitable for tag-switch. We
envisioned that a reagent, upon reaction with persulfides to
give a mixed aromatic disulfide linkage, could meet the
reactivity criteria. One potential candidate is methylsulfonyl
benzothiazole (MSBT), a thiol-blocking reagent recently
developed by our group.[7] We expected the disulfides
generated from MSBT and persulfides should be highly
activated and exert a unique reactivity with certain nucleo-
philes, in particular, enolates.[8]
With this idea in mind, we first tested the reaction
between MSBT and persulfide substrates. Since MSBT is
a very effective SH-blocking reagent[7] and persulfides
(-S-SH) are known to have very similar reactivity to thiols,[4]
we expected MSBT should effectively block persulfides.
However, it is known that small-molecule persulfides are
very unstable species.[9] We could not use purified/isolated
persulfides in the experiments. Instead we attempted several
approaches to generate persulfides in situ from precursors
like 1 (Scheme S1 in the Supporting Information) and used
the persulfide intermediates directly in MSBT blocking.
Indeed we obtained the desired product 3, although in low
yield (13%). This result demonstrated that MSBT can react
with persulfides to form R-S-S-BT adducts. The major
product in the reaction was found to be polysulfides derived
[a] Bn=Benzyl.
are quite stable. They do not react with potential nucleophilic
groups such as -OH and -NH2 (Scheme S3 in the Supporting
Information). We screened a series of carbon-based nucleo-
philes as potential candidates. As shown in Table 1, three
reagents (dimedone, malononitrile, and methyl cyanoacetate)
proved to be effective and the corresponding products 5b, 5e,
and 5 f were obtained in valuable yields. The reactions were
also found to be fast (completing within 20 min). Among
these candidates, methyl cyanoacetate (MCA; Table 1,
entry 6) was particularly attractive as the ester group could
allow easy installation of reporting molecules. Therefore
MCA was selected in subsequent studies.
Given the dramatic structure changes in protein persul-
fide substrates, we wondered whether MCA could effectively
react with different R-S-S-BT substrates. The reaction scope
was then studied using a series of cysteine-S-S-BT derivatives
(Scheme 3). Pleasingly, the reaction was found to be highly
effective. In all cases the substitution products were afforded
in good yields.
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Scheme 3. Scope of the reaction of MCA with R-S-S-BT derivatives.
Cbz=carbobenzyloxy.
If MCA is used to specifically label protein persulfide
derived R-S-S-BT moieties, it is critical to prove that MCA is
inert towards common disulfides. We thus carried out several
control experiments (Scheme S5 in the Supporting Informa-
tion). We first examined the reactivity of MCA against Cys
disulfide 8 (Boc = tert-butyloxycarbonyl). Under the tag-
switch reaction conditions the corresponding product was
not observed, even after hours. We also checked the reactivity
of MCA toward S-nitrosothiol 9, which represents another
well-known thiol modification in proteins. Again, no reaction
was observed. Finally a crossover experiment using both
R-S-BT 10 (derived from thiols) and R-S-S-BT 4 (derived
from persulfides) was tested. We only observed product 5 f
(from 4). The thiol-derived substrate 10 was unreactive and
could be fully recovered. These results suggested that the
proposed tag-switch method was selective for persulfides.
Figure 1. MS analysis of the tag-switch assay with Gpx3-persulfide.
A) The control reaction between Gpx3 persulfide and ethyl cyanoace-
tate (without MSBT-A). B) The reaction between Gpx3 persulfide and
ethyl cyanoacetate (with MSBT-A).
detected by dot blot or ESI-TOF MS (Figure 2C and
Figure S1 in the Supporting Information).
Although a few studies have suggested S-sulfhydration to
be a potential posttranslational modification mediated by H2S
that could regulate protein function,[5] there is no information,
to date, dealing with the mechanism(s) underlying its
formation. From a chemistry perspective, the direct reaction
of protein thiols (-SH) with H2S would not be possible.
However, the intermediate role of oxygen and metal centers
as well as the reactions with other posttranslational modifi-
cations of cysteine could be possible.
