Minireview:
The Redox Biochemistry of Protein
Sulfenylation and Sulfinylation
Mauro Lo Conte and Kate S Carroll
J. Biol. Chem. published online July 16, 2013
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The Redox Biochemistry of Protein Sulfenylation and Sulfinylation*
Mauro Lo Conte, Kate S. Carroll1
From the Department of Chemistry, The Scripps Research Institute, Jupiter, Florida, 33458
1
To whom correspondence should be addressed: Kate S. Carroll, The Scripps Research Institute, Florida
130 Scripps Way, #2B2, Jupiter, FL 33458, Email: kcarroll@scripps.edu, Phone: (561) 228-2460, Fax:
(561) 228-2919
* This work was supported by National Institutes of Health grant R01GM102187 (to K.S.C.).
nucleophilic
thiolate
(RS–).
Accordingly,
susceptibility to oxidation is usually correlated
with pKa, although for cysteines having pKa<7, the
RS– becomes less nucleophilic with the decrease
of pKa value (3). In proteins, microenvironments
can influence Cys acidity through the presence of
polar amino acids or specific hydrogen bonds,
which contribute to a decrease in pKa by balancing
the negative charge on the sulfur atom (4). The
same interactions, which affect the pKa of Cys
thiol, also influence the stability of the related
sulfenic acid. The microenvironment can also help
to stabilize the leaving group by lowering the
transition-state energy barrier (2). However, these
parameters are not sufficient to rationalize the
selective oxidation of specific proteins. Increasing
evidence shows that ROS signaling responses are
compartmentalized, and the proximity of the target
protein to the ROS source is a key aspect of spatial
regulation of Cys oxidation (5,6).
By virtue of the transient nature of RSOH, the
study of its chemical-physical properties has been
rendered extremely challenging. The pKa of RSOH
has been determined in only a few proteins (7,8).
The experimentally determined pKa of some smallmolecule sulfenic acids is one/two orders of
magnitude lower than the corresponding thiols
(9,10); however, it is not clear whether such
compounds are appropriate models of cysteine
sulfenic acid in proteins. From the chemical point
of view, RSOH exhibits both electrophilic and
nucleophilic behavior. Thiosulfinate formation
clearly exemplifies this dual nature (11), although
this self-condensation has little biological
relevance due to high abundant thiols and steric
hindrance which make this reaction negligible in
cells. Therefore, oxidation to Cys-SO2H appears to
be the only significant reaction in which RSOH
Controlled generation of reactive oxygen
species
(ROS)
orchestrates
numerous
physiological signaling events (1). A major
cellular target of ROS is the thiol side-chain
(RSH) of cysteine (Cys), which may assume a
wide range of oxidation states (i.e., -2 to +4).
Within this context, Cys sulfenic (Cys-SOH)
and sulfinic (Cys-SO2H) acids have emerged as
important mechanisms for regulation of protein
function. Although this area has been under
investigation for over a decade, the scope and
the biological role of sulfenic / sulfinic acid
modifications have been recently expanded
with the introduction of new tools for the
monitoring of cysteine oxidation in vitro and
directly in cells. This review discusses selected
recent examples of protein sulfenylation and
sulfinylation from the literature, highlighting
the
role
of
these
post-translational
modifications (PTMs) in cell signaling.
SULFENIC ACID
REACTIVITY
FORMATION
AND
RSOH is directly generated by the oxidation of
RSH with two-electron oxidants (Figure 1A).
Hydrogen peroxide (H2O2) reacts with smallmolecule thiols at a constant rate of around 20 M-1
s-1, but this reaction can take place up to eight
orders of magnitude faster (105 - 108 M-1 s-1) with
specific Cys residues within proteins (2). The
propensity of Cys residues to undergo oxidation is
mainly influenced by three general factors: thiol
nucleophilicity,
surrounding
protein
microenvironment, and proximity of the target
thiol to the ROS source. Peroxide-mediated thiol
oxidation is an SN2 reaction (Figure 1B) whereby
the actual reactive species is the much more
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exhibits its nucleophilic nature. On the other hand,
this species shows high reactivity toward
nucleophiles. Intramolecular or low-molecular
weight thiols may react with RSOH to generate a
mixed disulfide, which constitutes the principal
mechanism for disulfide bond formation in
proteins (12). In the absence of adjacent thiols,
RSOH can also react with nitrogen nucleophiles to
form a sulfenamide, though this species has been
identified in only a few proteins (13,14).
