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