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S-glutathionylated biomarker plasma proteins

S-glutathionylated biomarker plasma proteins. S-Glutathionylation Cycle. Oxidation. Reduction. [O]. [N]. Cysteinyl radical. Low pK cysteine residue. Sulfenic acid. Sulfinic acid. Sulfonic acid. S-glutathionylation. (GSTP). Glutaredoxin Sulfiredoxin. Grx. degrade.

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S-glutathionylated biomarker plasma proteins

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  1. S-glutathionylated biomarker plasma proteins

  2. S-Glutathionylation Cycle Oxidation Reduction [O] [N] Cysteinylradical Low pK cysteine residue Sulfenic acid Sulfinic acid Sulfonic acid S-glutathionylation (GSTP) Glutaredoxin Sulfiredoxin Grx degrade Deglutathionylation Protected 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) • Protein folding (e.g. PDI, HSP65, 20S proteosome) • 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 antitrypsin antichymotrypsin

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

  6. 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% NOV-002 (25 mg/kg, iv) 0 ¼ ½ 1 4 24 h Kda 150 100 75 50 37 A B C D WB: PSSG A WB: albumin Mice treated in vivo Mouse plasma treated ex vivo

  7. S-glutathionylation of SerpinsA1 & A3 is time and dose dependent and impacts protein structure A C WB:PSSG WB:PSSG WB: Serpin A1 WB:SerpinA1 0 5 10 25 50 75 100 μM PABA/NO 0 1 2.5 5 10 20 30 min WB:PSSG WB:PSSG WB: Serpin A3 WB:Serpin A3 0 5 10 25 50 75 100μM PABA/NO 0 1 2.5 5 10 20 30 min B D Figure 2

  8. liquid chromatography (LC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) RLGMFNIQHCK RLGMFNIQHC*K Figure 3A : Serpin A1 is S-glutathionylated at Cys256 y1* = cysteine + 305 + H2O, this ion in absent in the unmodified peptide

  9. liquid chromatography (LC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) DEELSCTVVELK DEELSC*TVVELK Figure 3B : Serpin A3 is S-glutathionylated at Cys263 y7* = cysteine + 305, this ion in absent in the unmodified peptide

  10. A. 150 100 75 50 WB: PSSG Relative Intensity a B. b 250 150 100 c 75 50 WB: PSSG WB: SerpinA1 WB: SerpinA3 WB: PSSG 37 WB:albumin Time 250 0 2.5 5 15 30 60 120 240 min 150 Immunoprecipitationhuman plasma treated with 40 mmol/L NOV-002 for 1h and biotinylatedserpin A1 and A3 antibodies were used to pull-down total unmodified and modified serpins. 100 75 50 37 - A1 A1 A1 A1 IP: A3 A3 IP: A3 A3 NOV-002: + - + - + - + - + NOV-002: Figure 4 40μM NOV-002; 20μg plasma

  11. A. B. WB:PSSG PSSGa IA1) PSSGb(A3) PSSGc(A1) WB: Serpin A1 WB: Serpin A3 WB: albumin 1 2 3 4 5 6 C. Unmodified serpins are elevated in certain cancers while the ratio of S-glutathionylated to unmodified serpin is decreased Figure 5

  12. A. PSSGa IA1) PSSGb(A3) PSSGc(A1) WB:PSSG WB: Serpin A1 WB: Serpin A3 WB: albumin 1 2 3 4 1 2 34 1 5 6 7 8 1 5 6 7 8 - - -- + + + + - - - - - + + + + +100μM NOV-002 Ex vivotreatment 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. B. 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. D. 66kDa WB: SERPIN A3 52kDa WB: SERPIN A1 WB: Albumin C. E. 0 2.5 5 15 30 60 120 240 min

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

  15. Cancer patient plasma have decreased GSTP and elevated Grx1. WB:GSTp WB: Grx1 WB: albumin 1 2 3 4 Figure 7

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

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