DBPs, have identified many other proteins that are
able to generate an RSOH-transient species in
cells (18,19), although the biological relevance of
these oxidations remains unclear. Table 1 clearly
demonstrates that many redox-regulated proteins
are directly involved in cell signaling.
Protein Tyrosine Phosphatases
Tyrosine phosphorylation levels are maintained
by the balanced action of protein tyrosine kinases
(PTKs)
and
phosphatases
(PTPs).
The
sulfenylation of the PTPs’ catalytic Cys (pKa
ranges from 4 to 6) has emerged as a dynamic
mechanism for inactivation of this protein family
(20). The half-life of RSOH is generally quite low
in PTPs. In fact, a neighboring cysteine residue
(e.g., in PTEN) or the backbone amide nitrogen
(PTP1B) readily reacts with RSOH to yield,
respectively, an intramolecular disulfide (21) or a
cyclic sulfenamide species (14).
Recently, an alternative mechanism of
inactivation emerged in SH2 domain-containing
PTPs (SHP-1 and SHP-2) (22), which possess two
highly conserved distal cysteines, both of which
can generate a disulfide with the oxidized catalytic
Cys. This intermediate disulfide typically
rearranges into the more stable disulfide formed
by the two backdoor cysteines to regenerate the
free catalytic Cys residue. Surprisingly, the
conformational change produced by the -backdoor
disulfide leads to an increased catalytic Cys pKa
value (~ 9) with resultant inhibition.
Although SHP-1 and SHP-2 have structural
similarities, they are regulated by different cellsignaling pathways. SHP-2, for example, exhibits
selective oxidation in response to platelet-derived
growth factor (PDGF) in association with a PDGF
receptor (23). However, these regulatory
differences can be influenced by the method used
to analyze their oxidation state. Employing an
indirect RSOH detection method, T-cell
activation, which induces H2O2 production, has
shown transient oxidation of SHP-2 but not of
SHP-1 (24). In a later work in which a DBP was
used, both SHP-1 and SHP-2 showed Cys
oxidation after T-cell activation, although with a
different response time (25). These results could
be rationalized by the greater sensitivity of direct
RSOH analysis.
In vitro experiments have demonstrated that
H2O2 deactivates SHP-1 and SHP-2 with second-
Sulfenic Acid as a Post-Translational
Modification
Disulfide and sulfenamide formations protect
Cys-SOH from further oxidation and lay the
foundation for redox signaling. In fact, these
PTMs can generate conformational changes in
protein structure and subsequent modulation of
protein activity. In addition, as a result of its
intrinsic nucleophilicity, Cys is present in the
active site of many enzymes. Transient oxidation
of these Cys residues is a well-established process
through which proteins can be spatially and
temporally inhibited.
From the first evidence of its existence reported
in 1976 (15) to the present, RSOH has been
identified in a relatively small number of proteins.
In fact, the identification of this elusive
modification remains difficult. In 2008, Fetrowet
al. published a review that included a list of 47
proteins in which Cys-SOH was identified by
crystal-structure
analysis
(16).
Because
identification of the crystal structure of proteinSOH is problematic, Fetrow et al.’s list represents
only the tip of the iceberg. Direct mass analysis
shows similar issues, making the use of chemical
probes the only suitable technique to monitor
RSOH formation (17).
Table 1 provides a list of the principal proteins
in which Cys-SOH has been identified using
chemical-trapping reagents. In addition to NBD-Cl
(4-Chloro-7-nitrobenzofurazan), which can be
employed only in vitro, dimedone-based probes
(DBPs) are emerging as the most promising tool
for RSOH trapping. These reagents are capable of
crossing the cellular membrane, capturing RSOHs
directly in the cell (17). We concentrated our
attention exclusively on those proteins where the
formation of Cys-SOH has been experimentally
substantiated and shown to play a regulatory role.
