Thiol Workshop- SFRBM '061 Protein Disulfide Isomerase (PDI) in Cell Signaling Bulent Mutus Dept. Chemistry & Biochemistry University of Windsor, Windsor, Canada

Thiol Workshop- SFRBM '062 The PDI family: PDI + PDI-like proteins. >10 PDI-family proteins in human tissues: PDI, ERp57, ERp72, ERp28, PDIp, PDIR and P5. PDI (PDI-1; EC ) is the most abundant: ~0.8% of the total cellular protein in yeast and mammalian cells. PDI- Primary function: Redox catalyst Gruber, CW et al. TIBS, 2006, 31: ox red

Thiol Workshop- SFRBM '063 Tian, G. et al. (2006) Cell 124:61–73.

Thiol Workshop- SFRBM '064 Gruber, CW et al. TIBS, 2006, 31:

Thiol Workshop- SFRBM '065 PDI- other functions (postulated from in vitro studies): Chaperone role: prevents protein aggregation and retention in ER PDI leaks out and is attached via hydrophobic interactions to cell surfaces (csPDI) Mediation of platelet function by psPDI: Essex DW, Chen K, Swiatkowska M. Localization of protein disulfide isomerase to the external surface of the platelet plasma membrane. Blood. 1995, 86: Essex DW, et al. Protein disulfide isomerase catalyzes the formation of disulfide- linked complexes of vitronectin with thrombin-antithrombin. Biochemistry. 1999, 38: Essex DW, Li M. Protein disulphide isomerase mediates platelet aggregation and secretion. Br J Haematol. 1999, 104: Milev Y, Essex DW. Links Protein disulfide isomerase catalyzes the formation of disulfide-linked complexes of thrombospondin-1 with thrombin-antithrombin III. Arch Biochem Biophys. 1999, 361: Lahav J, et al. Protein disulfide isomerase mediates integrin-dependent adhesion. FEBS Lett : Lahav J, et al. Sustained integrin ligation involves extracellular free sulfhydryls and enzymatically catalyzed disulfide exchange. Blood. 2002,100: Burgess JK, et al. Physical proximity and functional association of glycoprotein 1balpha and protein-disulfide isomerase on the platelet plasma membrane. J Biol Chem. 2000,27:

Thiol Workshop- SFRBM '066 PDI- other functions (postulated from in vitro studies): Mediation of platelet function by psPDI: (continued) Lahav J, et al. Enzymatically catalyzed disulfide exchange is required for platelet adhesion to collagen via integrin alpha2beta1. Blood. 2003, 102: Mediation of the transport of NO-equivalents (RSNO) across membranes: A. Zai, M.A. Rudd, A.W. Scribner, J. Loscalzo, Cell-surface proteindisulfide isomerase catalyzes transnitrosation and regulates intracellular transfer of nitric oxide, J. Clin. Invest. 1999, 103: 393–399.  N. Ramachandran, P. Root, X.M. Jiang, P.J. Hogg, B. Mutus, Mechanism of transfer of NO from extracellular S-nitrosothiols into the cytosol by cell-surface protein disulfide isomerase, Proc. Natl. Acad. Sci. USA 2001, 98:9539–9544. Root, P et al. Platelet cell-surface protein disulphide-isomerase mediated S- nitrosoglutathione consumption. Biochem J. 2004, 382:  Sliskovic I, Raturi A, Mutus B. Characterization of the S-denitrosation activity of protein disulfide isomerase. J Biol Chem. 2005, 280: Shah CM, Bell SE, Locke IC, Chowdrey HS, Gordge MP. Interactions between cell surface protein disulphide isomerase and S-nitrosoglutathione during nitric oxide delivery. Nitric Oxide Aug 11; [Epub ahead of print]

Thiol Workshop- SFRBM '067 Presentation Focus: Methodology utilized to demonstrate cell signaling roles of csPDI. 1)Mechanism of transfer of NO from extracellular S-nitrosothiols into the cytosol by cell-surface protein disulfide isomerase. 2)Characterization of the S-denitrosation activity of protein disulfide isomerase. 3)Redox regulation of PDI implications for platelet function normal vs. disease (T2D) (new work).

Thiol Workshop- SFRBM '068 1)Mechanism of transfer of NO from extracellular S-nitrosothiols into the cytosol by cell-surface PDI: 1.1- A probe sensitive to S-nitrosation: N-dansylHCYS-SH Fluorescence N-dansylHCYS-SH + NO + - NO + N-dansylHCYS-S-NO (N-dansylHCYS-S) 2 cell permeable: reduced to N-dansylHCYS-SH in cytosol

Thiol Workshop- SFRBM '069 1)Mechanism of transfer of NO from extracellular S-nitrosothiols into the cytosol by cell-surface PDI: 1.1- A probe sensitive to S-nitrosation: N-dansylHCYS-SH cont.

