Thiol Workshop- SFRBM '06 1

Protein Disulfide Isomerase (PDI) in Cell Signaling

Bulent MutusDept. Chemistry & BiochemistryUniversity of Windsor, Windsor, Canada

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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

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Tian, G. et al. (2006) Cell 124:61–73.

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Gruber, CW et al. TIBS, 2006, 31: 455-464.

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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.

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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]

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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).

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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

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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.

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1) Mechanism of transfer of NO from extracellular S-nitrosothiols into the cytosol by cell-surface PDI:

1.2- HT1080 fibroblastomas underexressing/overexpressing PDI

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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

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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

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1) Mechanism of transfer of NO from extracellular S-nitrosothiols into the cytosol by cell-surface PDI:

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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

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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

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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

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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

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2. Characterization of the S-denitrosation activity of PDI:

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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.

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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

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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

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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.

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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

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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

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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

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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

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-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]

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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

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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

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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