S-glutathionylated biomarker
plasma proteins
S-Glutathionylation Cycle
Oxidation
Reduction
[O]
[N]
Low pK cysteine
residue
Cysteinyl radical
Sulfenic acid
S-glutathionylation
Glutaredoxin
Sulfiredoxin
Sulfinic acid
(GSTP)
Grx
Sulfonic acid
degrade
Deglutathionylation
Protected
From: Tew, Biochem. Pharmacology 2007
Protein clusters subject to S-glutathionylation
1.
Cytoskeletal (actin)
2.
Glycolysis/energy metabolism (e.g. GAPDH, pyruvate kinase, triose
phosphoisomerase,phosphoglycerate kinase, aldolase, ketoglutarate
dehydrogenase, isocitrate dehydrogenase)
3.
Signaling pathways (e.g. MEKK1, NfB, STAT3, PKC, cAMP dependent
PKA, p53, GTPase p21 ras, Trx, GSTP)
4.
Calcium homeostasis (e.g. S100A1, SERCA, calmodulin, ryanodine
receptor)
5.
Phosphatases (e.g. PTP1B, PTEN, PP1, Cdc2, Cdc25a/b)
6.
Protein folding (e.g. PDI, HSP65, 20S proteosome)
6.
Serine protease inhibitors (SERPIN’s)
From: Townsend, Molecular Interventions 2008
SERPIN’s
• Serine Protease Inhibitors
• Inactivate enzymes by binding them covalently
• Have a characteristic secondary structure of beta sheets and alpha helices
antitrypsin
antichymotrypsin
Possible pathway of activation of proteases by serpinA1 S-glutathionylation
Site of S-glutathionylation?
From: Silverman et al, 2006
MALDI-TOF identification of S-glutathionylated plasma proteins
following NOV-002 treatment
Protein
(A) Complement C3
(B, D) Serpin A1
(C) Contrapsin (Serpin A3)
NCBI Accession #
1352102
Confidence Interval
100%
6678087
100%
54173
100%
NOV-002 (25 mg/kg, iv)
Kda 0 ¼ ½ 1 4 24 h
150
100
75
50
37
A WB: PSSG
B
C
D
WB: albumin
Mice treated in vivo
Mouse plasma treated ex vivo
S-glutathionylation of Serpins A1 &
and impacts protein structure
A3 is time and dose dependent
A
C
WB:PSSG
0
WB:Serpin A1
1 2.5 5 10 20 30 min
WB:PSSG
WB:Serpin A3
0 1 2.5 5 10 20 30 min
B
WB:PSSG
WB: Serpin A1
0 5 10 25 50 75 100 μM PABA/NO
0
D
Figure 2
WB:PSSG
WB: Serpin A3
5 10 25 50 75 100 μM PABA/NO
liquid chromatography (LC)-electrospray ionization (ESI)-tandem
mass spectrometry (MS/MS)
RLGMFNIQHCK
RLGMFNIQHC*K
y1* = cysteine + 305 + H2O, this ion in absent in the unmodified peptide
Figure 3A : Serpin A1 is S-glutathionylated at Cys256
liquid chromatography (LC)-electrospray ionization (ESI)-tandem
mass spectrometry (MS/MS)
DEELSCTVVELK
DEELSC*TVVELK
y7* = cysteine + 305, this ion in absent in the unmodified peptide
Figure 3B : Serpin A3 is S-glutathionylated at Cys263
A.
a
b
c
1
15
007
a
b
05
5
0
c
Relative Intensity
WB: PSSG
WB:albumin
0 2.5 5 15 30 60 120 240 min
B.
WB: PSSG
18
16
14
12
10
8
6
4
2
0
0
WB: SerpinA1
100
75
100
75
50
50
37
37
A1 A1
+ - +
A1 A1
-
+
15
WB: PSSG
250
150
-
5
30
60
120 240
Time
250
150
IP:
NOV-002:
2.5
IP:
NOV-002:
A3 A3
-
+
WB: SerpinA3
A3 A3
- +
Immunoprecipitation human plasma treated with 40 mmol/L NOV-002 for 1h and
biotinylated serpin A1 and A3 antibodies were used to pull-down total unmodified and
modified serpins.
Figure 4
B.