Proteomic studies, based on the employment of
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order rate constants of 2.0 M-1 s-1 and 2.4 M-1 s-1
respectively (22). These values are similar to those
observed with other PTPs and are apparently too
low to justify their oxidation within the cellular
context. Recently, our group has observed that,
following epidermal growth factor (EGF)
stimulation, SHP-2 forms a complex with the EGF
receptor (EGFR) and Nox2 (6), which could
provide an explanation to its highly propensity to
oxidation. In a similar manner, PTP1B, which is
localized exclusively on the cytoplasmic face of
the endoplasmic reticulum (ER), appears to be
oxidized through the H2O2 generated by Nox4, an
NADPH oxidase highly abundant in the ER (5).
These two examples highlight the importance of
the proximity of the target protein to the ROS
source in explaining PTP oxidation.
domain, was found to be susceptible to
sulfenylation. Cys124-SOH can generate a
disulfide with two distinct Cys residues – Cys297
and Cys311 – located in the kinase domain. This
modification negatively modulates Akt2, although
in vitro experiments showed that disulfide
formation has no direct effect on kinase activity.
The inhibition mechanism remains unclear, but a
previous work showed that Akt oxidation
enhances its association with protein phosphatase
2A
(PP2A),
which
could
promote
dephosphorylation of Akt (29).
Transcription Factors
In addition to the redox switch of PTKs and
PTPs activities, which indirectly regulate
transcription factors (TFs), H2O2 can directly
modulate several TFs through the formation of
intra- and intermolecular disulfide bonds(30). The
first evidence of a redox-sensitive TF was
identified in OxyR, a bacterial transcription factor,
in which Cys-SOH mediated disulfide bond
formation between Cys199 and Cys208 (31).
Many other TFs are redox-regulated in
prokaryotes (32-36), but relatively few cases have
been identified in eukaryotes. In yeast, the
activation of Yap1 represents an interesting case
of TF redox regulation in which Gpx3-SOH
mediates the oxidation of Yap1 through the
formation of Gpx3-Yap1 intermolecular disulfide
(37,38).
The anti-apoptotic NF-kB remains the only
mammalian TF in which formation of Cys-SOH
has been verified experimentally; however, this
modification may also have a role in other
peroxide-sensitive pathways of gene activation,
such as the Nrf2/Keap-1 system (39). H2O2
negatively switches NF-κB’s DNA affinity –
directly through the oxidation of its p50 subunit at
Cys62 (40) and indirectly via Cys179
sulfenylation of the β subunit of the IKK complex
(IKKβ), the kinase that is responsible for the NFκB activation along the canonical pathway (41).
Kinases
Increasing research has highlighted the key role
of H2O2 in the modulation of PTKs activity. In
comparison with PTPs, which are always inhibited
by ROS, the oxidation of PTKs can lead to both
enhancement and inhibition of kinase activity (26,
27).
The central role of Cys oxidation in PTK
activity is exemplified by the redox control of
EGFR signaling. EGFR is a receptor tyrosine
kinase (RTK) activation of which is involved in
the
regulation
of
cellular
proliferation,
differentiation, and survival. In addition to
promoting the tyrosine phosphorylation of protein
targets, EGFR stimulation triggers the production
of endogenous H2O2 by Nox activation. This
localized increase in H2O2concentration leads to
the sulfenylation of a conserved Cys residue
located within the intracellular kinase domain of
EGFR (Cys797), which enhances its tyrosine
kinase activity (6). The redox regulation of EGFR
could represent a more general mechanism for the
modulation of other RTK activity. In fact, nine
additional members of this family show a Cys
structurally analogous to EGFR Cys797, although
further studies are needed in this direction.
Recently, Akt (a serine/threonine protein
kinase) was also identified as a redox target.
PDGF stimulation of fibroblasts induced H2O2
production, which led to isoform-specific
regulation of Akt2 (28). A cysteine (Cys124),
positioned in the linker region connecting the
pleckstrin homology (PH) domain to the kinase
Cysteine Proteases
Protein ubiquitination has emerged as a central
PTM whereby lysine residues are conjugated to
ubiquitin (Ub), a 76 amino acid polypeptide (42).