Based on the mechanistic studies for S-glutathionyla-
tion[3,11] we addressed the following hypothetical reactions, as
potential paths for P-SSH/PSSꢀ generation under physiolog-
ical conditions [Eq. (1)–(6)].
The results shown above demonstrate the chemical
foundation of the tag-switch method. We then tested it in
protein samples. Gpx3, an established protein-S-SH model,[4]
was used in this experiment. Freshly prepared Gpx3 persul-
fide was treated with MSBT-A, a water-soluble MSBT
derivative,[7] followed by the addition of cyanoacetate. The
protein was then purified and analyzed by LC-MS. As shown
in Figure 1B, cyanoacetate-labeled protein was clearly iden-
tified by MS. In the control (Figure 1A, without MSBT-A),
we did not observe the peak for the cyanoacetate-labeled
protein. An oxidative byproduct (P-S-SO3H) was observed in
both samples and this is common for Gpx3 based on our
previous experience.[4]
P-SH þ H2S þ O2 ! P-S-SH þ H2O2
P-S-S-R þ H2S ! P-S-SH þ RSH
P-S-OH þ H2S ! P-S-SH þ H2O
P-S-NO þ H2S ! P-S-SH þ HNO
P-SðHÞNOH þ H2S ! P-S-SH þ NH2OH
ð1Þ
ð2Þ
ð3Þ
ð4Þ
ð5Þ
ð6Þ
We next tested the selectivity of tag-switch assay towards
different oxPTMs. A biotin-linked cyanoacetate (CN-biotin)
was prepared and used in this study. A relatively stable
sulfenic acid derivative of bovine serum albumin (BSA-SOH)
was prepared[10] and in its reactions with glutathione[10,11] and
H2S the corresponding glutathionylated and S-sulfhydrated
derivatives were generated (Figure 2A,B). Neither BSA-SH,
BSA-SOH, nor BSA-SSG gave positive signals in the tag-
switch assay. Only in the case of BSA-SSH could biotinylated
product be pulled down by streptavidin agarose beads and
C
C
P-SH þ HS þ O2 ! P-S-SH þ HO2
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We tested the reaction
pathways (1), (3), (5), and
(6) using glyceraldehyde
3-phosphate dehydrogen-
ase
a
(GAPDH)
model. Angeliꢀs salt
as
was used as a donor of
HNO[18] to form the
(hydroxyamino)sulfanyl
derivative. Formation of
P-SOH was induced by
reaction with H2O2. Pro-
teins were also treated
with supraphysiological/
pharamcological concen-
trations of H2S alone and
H2S in combination with
rigorous
shaking
to
Figure 2. Testing the selectivity of the tag-switch assay. A) Schematic description of the tag-switch assay.
B) Preparative procedure for generating different oxPTMs of BSA. C) Percentage of the unbound protein after
treatment with streptavidin agarose beads. Samples 1, 3, 5, and 7 are untreated samples of BSA-SH, BSA-SOH,
BSA-SSG, and BSA-SSH, respectively. Samples 2, 4, 6, and 8 are BSA-SH, BSA-SOH, BSA-SSG and BSA-SSH,
respectively, treated with tag-switch reagents. n=3, *p<0.001. Inset shows the dot blot detection of successful
biotinylation.
increase the oxygenation
of the solution. As
a source of HSC we used
a combination of water-
soluble ferric porphyrin
and H2S.[19] Only when
The reaction shown in Equation (1) is only the sum of the
multiple reaction steps that could occur during the sponta-
neous oxidation of H2S where HSC is a possible intermedi-
ate[12] that would then lead to formation of S-sulfhydrated
protein by means of the reaction shown in Equation (6).
