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Structural Interaction of Thioredoxin (Trx) and Thioredoxin-interacting Protein (Txnip)

Dr. Richard Lee’s Lab, BWH. Structural Interaction of Thioredoxin (Trx) and Thioredoxin-interacting Protein (Txnip). Haydeliz Martínez-Ruiz University of Puerto Rico at Mayagüez. Outline. Thioredoxin and Txnip are important in regulating both redox and glucose metabolism

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Structural Interaction of Thioredoxin (Trx) and Thioredoxin-interacting Protein (Txnip)

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  1. Dr. Richard Lee’s Lab, BWH Structural Interaction of Thioredoxin (Trx) and Thioredoxin-interacting Protein (Txnip) Haydeliz Martínez-Ruiz University of Puerto Rico at Mayagüez

  2. Outline • Thioredoxin and Txnip are important in regulating both redox and glucose metabolism • Structural information about Txnip is not known • Generation of pure recombinant Txnip protein for crystallography studies

  3. One of the major cellular antioxidant systems Reduces protein disulfides through a cysteine disulfide at its active site (C32 and C35) Promotes growth and protects cells from apoptosis C32 C73 C35 C62 C69 Thioredoxin (Trx) ."Yoshioka, J., "Schreiter , E. R. "., & "Lee, R. T. ". (2006). Role of thioredoxin in cell growth through interactions with signaling molecules. Antioxidant and Redox Signaling, 8, 2143-2151.

  4. Thioredoxin-interacting protein(Txnip) • Inhibits thioredoxin reducing activity. • Binding to thioredoxin requires two cysteines, suggesting an interaction through a disulfide bond • Regulates glucose metabolism in humans.

  5. Trx - Txnip mechanism "Patwari, P., "Higgins, L., " Chutkow, William A.", " Yoshioka, J., & " Lee, R. T. ". (2006). The interaction of thioredoxin with txnip: Evidence for formation of a mixed disulfide by disulfide exchange. J Biol Chem., 281(31), 21884-21891.

  6. Aims of the project • Define the structure of Txnip and the mechanism of the Txnip-Trx interaction by crystallization • Test methods to produce pure protein and eliminate aggregation of Txnip

  7. Why crystallize Txnip? • Understand the Complex (Txnip-Trx) • Test Hypothesis • Drug Development • Diabetes • Cancer

  8. Could incubating Thioredoxin with Txnip reduce aggregation? • Hypotesis: Incubating Txnip with GST-Thioredoxin would prevent disaggregation • Mutated Trxs: 35S,73; 73S; 32S,35S,73S ."Yoshioka, J., "Schreiter , E. R. "., & "Lee, R. T. ". (2006). Role of thioredoxin in cell growth through interactions with signaling molecules. Antioxidant and Redox Signaling, 8, 2143-2151.

  9. Protocol for Protein Purificationfor human-Txnip TB Media with Bacteria (OD 0.6-1) 1 Thrombin to cleavage 6 Centrifugation And Sonication 2 Concentrate (Polyethylene Glycol) 5 Dialysis 4 Column Purification (Ni-NTA) 3

  10. Protocol for Protein Purificationfor human-Txnip HPLC Size Exchange Chromatography 7 Coomassie Blue Western Blot 9

  11. h-Txnip protein can be purified as a fusion protein in native conditions Western Blot Coomasie kDa 62 49 Fusion protein ?

  12. Txnip E. coli Trx His hTxnip thrombin 15 kDa 47 kDa 62 kDa Thrombin cleaves the fusion protein creating hTxnip and E coli. thioredoxin Coomasie Western Blot kDa 62 47 15 Fusion hTxnip ? E. ColiTrx

  13. Size exclusion chromatography (TXNIP) after thrombin cleavage Txnip Aggregates 44kDa Txnip? E.coli-Trx 17kDa

  14. Generation of Purified Recombinant Txnip 47kDa h-TXNIP

  15. Protocol for Protein PurificationTrx Centrifugation (Supernatant) 3 LB Media with Bacteria (OD 0.6-1) 1 Centrifugation (Pellet) And Sonication 2 Thrombin to cleavage GST-Trx 5 Wash Beads (Glutathione-Sepharose Beads and Centrifuge 4 Centrifuge and save the supernatant 6 Coomassie Blue Western Blot 8

  16. Trx protein can be purified as a fusion protein in native conditions GST=26kDa Trx=12kDa CoomassieBlue Stain 32,35,73 Trx Wild Type Trx 35STrx 73S Trx GST-Trx (38kDa)

  17. Trx GST Trx thrombin 12kDa 26kDa 38kDa Thrombin cleaves the fusion protein creating GST and Trx Coomassie after thrombin cleavage 32,35S,73S 35S, 73 Wild Type 73S GST Trx

  18. Dynamic Light Scattering Studies(DLS) • Allows to know if there is aggregation by mass spectra • GST-Trx, h-Txnip, Mutated GST-Trx, GST-Trx and h-Txnip complex

  19. DLS Results GST-Trx h-Txnip

  20. DLS Results (h-Txnip/GST-Trx interaction) h-Txnip /GST-Wild type Trx

  21. DLS Results Table 1. DLS Results for Mass Spectra for each protein

  22. DLS Results h-Txnip with Mutated GST-Trx Table 2. DLS Results for Mass Spectra for each complex

  23. Conclusion • Successfully purified recombinant Txnip protein • Incubating Txnip with GST-Thioredoxin did not prevent disaggregation

  24. Recommendations • Concentrate GST-Trx without loosing protein during Size Exclusion • Eliminating GST from the Trx by Size Exclusion Chromatography • Try DLS with Trx-Txnip complex

  25. Acknowledgements • Dr. Richard Lee (P.I.) • Dr. Jun Yoshioka (Mentor) • Dr. Parth Patwari • Dr. Alexander Sigalov • Dr. Zarixia Zavala • Dr. Eric Schreiter • Dr. Bruce Birren • Shawna Young • Dr. Neal Lerner • BROAD Institute

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