Deubiquitinating
enzymes
(DUBs)
cleave
ubiquitin or ubiquitin-like proteins from the target,
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contributing to the balance of the Ub system. Four
of the five different families of DUBs are cysteine
proteases, which share in common a low-pKa Cys
residue essential for the catalytic mechanism.
Recently, three distinct works have shown that
Cys oxidation can modulate DUB activity. CottoRios et al. reported transient sulfenylation of
catalytic Cys for several members of the Ubspecific protease (USP) family and for UCH-L1
(43). In particular, the authors were able to
establish that USP-1, a DUB involved in DNA
damage-response
pathways,
is
reversibly
inactivated following the induction of oxidative
stress in cells. Additionally, Komander, in
collaboration with our group, demonstrate that
many members of the ovarian tumor (OUT) DUBs
also undergo Cys oxidation upon H2O2 treatment
(44), including the tumor suppressor A20. Crystalstructure analysis of oxidized A20 showed that
transient RSOH can be stabilized by the formation
of hydrogen bonds with the highly conserved
residues located in the loop preceding catalytic
Cys. Both works noted that each DUB member
exhibits a distinct level of sensitivity to oxidation.
Differences in behavior can reflect various ranges
of catalytic activation in which the conformational
inactive enzyme could be less susceptible to
oxidation. Lee et al. confirmed this hypothesis by
showing that pre-incubation of USP7 with
ubiquitin, which behaves as an allosteric activator,
increased USP7 sensitivity to ROS (45).
An analogous inhibition has been found in
small Ub-like modifier (SUMO) proteases. H2O2
treatment induces RSOH-mediated formation of
an intermolecular disulfide in the yeast SUMO
protease Ulp1 as well as in its human equivalent,
SENP1 (46). Interestingly, SUMOylation also
appears to be redox-regulated by reversible
oxidation of the catalytic Cys of SUMO
conjugating enzymes (47), although no clear
evidence of Cys-SOH formation has been
provided.
oxidation of the extracellular Cys195 (49),
although the nature of this Cys oxidation remains
unknown.
One exception is represented by the redoxregulation of Kv1.5, a potassium voltage-gated
channel expressed in the heart and in pulmonary
vasculature. Several studies have highlighted the
fact that increased ROS concentration in cells is
correlated to a reduction in Kv1.5 expression but
have not provided a clear relationship between the
two events. In collaboration with Martes’
laboratory, we were recently able to elucidate the
specific mechanism for Kv1.5 channel redoxregulation (50). Labeling studies with DBPs have
shown that a single Cys residue, located in the
extracellular C-terminal domain of Kv1.5
(Cys581), forms a Cys-SOH after H2O2 exposure.
This modification triggers channel internalization,
blocking its recycling to the cell membrane and
promotes Kv1.5 degradation.
Cellular Lifetime of Sulfenic Acid
Although limited solvent access and nearby
hydrogen bond acceptors would contribute to
RSOH stabilization, the absence of proximal thiols
capable of generating an intramolecular disulfide
is considered a major stabilizing factor.
In the absence of neighboring Cys residues,
RSOH can be directly reduced to RSH by Trx
(Figure 2A - Cycle 1) or may react with GSH to
generate a mixed disulfide, which is later reduced
by glutaredoxin (Figure 2A - Cycle 2). For
example, human serum albumin (HSA) has only
one free cysteine (Cys34), which is susceptible to
H2O2 oxidation (rate constant 2.5 M-1 s-1). We can
estimate the half-life of HSA-SOH based on its
reaction with GSH. Using the known second-order
rate constant for this reaction (~3 M-1 s-1) (51) and
estimating GSH concentration at 1 mM, the firstorder rate constant would be 0.003 s-1. Substituting
this value in the equation t1/2 = ln2/k, the estimated
half-life of HSA-SOH would be ~4 minutes.
On the other hand, many redox-regulated
proteins have a second proximal Cys that can form
an internal disulfide with RSOH (Figure 2B). In
Cdc25c, for example, Cys377-SOH reacts with
“backdoor” Cys330 at a rate constant of 0.012 s-1
(52). Applying the same calculations as above, the
half-life of Cdc25c-SOH would be ~1 minute.
