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Thiol Workshop- SFRBM '06 1
Protein Disulfide Isomerase (PDI) in Cell Signaling
Bulent MutusDept. Chemistry & BiochemistryUniversity of Windsor, Windsor, Canada
Thiol Workshop- SFRBM '06 2
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 5.3.4.1) 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: 455-464.
ox
red
Thiol Workshop- SFRBM '06 3
Tian, G. et al. (2006) Cell 124:61–73.
Thiol Workshop- SFRBM '06 4
Gruber, CW et al. TIBS, 2006, 31: 455-464.
Thiol Workshop- SFRBM '06 5
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, 86:2168-73.
• Essex DW, et al. Protein disulfide isomerase catalyzes the formation of disulfide-linked complexes of vitronectin with thrombin-antithrombin. Biochemistry. 1999, 38:10398-405.
• Essex DW, Li M. Protein disulphide isomerase mediates platelet aggregation and secretion. Br J Haematol. 1999, 104:448-54.
• 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, 361:120-6.
• Lahav J, et al. Protein disulfide isomerase mediates integrin-dependent adhesion. FEBS Lett. 2000 475:89-92.
• Lahav J, et al. Sustained integrin ligation involves extracellular free sulfhydryls and enzymatically catalyzed disulfide exchange. Blood. 2002,100:2472-8.
• Burgess JK, et al. Physical proximity and functional association of glycoprotein 1balpha and protein-disulfide isomerase on the platelet plasma membrane. J Biol Chem. 2000,27:9758-66.
Thiol Workshop- SFRBM '06 6
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, 102:2085-92.
Mediation of the transport of NO-equivalents (RSNO) across membranes:• A. Zai, M.A. Rudd, A.W. Scribner, J. Loscalzo, Cell-surface protein disulfide
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, 382:575-80.
Sliskovic I, Raturi A, Mutus B. Characterization of the S-denitrosation activity of
protein disulfide isomerase. J Biol Chem. 2005, 280:8733-41. • 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. 2006 Aug 11; [Epub ahead of print]
Thiol Workshop- SFRBM '06 7
Presentation Focus:
Methodology utilized to demonstrate cell signaling roles of csPDI.
• Mechanism of transfer of NO from extracellular S-nitrosothiols into the cytosol by cell-surface protein disulfide isomerase.
• Characterization of the S-denitrosation activity of protein disulfide isomerase.
• Redox regulation of PDI implications for platelet function normal vs. disease (T2D) (new work).
Thiol Workshop- SFRBM '06 8
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
FluorescenceFluorescence
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 '06 9
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 '06 10
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 '06 11
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
NH
O
As(OH)2
Thiol Workshop- SFRBM '06 12
1) Mechanism of transfer of NO from extracellular S-nitrosothiols into the cytosol by cell-surface PDI:
1.3- a N2O3 quencher: -tocopherol
+ -tocopherol
- -tocopherol
Thiol Workshop- SFRBM '06 13
1) Mechanism of transfer of NO from extracellular S-nitrosothiols into the cytosol by cell-surface PDI:
Thiol Workshop- SFRBM '06 14
2. Characterization of the S-denitrosation activity of PDI:
2.1- Is NO produced upon interaction of GSNO with PDI?
2.1.1 OxyHb as an NO detector
Thiol Workshop- SFRBM '06 15
2. Characterization of the S-denitrosation activity of PDI:
2.1- Is NO and thiyl/dithiyl radicals produced upon interaction of GSNO with PDI?
2.1.1 OxyHb as an NO detector2.1.2 Ac-Tempo fluorogenic thiyl/dithiyl radical probe
Thiol Workshop- SFRBM '06 16
2. Characterization of the S-denitrosation activity of PDI:
2.1- Is NO produced upon interaction of GSNO with PDI?
2.1.3 NO electrode
Thiol Workshop- SFRBM '06 17
2. Characterization of the S-denitrosation activity of PDI:
2.2- PDI Thiol reactivity and PDI-SNO formation
2.2.1- DTNB
2.2.2- UV/vis
Thiol Workshop- SFRBM '06 18
2. Characterization of the S-denitrosation activity of PDI:
Thiol Workshop- SFRBM '06 19
3. Redox regulation of PDI implications for platelet function normal vs. disease (T2D).
Background: PDI essential for platelet aggregation
Essex et al. Biochemistry. 2001, 40:6070-5.
Thiol Workshop- SFRBM '06 20
3. 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 21. Blood. 2003, 102:2085-92.
21-SH + S-S-collagen
PDI ox
21-S-S-collagen
PDI red
Thiol Workshop- SFRBM '06 21
3. 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-PDIii) competing for the PDI active site (i.e. competitive inhibitor)
21-SH + S-S-collagen
PDI ox
21-S-S-collagen
PDI red
+ GSNO
N=O
Thiol Workshop- SFRBM '06 22
3. 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-PDIii) competing for the PDI active site (i.e. competitive inhibitor)
21-SH + S-S-collagen
PDI ox
21-S-S-collagen
PDI red
+ GSNO
N=O
PDI ox
•Biochem J. 2004, 382:575-80.
Thiol Workshop- SFRBM '06 23
3. 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
eosinEGSH
diEGSSG
~70-fold enhancement in fluorescence upon disulfide reduction
Thiol Workshop- SFRBM '06 24
3. 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.
KM
GSSG 300 M
diEGSSG 0.65 M
Thiol Workshop- SFRBM '06 25
3. 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
pla
sm
a
cy
tos
ol
Thiol Workshop- SFRBM '06 26
3. 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
0
5
10
15
20
25
30
35
1Control T2DPDI dentirosation activity
0
20
40
60
80
100
120
140
160
180
1Control T2D
vo aggregation rates
0
500
1000
1500
2000
2500
1Control T2D
PDI thiol reductase
activity
Thiol Workshop- SFRBM '06 27
-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 '06 28
0
2
4
6
8
10
12
100:0 100:25 100:50 100:100 0:100
GSH:GSSG
v o a
ggre
gatio
n (/
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 '06 29
3. Redox regulation of PDI implications for platelet function normal vs. disease (T2D).
PDI oxPDI red
T2D promotes PDI oxidation and platelet activation
Normal conditions promote PDI reduction and platelet inhibition
Thiol Workshop- SFRBM '06 30
Thiol Workshop- SFRBM '06 31
Acknowledgements:
Dr. Niro RamachandranArun RaturiInga SliskovicShane Miersch
Ruchi ChaubeHarman KaurKhaled Elmosrati
Funding:Canadian Institutes of Health ResearchCanadian Diabetes AssociationNatural Sciences and Engineering Research CouncilU. Windsor Research Chair Funds