Thiol Workshop- SFRBM '0610 1)Mechanism of transfer of NO from extracellular S-nitrosothiols into the cytosol by cell-surface PDI: 1.2- HT1080 fibroblastomas underexressing/overexpressing PDI

Thiol Workshop- SFRBM '0611 1)Mechanism of transfer of NO from extracellular S-nitrosothiols into the cytosol by cell-surface PDI: 1.3- a vicinal thiol-specific agent: GSAO +GSAO -GSAO

Thiol Workshop- SFRBM '0612 1)Mechanism of transfer of NO from extracellular S-nitrosothiols into the cytosol by cell-surface PDI: 1.3- a N 2 O 3 quencher:  -tocopherol +  -tocopherol -  -tocopherol

Thiol Workshop- SFRBM '0613 1)Mechanism of transfer of NO from extracellular S-nitrosothiols into the cytosol by cell-surface PDI:

Thiol Workshop- SFRBM ' Characterization of the S-denitrosation activity of PDI: 2.1- Is NO produced upon interaction of GSNO with PDI? OxyHb as an NO detector

Thiol Workshop- SFRBM ' Characterization of the S-denitrosation activity of PDI: 2.1- Is NO and thiyl/dithiyl radicals produced upon interaction of GSNO with PDI? OxyHb as an NO detector Ac-Tempo fluorogenic thiyl/dithiyl radical probe

Thiol Workshop- SFRBM ' Characterization of the S-denitrosation activity of PDI: 2.1- Is NO produced upon interaction of GSNO with PDI? NO electrode

Thiol Workshop- SFRBM ' Characterization of the S-denitrosation activity of PDI: 2.2- PDI Thiol reactivity and PDI-SNO formation DTNB UV/vis

Thiol Workshop- SFRBM ' Characterization of the S-denitrosation activity of PDI:

Thiol Workshop- SFRBM ' Redox regulation of PDI implications for platelet function normal vs. disease (T2D). Background: PDI essential for platelet aggregation Essex et al. Biochemistry. 2001, 40:

Thiol Workshop- SFRBM ' Redox regulation of PDI implications for platelet function normal vs. disease (T2D). Background: PDI essential for platelet aggregation Lahav J, et al. PDI catalyzed disulfide exchange is required for platelet adhesion to collagen via integrin  2  1. Blood. 2003, 102:  2  1 -SH + S-S-collagen PDI ox  2  1 -S-S-collagen PDI red

Thiol Workshop- SFRBM ' Redox regulation of PDI implications for platelet function normal vs. disease (T2D). Background: PDI essential for platelet aggregation We have shown that GSNO can block aggregation by: i) via PDI mediated NO release >cGMP pathway requires red-PDI ii) competing for the PDI active site (i.e. competitive inhibitor)  2  1 -SH + S-S-collagen PDI ox  2  1 -S-S-collagen PDI red + GSNO N=O

Thiol Workshop- SFRBM ' Redox regulation of PDI implications for platelet function normal vs. disease (T2D). Background: PDI essential for platelet aggregation We have shown that GSNO can block aggregation by: i) via PDI mediated NO release >cGMP pathway requires red-PDI ii) competing for the PDI active site (i.e. competitive inhibitor)  2  1 -SH + S-S-collagen PDI ox  2  1 -S-S-collagen PDI red + GSNO N=O PDI ox Biochem J. 2004, 382:

Thiol Workshop- SFRBM ' Redox regulation of PDI implications for platelet function normal vs. disease (T2D). Our interest: how does redox status of PDI affect platelet function can this affect platelet hyperactivity observed in T2D. 3.1 A sensitive assay for PDI thiol reductase activity: Eosin-GS-SG-Eosin: diEGSSG eosin EGSH diEGSSG ~70-fold enhancement in fluorescence upon disulfide reduction

Thiol Workshop- SFRBM ' Redox regulation of PDI implications for platelet function normal vs. disease (T2D). Our interest: how does redox status of PDI affect platelet function can this affect platelet hyperactivity observed in T2D. 3.1 A sensitive assay for PDI thiol reductase activity: cont. KMKM GSSG 300  M diEGSSG 0.65  M

Thiol Workshop- SFRBM ' Redox regulation of PDI implications for platelet function normal vs. disease (T2D). Reductase activity of PDI highly sensitive to GSH:GSSG ratio: GSH (200  M- 4mM) GSSG (200  M-fixed) ER or plasma cytosol

Thiol Workshop- SFRBM ' Redox regulation of PDI implications for platelet function normal vs. disease (T2D). compare platelets from control and T2D human subjects (n=22) for: platelet- initial rates of aggregation: PDI- denitrosation activity PDI- disulfide reductase activity

Thiol Workshop- SFRBM '0627 -as platelets are exposed to larger [GSSG] PDI thiol reductase activity is lost 3. Redox regulation of PDI implications for platelet function normal vs. disease (T2D). -expose control platelets to increasingly more oxidizing conditions [GSSG]

Thiol Workshop- SFRBM '0628 v o aggregation (/min) -as platelets are exposed to larger [GSSG] they aggregate faster 3. Redox regulation of PDI implications for platelet function normal vs. disease (T2D). -expose control platelets to increasingly more oxidizing conditions

Thiol Workshop- SFRBM ' Redox regulation of PDI implications for platelet function normal vs. disease (T2D). PDI ox PDI red T2D promotes PDI oxidation and platelet activation Normal conditions promote PDI reduction and platelet inhibition

Thiol Workshop- SFRBM '0630

Thiol Workshop- SFRBM '0631 Acknowledgements: Dr. Niro Ramachandran Arun Raturi Inga Sliskovic Shane Miersch Ruchi Chaube Harman Kaur Khaled Elmosrati Funding: Canadian Institutes of Health Research Canadian Diabetes Association Natural Sciences and Engineering Research Council U. Windsor Research Chair Funds