Taken together, these estimated protein-SOHs
half-lives correlate well to the sulfenylation
Ion Channels
It is well established that ROS plays a
regulatory role for some ion channels (48), but
little is known about the molecular mechanism
through which this modulation is explicated. For
example, human T-helper lymphocyte ORAI1
channels, a family member of Ca2+ releaseactivated Ca2+ (CRAC) channels, are inhibited by
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studies published by our group and they appear
similar to the cellular lifetimes of many other
PTMs such as phosphorylation. In A431 cells, we
observed a peak of protein sulfenylation around 5
minutes after EGF stimulation, with a subsequent
decay over 30 minutes (6).
shown that around 5% of Cys residues exist as
Cys-SO2H (55). Finally, the discovery of
Sulfiredoxin (Srx), an ATP-dependent protein that
specifically
reduces
Cys-SO2H
in
the
peroxiredoxin (Prx) family, has opened the door to
an additional layer of redox regulation and
increased interest in this specific modification
(56).
Table 2 provides a list of proteins in which a
biological functional role has emerged for RSO2H.
In comparison with Table 1, the number of
reported proteins is decidedly exiguous. This does
not necessarily indicate that RSO2H plays a
negligible role in protein redox-regulation but
rather reflects the lack of robust methods for
monitoring the formation of such modifications
within proteins. Although, RSO2H shows higher
stability in comparison to RSOH, mass and
crystal-structure analyses can introduce a high
percentage of artifacts. In addition, the emerging
relevance of persulfide modification (RSSH) –
which has the same nominal mass shift as 32 Da –
makes the use of high-resolution mass
spectroscopy essential (57). We believe that the
development of chemical probes capable of
specifically trapping RSO2H will push this Cys
modification from the minor role to which it has
been relegated. In this connection, we recently
proposed the use of aryl-nitroso compounds as
chemoselective probes for RSO2H (58).
SULFINIC ACID FORMATION AND
REACTIVITY
RSOH may be over-oxidized to RSO2H by
two-electron oxidants (Figure 1A). This reaction
requires nucleophilic attack by RSOH on the
peroxide species. Although the H2O2-mediated
oxidation of RSOH can proceed through two
possible pathways (Figure 1C), the pH profile
indicates that sulfenate anion (RSO–) is the
reacting species. Therefore, the pKa value of
RSOH should influence this reaction (7). As we
emphasized above, the formation of a more stable
disulfide (or sulfenamide) should prevent RSOH
oxidation. Taking Cdc25c as an example, the
oxidation of Cys377-SOH to RSO2H has a rate
constant of 110 M-1 s-1 (52); this value is on par
with the general tendency of protein-SOHs to
oxidation, which is generally in the range of 10102M-1 s-1 (7,51,52). Since internal disulfide
formation has a rate constant of 0.012 s-1, the
oxidation of Cys377-SOH has significance only
over 100 µM of H2O2.
With a pKa value of around 2, RSO2H exists
exclusively in deprotonated form at physiological
pH. The sulfinate group (RSO2–), which behaves
primarily as a soft nucleophile (53), shows low
spontaneous reactivity in cells and, because it is
not reducible by typical cellular reductants, its
oxidation to sulfonic acid (RSO3H - Figure 1A)
appears to be the only relevant reaction in cells.
The considerations adduced above for RSOH
stability can also be applied to RSO2H; therefore,
the formation of hydrogen bonds and steric
hindrance may stabilize Cys-SO2H within
proteins, reducing its propensity to oxidation (54).
Peroxiredoxins and Sulfiredoxin
Prxs are a family of cysteine-based peroxidases
that remove H2O2 and other peroxides from cells.
Being highly abundant and exceptionally efficient
(second constant rate of 105–107 M-1 s-1), Prxs
maintain the cytosolic concentration of H2O2
under 100 nM (59). Therefore, regulation of Prx
activity is required to trigger H2O2-mediated
intracellular signaling.
