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?