s-glutathionylated biomarker plasma proteins. low pk cysteine residue s-glutathionylation...
TRANSCRIPT
S-glutathionylated biomarker plasma proteins
Low pK cysteine residue
S-glutathionylation
Deglutathionylation
Cysteinyl radical Sulfinic acid Sulfonic acidSulfenic acid
[O]
[N]
S-Glutathionylation Cycle
Protected
Glutaredoxin
Sulfiredoxin
(GSTP)
OxidationReduction
degradeGrx
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, NfB, 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)
7. 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
From: Silverman et al, 2006
Possible pathway of activation of proteases by serpinA1 S-glutathionylation
Site of S-glutathionylation?
MALDI-TOF identification of S-glutathionylated plasma proteins following NOV-002 treatment
Protein NCBI Accession # Confidence Interval (A) Complement C3 1352102
100%
(B, D) Serpin A1 6678087 100%
(C) Contrapsin (Serpin A3) 54173 100%
A B C
D
Kda150100
75 50
37
NOV-002 (25 mg/kg, iv) 0 ¼ ½ 1 4 24 h
WB: PSSG
WB: albumin
Mice treated in vivo Mouse plasma treated ex vivo
D
Figure 2
WB:PSSG
WB:Serpin A3 0 1 2.5 5 10 20 30 min
WB:PSSG
WB:Serpin A1 0 1 2.5 5 10 20 30 min
A
B
0 5 10 25 50 75 100 μM PABA/NO
0 5 10 25 50 75 100 μM PABA/NO
WB:PSSG
WB: Serpin A1
WB:PSSGWB: Serpin A3
C
S-glutathionylation of Serpins A1 & A3 is time and dose dependent and impacts protein structure
y1* = cysteine + 305 + H2O, this ion in absent in the unmodified peptide
RLGMFNIQHC*K
RLGMFNIQHCK
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
liquid chromatography (LC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS)
0 2.5 5 15 30 60 120 24002468
1012141618
abca
c
b
WB: PSSG
WB:albumin
0 2.5 5 15 30 60 120 240 min
75 50
100
150
Figure 4
Time
Rela
tive
Inte
nsity
A3 A3IP:
WB: SerpinA1
NOV-002: - + - +
250
75
50
100
150
37
A1 A1-IP:
WB: PSSG
A3 A3NOV-002: + - + - +
A1 A1
WB: PSSG WB: SerpinA3
A.
B.250
75
50
100
150
37
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 5
1 2 3 4 5 6WB: albumin
WB: Serpin A1
WB: Serpin A3
WB:PSSG
A. B.
C.
PSSGa IA1)
PSSGb(A3)
PSSGc(A1)
Unmodified serpins are elevated in certain cancers while the ratio of S-glutathionylated to unmodified serpin is decreased
WB:PSSG
WB: albumin 1 2 3 4 1 2 3 4 1 5 6 7 8 1 5 6 7 8 - - - - + + + + - - - - - + + + + + 100μM NOV-002
WB: Serpin A3
WB: Serpin A1
A.PSSGa IA1)
PSSGb(A3)
PSSGc(A1)
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 disease-
free +/- SEM.
B. C.
WB: SERPIN A152kDa
WB: SERPIN A366kDa
WB: Albumin
0 2.5 5 15 30 60 120 240 min
D. E.
Low pK cysteine residue
S-glutathionylation
Deglutathionylation
Cysteinyl radical Sulfinic acid Sulfonic acidSulfenic acid
[O]
[N]
S-Glutathionylation Cycle
Protected
Glutaredoxin
Sulfiredoxin
(GSTP)
OxidationReduction
degradeGrx
From: Tew, Biochem. Pharmacology 2007
1 2 3 4
WB:GSTp
WB: albumin
WB: Grx1
Figure 7
Cancer patient plasma have decreased GSTP and elevated Grx1.
What next?• Insert biomarkers into a protocol
where treatments are likely to modulate redox homeostasis.
• Volunteers?