The reduction of a disulfide bond by H2S [Eq. (2)] is
thermodynamically unfavorable.[13] Based on the calculation
of the bond energies of GSSG and GSSH, the bonding energy
in the latter is roughly 18 kJmolꢀ1 lower.[14] The reaction with
sulfenic acids [Eq. (3)] does occur, as demonstrated in
Figure 2B. S-Nitrosothiols would react with H2S to give
HSNO, rather than to form HNO and the corresponding S-
sulfhydrated protein as we previously demonstrated (with
Drxn1G8 =+ 40 kJmolꢀ1).[14] However, a recent computational
H2S reacted with P-SOH or when HSC was generated with
the iron center was PS-SH detectable (Figure S2 in the
Supporting Information). When BSA was used as a model of
the protein with intramolecular disulfide bonds, no S-sulfhy-
dration was observed upon addition of H2S (data not shown),
confirming the low reducing power of free H2S. It is worth
mentioning that an alternative mechanism could lead to
protein persulfide formation, such as the reaction with
polysulfides,[5g] although their physiological relevance
remains to be elucidated.
As a proof of concept that the method can be applied to
more complex systems such as the intracellular environment,
we attempted to label proteins by the tag-switch technique in
cell extracts. Protein extracts from control and Jurkat cells
treated with 200 mm H2S (using Na2S as the equivalent) for
30 min at 378C were labeled by the tag-switch method,
resolved by SDS-PAGE, transferred to a nitrocellulose mem-
brane, and identified by anti-biotin horse radish peroxidase
(HRP)-conjugated antibodies. Representative Western blots
(Figure 3A–C) demonstrated that a small number of proteins
showed positive signals, confirming the existence of endog-
enous S-sulfhydration. The treatment with Na2S increased the
signal intensity, but did not significantly affect the total
number of S-sulfhydrated proteins. Importantly, when cell
lysates were treated with streptavidin agarose beads and
subsequently analyzed, no biotinylated proteins could be
detected, suggesting that all modified proteins could be pulled
down and the method used for the further proteomic analysis
(for example, see Figure S3A in the Supporting Information).
It has been reported previously that GAPDH could be
one of the major targets for S-sulfhydration.[5a,b] Indeed, when
we attempted to identify GAPDH with specific antibodies we
found that this protein was endogenously S-sulfhydrated,
although the signal was much stronger after the treatment
with H2S (Figure 3C).
ꢀ
study pointed out that the surrounding of the S NO bond
could significantly affect the thermodynamic feasibility of the
reaction shown in Equation (4), making the reaction possible
for certain proteins that have positively charged amino acids
[15]
ꢀ
in close proximity to the S NO bond.
The reaction of nitroxyl (HNO), a redox sibling of NO
with distinct signaling pathways,[16] with a protein thiol leads
to the formation of a (hydroxyamino)sulfanyl derivative. It is
that this (hydroxyamino)sulfanyl derivative then reacts with
other thiols with elimination of hydroxylamine as shown in
Equation (5).[16]
Finally, a possible way to form S-sulfhydrated proteina is
the reaction of HSC with protein thiols, where initially PSSHC is
formed, which subsequently reacts quickly with O2 to give
O2Cꢀ and PSSH [Eq. (6)], as observed in the formation of S-
glutathionylated proteins.[11] Formation of HSC under physio-
logical conditions would require interaction with oxidized
metal centers, such as those in ferrioc heme porphyrins, which
would be reduced forming HSC by means of inner-sphere
electron transfer.[17]
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lysates used in the experiments shown in Figure 3 were
additionally exposed to 200 mm H2S, much stronger overall
sulfhydration was detected than when living cells were treated
(Figure S3B in the Supporting Information).
Finally, in a preliminary set of experiments we tried to
adapt the tag-switch technique for the in situ labeling of
methanol- or paraformaldehyde-fixed cells. Human umbilical
vein cells (HUVECs), previously exposed to 100 mm Na2S for
30 min or to 2 mm propargylglycine, a cystathionine gamma
lyase inhibitor,[14] for 2 h, were fixed with ice-cold methanol.
Free thiols were blocked with MSBT-A and the protein S-
sulfhydration (protein persulfides) tagged with CN-biotin.