Typical Prxs exist in antiparallel dimeric or
decameric forms and possess two Cys residues:
“peroxidatic” Cys (Cp), which reacts directly with
H2O2 to generate Cys-SOH, and “resolving” Cys
(Cr), which forms an intramolecular disulfide with
transient sulfenic acid. Finally Trx reduces
disulfide, restoring the catalytic cycle. Eukaryotic
2-Cys Prxs possess two sequence motifs (GGLG
and YF) in their C-termini that reduce the ability
of Cr to approach Cp-SOH (60). The resulting
decrease in the disulfide-formation rate allows a
Sulfinic Acid as a Post-Translational
Modification
Cys-SO2H was long considered merely an
artifact of protein purification. However,
increasing evidence indicates that hyperoxidation
to RSO2H is not a rare event. Indeed, quantitative
analysis of soluble proteins from rat liver has
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second molecule of H2O2 to react with Cp-SOH
(Figure 2C), generating a Cys-SO2H. Such overoxidation leads to the deactivation of peroxidase
activity and the formation of high-molecularweight aggregates, which exhibit molecular
chaperone activity (61). Although just 0.1% of the
CP in human PrxI is oxidized to Cys-SO2H during
each turnover (62) at low concentrations of H2O2,
recent kinetic studies demonstrate that Prxs 2 and
3 can undergo appreciable hyperoxidation without
requiring recycling of the disulfide (63).
The peroxidase activity of 2-Cys Prxs is
restored by Srx (64). The first step in the proposed
catalytic mechanism involves the oxygen attack of
RSO2– on the γ-phosphate of ATP and the
resulting generation of a sulfinic phosphoryl ester
(Figure 2C). This species represents a sort of
activated SO2H, which collapses to a thiosulfinate
intermediate (Prx-S(O)-S-Srx) after attack by a
conserved Cys residue in Srx (65). Thiosulfinate is
subsequently resolved by a third reducing species.
Kinetic studies show that Srx is an inefficient
enzyme. The rate of Prx-SO2H reduction is indeed
rather low (k2> 120 s-1, k3 ~ 85 s-1), suggesting that
Prx requires a slow reparation process in order to
allow H2O2 transient accumulation in response to
extracellular signals.
contrary, the structurally similar E18N mutant
shows increased propensity to oxidation even in
the absence of H2O2. More important, E18D
mutants fail to protect cells from ROS while E18N
showed similar levels of cell viability in
comparison to the wild type, demonstrating that
Cys106 oxidation to RSO2H is essential for
maintaining protective functions (69).
Considering Cys106’s high propensity to
oxidation, it has been proposed that DJ-1 acts
merely as a direct ROS scavenger. However, an
elegant new study reported that the C106DD DJ-1
mutant is still able to protect cells against
oxidative stress (70), excluding direct scavenger
action by Cys oxidation.
Cysteine oxidation and metal binding
properties
Cys residues are very common in metalbinding motif and can form coordinative bonds
with several metal ions, including zinc, copper,
and iron. Many proteins contain a Cys-Zn-Cys
complex, for example, which furnishes structural
rigidity. Oxidation of these cysteines causes Zn2+
release and a subsequent conformational change,
which can switch protein function. Although
oxidation is usually transient, through the
formation of a disulfide bond, in some cases it can
lead to irreversible Cys-SO2H (71).
Redox zinc switching is also involved in the
activation of matrix metalloproteinases (MMPs).
Matrilysis (MMP-7) contains a highly conserved
cysteine switch sequence, PRCGVPDVA, in its
pro-domain. The thiolate side-chain coordinates
the catalytic Zn2+, contributing to the maintenance
of enzyme inactivity. Fu et al. showed that
hypochlorous acid (HOCl), but not H2O2, can
activate the enzyme through the conversion of Cys
residue to RSO2H, which disrupts zinccoordination (72). An analogous redox-mechanism
also appears to be involved in the activation of
other MMPs (73).
The unique active site of nitrile hydratase
(NHase) offers a sort of compendium of thiol
oxidation states and metal coordinations.
Structural analysis reveals that NHase contains an
FeIII or CoIII active site, in which three Cys
residues, having three different oxidation states
(RSH, RSOH and RSO2H), contribute to the
coordination of the metal ion (74,75). The fully
reduced enzyme appears inactive, suggesting that
Parkinson’s Disease Protein DJ-1
DJ-1 is a homodimeric small protein that has
been associated with early onset Parkinson’s
Disease (66). Many studies demonstrate that DJ-1
protects cells against oxidative stress-mediated
apoptosis; however the mechanism of its
protective function remains largely unknown (54).