A1
A3
3
10
WB: Serpin A3
al
m
N
or
PSSGc/A1
2.0
0.0
0.0
rm
No
er
0.5
r
Ca
nc
e
No
r
m
al
0.0
1.0
0.5
Ca
nc
0.2
al
0.4
r
1.0
Mann Whitney test
P = 0.0002
1.5
Ca
nc
e
0.6
Mann Whitney test
P = 0.0088
1.5
PSSG/Serpin
Unpaired t test
P = 0.0026
2.0
PSSG/Serpin
0.8
PSSG/Serpin
PSSGb/A3
PSSGa/A1
al
C
. 1.0
5 6
m
3 4
No
r
1 2
2
Ca
nc
No
rm
WB: albumin
4
0
er
al
0
6
ce
r
1
Mann Whitney test
P = 0.0018
an
2
8
C
Unpaired t test
Welch's correction
P < 0.0001
Normalized Serpin Levels
W
B
:PSSGa IA1)
PPSSGb(A3)
SPSSGc(A1)
S
G
WB: Serpin A1
Normalized Serpin Levels
A.
Unmodified serpins are elevated in certain cancers while the ratio of Sglutathionylated to unmodified serpin is decreased
Figure 5
A.
WB:PSSG
PSSGa IA1)
PSSGb(A3)
PSSGc(A1)
WB: Serpin A1
WB: Serpin A3
WB: albumin
1
-
2
-
3
-
4
1
- +
2 3 4
+ + +
1
5 6
- -
-
7 8
- -
1 5
6 7 8
+ + + + +
100μM NOV-002
Ex vivo treatment with NOV-002 induces serpin A1 and A3 S-glutathionylation and results
in greater relative increases in Serpin A1 glutathionylation in normal human plasma
Parametric data were analyzed using T-tests and non-parametric data using Wilcoxon
matched-pairs signed rank test. Data are mean for 45 cancer samples and 8 diseasefree +/- SEM.
C.
B.
30
4
2
5
Untreated
Treated
20
10
10
0
ce
r
al
Ca
n
rm
Ca
nc
rm
No
ce
Ca
n
No
at
ed
rm
a
r
l
0
Tr
e
at
ed
Un
tre
at
ed
Tr
e
at
ed
20
Interaction P < 0.0001
0
0
Un
tre
Tr
e
Un
tre
at
ed
0
at
ed
0
Interaction P < 0.0001
Untreated
Two-way RM ANOVA
Treated
No
2
10
Two-way RM ANOVA
PSSGc/A1
30
6
al
1
4
PSSGc/A1
Two-way RM ANOVA
Interaction P < 0.0001
PSSG/Serpin
2
6
PSSGa/A1
PSSG/Serpin
3
15
Wilcoxon matched-pairs signed rank test
P > 0.0001
Pairing was effective: P > 0.05
er
4
PSSGc
Normalized PSSG Levels
8
Normalized PSSG Levels
E.
A1
3
5 15 30 60 120 240
min
Normalized Serpin Levels
0
8
6
4
2
0
at
ed
2.5
10
Wilcoxon matched-pairs signed rank test
P value is ns
Pairing was effective: P > 0.0001
at
ed
0
at
ed
WB: Albumin
1
at
ed
WB: SERPIN A1
2
U
nt
re
52kDa
Normalized Serpin Levels
WB: SERPIN A3
Tr
e
66kDa
A3
Paired t test
P value is ns
Pairing was effective: P > 0.0001
Tr
e
D.
Un
tre
5
Normalized PSSG Levels
PSSGb
Wilcoxon matched-pairs signed rank test
P > 0.0001
Pairing was effective: P > 0.01
PSSG/Serpin
PSSGa
Wilcoxon matched-pairs signed rank test
P > 0.0001
Pairing was effective: P > 0.05
Untreated
Treated
S-Glutathionylation Cycle
Oxidation
Reduction
[O]
[N]
Low pK cysteine
residue
Cysteinyl radical
Sulfenic acid
S-glutathionylation
Glutaredoxin
Sulfiredoxin
Sulfinic acid
(GSTP)
Grx
Sulfonic acid
degrade
Deglutathionylation
Protected
From: Tew, Biochem. Pharmacology 2007
Cancer patient plasma have decreased GSTP and elevated Grx1.
GSTpb
0.0
an
ce
r
C
or
m
m
al
0.0
N
Figure 7
0.5
or
4
NS
1.0
N
2 3
0.5
al
1
1.0
Normalized Densitometry
WB: albumin
P value = 0.0148
Mann-Whitney
an
ce
r
WB: Grx1
Grx
1.5
C
WB:GSTp
Normalized Densitometry
1.5
What next?
• Insert biomarkers into a protocol
where treatments are likely to
modulate redox homeostasis.
• Volunteers?