Finally, the cells were exposed to fluorescein-labeled strepta-
vidin. A similar protocol was used with cells treated with Na2S
or 2-ketobutyric acid (inhibitor of mercaptopiruvate S-trans-
ferase, a mitochondrial enzyme for H2S production), but with
the initial difference that they were fixed with paraformalde-
hyde.
As shown in Figure 4A,B, detectable S-sulfhydrated
proteins increased in HUVECs treated with H2S. Partial
inhibition, relative to the control, was achieved by treatment
with propargylglycine but it was almost completely abolished
by the use of 2-ketobutyric acid, implying the essential role of
mitochondrially produced H2S, as suggested previously.[21]
Almost complete absence of the signal in 2-ketobutyric acid
treated cells also confirms that the nonselective binding of the
fluorescent probe, incomplete blocking of free thiols, and/or
unselective background fluorescence are not contributors of
the main fluorescence signal. In addition, the pretreatment of
the fixed cells with dimedone did not affect the detection of
intracellular persulfides (Figure S4 in the Supporting Infor-
mation). These data suggest that the tag-switch assay is
selective for P-S-SH in the presence of P-S-OH. It should be
noted that the reactivity of sulfenic acids toward carbon
nucleophiles like cyanoacetate may change depending on the
protein enviroment. However, even if certain P-S-OH groups
would react with cyanoacetate, samples could always be
pretreated with dimedone to remove the false signals from P-
S-OH, as we previously demonstrated that dimedone reacts
with P-S-OH but not with P-S-SH.[4]
Higher magnification microscopy (100 ꢁ) gave us some
indication of the intracellular distribution of the signal
(Figure 4D). The perinuclear localization of the signal is
indicative of localization in the mitochondrion and/or endo-
plasmic reticulum (ER). Since the signal in the cells was less
diffused with methanol fixation we used it for co-localization
studies. Mitochondrial and ER trackers proved that most of
the detected persulfide signal is localized within these two
organelles, within the ER predominantly (Figure S5 in the
Supporting Information).
Figure 3. Detection of protein S-sulfhydration in cell lysates by the tag-
switch assay. A) Jurkat cell lysates of the control (lane 1) and H2S-
treated cells (lane 2) (200 mm Na2S, 30 min, 378C) analyzed by the tag-
switch assay. In parallel the same cell extracts were tested by Western
blot analysis for the presence of Hsp70, which appeared at exactly the
same position as the strongest S-sulfhydrated band. B) Quantification
of the S-sulfhydration levels in the control and H2S-treated cells based
on the intensity of the band at 70 kDa. C) Detection of GAPDH as
a standard S-sulfhydrated protein.
The most prominent S-sulfhydration was detected on
a protein with a molecular weight of roughly 70 kDa. Using
antibodies specific for heat shock protein 70 (Hsp70), we
demonstrated that this protein is most likely Hsp70 (Fig-
ure 3A). Hsp70 is of great pharmacological interest[20a] and
recent studies showed that it could serve as a redox sensor
through oxidation of its cysteines by sulfenylation (P-
SOH),[20b] which can also explain how Hsp70 could form
persulfides [Eq. (3)].
As we suggested in Figure S2 in the Supporting Informa-
tion and in Equation (6), metal-assisted generation of P-SSH
could be the predominant way for forming P-SSH, but also
a source of its artificial formation. Indeed when Jurkat cell
Taken together, these data offer a new, selective method
for the detection of protein S-sulfhydration. Our results
demonstrate that carbon-based nucleophiles such as cyanoa-
cetate do not react with common disulfides in proteins, but
with highly chemically activated disulfide species. Some
mechanistic insight into physiological mechanisms for the
formation of protein S-sulfhydration is presented, suggesting
that the metal-center-assisted oxidation of H2S could be
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Figure 4. In situ fluorescence detection of intracellular S-sulfhydration of proteins.
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a control. Treatments were as follows: 100 mm Na2S (30 min, 378C), 2 mm propargyl-
glycine (PG; 2 h, 378C), or 2 mm 2-ketobutyric acid (2 h, 378C). D) Fluorescent
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