A conserved Cys residue, Cys106, is extremely
sensitive to oxidation and tends to form a CysSO2H species generation of which appears to be
critical for DJ-1 function. The highly conserved
Glu18 residue facilitates the ionization of Cys106,
reduces its pKa, and helps to stabilize Cys106SO2H through the formation of an unusually short
and consequently strong hydrogen bond (67).
Wilson et al. have shown that small changes in
this position can drastically influence the oxidation
propensity of Cys106. For example, in the E18D
DJ-1 mutant, the distance between the thiolate and
the protonated carboxylic side chain is increased
and Cys106 is predominantly oxidized to sulfenic
acid (68). In fact, Asp18 tends to stabilize Cys106SOH, hampering further oxidation. On the
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Cys sulfenylation and sulfinylation are critical in
maintaining the catalytic activity of NHase (76),
probably by increasing the Lewis acidity of the
metal ion. An analogous motif was more recently
found in the catalytic site of thiocyanate hydrolase
(SCNase), which incorporates CoIII only after Cys
oxidation (77).
The active site of NHase and SCNase suggests
that the oxidation state may influence Cys-binding
properties, switching the affinity from zinc (for
RSH) to iron and cobalt (for oxygenated sulfur
species). This change could provide additional
redox control of protein functions (78).
may regulate levels of phosphorylation,
ubiquitination, and SUMOylation in cells. The
modulation of transcription factors, and channel
activity by Cys-SOH adds another level to the
redox-signaling cascade.
The role of protein sulfinylation in cell
signaling appears mainly confined in the Prx/Srx
pair. We believe that the development of specific
chemical probes for RSO2H may help to find new
Srx substrates or alternative reducing systems.
Generally speaking, there is an urgent need for
new protocols to analyze the full proteome and
identify new targets. A deeper exploration of Cys
oxidation in relation to metal-binding properties
could open up new vistas on redox signaling.
Finally, the development of drugs that specifically
target the oxidative state form of proteins would
appear to be a worthwhile goal (79).
CONCLUSIONS AND PERSPECTIVES
Protein sulfenylation influences a wide range
of PTMs both directly and especially indirectly
(through the switching of protein function). We
have seen how the oxidation of specific Cys
residues in PTPs, PTKs, and cysteine proteases
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Table 1 – Protein sulfenic acid modification
Function
Protein
Organism
Cys-SOH
References
Peroxidase
AhpC
M. tuberculosis
Cys165 a
(80)
Phosphatase
Prx
H. sapiens
Cys51
Orp1/GPx3
S. cerevisiae
Cys36 a
PTP1B
Transcription factor
Channel
Oxidoreductase
Transferase
Integrin
(60,81)
(38)
Cys215
a
(6,82)
a
(82)
Y. enterocolitica
Cys403
PTEN
H. sapiens
Cys124 a
Cdc25a
H. sapiens
Cys431
a
SHP-1
H. sapiens / M. musculus
Cys455 a
H. sapiens / M. musculus
Cys459
a
(6,22)
a
(6)
(6,21)
(83)
(22,84)
EGFR
H. sapiens
Cys797
JAK2
M. musculus
Cys866 / Cys917 a
(27)
a
(28)
Akt2
M. musculus
Cys124
IKK-β
RegB
H. sapiens
R. capsulatus
Cys179 a
(41)
a
(85)
Cys265
b
PGKase
P. tricornutum
Cys77
L-PYK
H. sapiens
Cys436 b, c
AphB
V. cholerae
Cys235
MgrA
S. aureus
Cys12 b
SarZ
Cysteine Protease
H. sapiens
(PrxI)
YopH
SHP-2
Kinase
a, c
S. aureus
(86)
(87)
b
(35)
(32)
Cys13
b,c
(36)
b
(34)
OhrR
B. subtilis
Cys15
OxyR
E. coli
Cys199 b
(88)
a
(33)
CrtJ
R. capsulatus
Cys420
p50 (NF-kB )
H. sapiens
Cys62 a
H. sapiens
a
USP1
Cys90
(40)
(43)
a
(43)
USP7
H. sapiens
Cys223
A20
H. sapiens
Cys103 a
(44)
Cathepsin K
H. sapiens
Cys25
a,b
(89)
Papain
P. latex
Cys25 a
Kv1.5
H. sapiens
(80)
Cys581
a
(50)
a
(81)
(90)
GAPDH
O. cuniculus
Cys298
Aldose reductase
H. sapiens
Cys298 a
MsrA
S. cerevisiae
MTAP
H. sapiens
α7β1
a, c
Cys72
Cys136 / Cys223 b
a
(91)
(92)
R. rattus
Cys923 / Cys928
(94)
(93)
Chaperone
Hsp70
H. sapiens
Cys306 a
Serum Protein
HSA
H. sapiens
Cys34 a,b
(51)
(95)
(96)
Oxygen carrier
Hemoglobin
H. sapiens
Cys 93 a
Apoptotic regulator
Bcl-2
H. sapiens
Cys158 / Cys229 a
Cytoskeleton Protein
a
β-actin
Cys272
H. sapiens
a
(97)
Identified using dimedone or dimedone-based probes; b Identified using NBD-Cl; c Identified by crystal structure.
