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S-glutathionylated biomarker plasma proteins. Low pK cysteine residue S-glutathionylation Deglutathionylation Cysteinyl radical Sulfinic acidSulfonic.

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Presentation on theme: "S-glutathionylated biomarker plasma proteins. Low pK cysteine residue S-glutathionylation Deglutathionylation Cysteinyl radical Sulfinic acidSulfonic."— Presentation transcript:

1 S-glutathionylated biomarker plasma proteins

2 Low pK cysteine residue S-glutathionylation Deglutathionylation Cysteinyl radical Sulfinic acidSulfonic acidSulfenic acid [O] [N] S-Glutathionylation Cycle Protected Glutaredoxin Sulfiredoxin (GSTP) Oxidation Reduction degrade Grx From: Tew, Biochem. Pharmacology 2007

3 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

4 SERPIN’s Serine Protease Inhibitors Inactivate enzymes by binding them covalently Have a characteristic secondary structure of beta sheets and alpha helices antitrypsinantichymotrypsin

5 From: Silverman et al, 2006 Possible pathway of activation of proteases by serpinA1 S-glutathionylation Site of S-glutathionylation?

6 MALDI-TOF identification of S-glutathionylated plasma proteins following NOV-002 treatment Protein NCBI Accession # Confidence Interval (A) Complement C % (B, D) Serpin A % (C) Contrapsin (Serpin A3) % A B C D Kda NOV-002 (25 mg/kg, iv) 0 ¼ ½ h WB: PSSG WB: albumin A Mice treated in vivo Mouse plasma treated ex vivo

7 D Figure 2 WB:PSSG WB:Serpin A min WB:PSSG WB:Serpin A min A B μM PABA/NO μM PABA/NO WB:PSSG WB: Serpin A1 WB:PSSG WB: Serpin A3 C S-glutathionylation of Serpins A1 & A3 is time and dose dependent and impacts protein structure

8 y1* = cysteine H 2 O, this ion in absent in the unmodified peptide RLGMFNIQHC*K RLGMFNIQHCK Figure 3A : Serpin A1 is S-glutathionylated at Cys 256 liquid chromatography (LC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS)

9 DEELSCTVVELK DEELSC*TVVELK y7* = cysteine + 305, this ion in absent in the unmodified peptide Figure 3B : Serpin A3 is S-glutathionylated at Cys 263 liquid chromatography (LC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS)

10 a c b WB: PSSG WB:albumin min Figure 4 Time Relative Intensity 40μM NOV-002; 20μg plasma A3 IP: WB: SerpinA1 NOV-002: A1 - IP: WB: PSSG A3 NOV-002: A1 WB: PSSG WB: SerpinA3 A. B Immunoprecipitation human plasma treated with 40  mol/L NOV-002 for 1h and biotinylated serpin A1 and A3 antibodies were used to pull-down total unmodified and modified serpins.

11 Figure WB: albumin WB: Serpin A1 WB: Serpin A3 WB:PSSGWB:PSSG A. B. C.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

12 WB:PSSG WB: albumin μM 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

13 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 A1 52kDa WB: SERPIN A3 66kDa WB: Albumin min D. E.

14 Low pK cysteine residue S-glutathionylation Deglutathionylation Cysteinyl radical Sulfinic acidSulfonic acidSulfenic acid [O] [N] S-Glutathionylation Cycle Protected Glutaredoxin Sulfiredoxin (GSTP) Oxidation Reduction degrade Grx From: Tew, Biochem. Pharmacology 2007

15 WB:GSTp WB: albumin WB: Grx1 Figure 7 Cancer patient plasma have decreased GSTP and elevated Grx1.

16 What next? Insert biomarkers into a protocol where treatments are likely to modulate redox homeostasis. Volunteers?


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