14 Downloaded from http://www.jbc.org/ at The Scripps Research Institute on July 17, 2013
Table 2 – Protein sulfinic acid modification
Function
Protein
Organism
Cys-SOH
References
d
Peroxidase
Prx
H. sapiens
Cys51 (PrxI)
(60)
Chaperone (?)
DJ-1
H. sapiens
Cys106 c
(67-69)
Oxidoreductase
Protease
Hydratase
S-transferase
c
c
YaiL
E. coli
Cys106
D-Amino Acid Oxidase
T. variabilis
Cys108 d
MMP-7
H. sapiens
Pro-domain Cys
(77)
Cys131
R. erythropolis
Cys133 c
H. sapiens
Cys65
(72)
(74)
P. thermophila
SNCase
L-PGDS
(99)
c
c
NHase
d
(98)
(100)
Identified by crystal structure; d Identified by mass.
15 Downloaded from http://www.jbc.org/ at The Scripps Research Institute on July 17, 2013
Figure Legends
Figure 1. Main oxidative modifications of protein cysteine residues. (A) The diagram shows the main
oxidative modifications of protein cysteine residues. The initial reaction of cysteine with oxidants, [O] =
ROS / RNS, yields a sulfenic acid (SOH). Once formed, the SOH can be reduced to thiol or further
oxidized to generate cysteines SO2H and SO3H. (B) Thiolate anion is much more nucleophilic of the
corresponding protonated form and can be readily oxidized to sulfenic acid. The protein
microenvironment can help to stabilize the poor hydroxide-leaving group and thus, accelerate the reaction
rate. (C) Two possible mechanisms have been proposed for the H2O2-mediated oxidation of RSOH to
RSO2H: a first pathway, which involves the direct participation of a sulfenate anion, or a second
concerted mechanism, which is mediated by a hydrogen bond.
Figure 2. Sulfenic and sulfinic acid redox cycles. (A) R-SOH can be directly reduced to free thiol by
Trx, although the importance of this pathway in cells is still debated (Cycle 1). RSOH can also react with
GSH to generate a mixed disulfide (although not all protein-SOHs are accessible to GSH), which is
subsequently reduced by Grx (Cycle 2). (B) In the presence of a neighboring Cys, RSOH forms an
internal disulfide that is later reduced by Trx (Cycle 3). (C) Typical eukaryotic 2-Cys Prx are inactivated
by over-oxidation to sulfinic acid (Step 1). Srx restores the sulfenic acid group using an ATP-dependent
mechanism in which an activated sulfinic phosphoryl ester is generated (Step 2). This intermediate
collapses to form a thiosulfinate moiety with Cys99 of Srx (Step 3). It has been proposed that this
intramolecular thiosulfinate is finally resolved by a common cellular reductant, such as GSH or Trx, with
consequent release of Prx-SOH (Step 4).
16 Downloaded from http://www.jbc.org/ at The Scripps Research Institute on July 17, 2013
Figure 1
17 Downloaded from http://www.jbc.org/ at The Scripps Research Institute on July 17, 2013
Figure 2
18 Downloaded from http://www.jbc.org/ at The Scripps Research Institute on July 17, 2013