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

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. Thioredoxin (Trx) – –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 C35 C73 C62 C69."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) (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 Purification for human-Txnip 4 Dialysis 4 Centrifugation 2 And Sonication 2 Column Purification 3 (Ni-NTA) 3 TB Media with Bacteria 1 (OD 0.6-1) 1 Concentrate 5 (Polyethylene Glycol) 5 6 Thrombin to cleavage 6

10. Protocol for Protein Purification for 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 Coomasie Western Blot kDa 62 49 Fusion protein ?

12. Thrombin cleaves the fusion protein creating hTxnip and E coli. thioredoxin hTxnip E. coli Trx His thrombin 15 kDa47 kDa 62 kDaTxnip Coomasie kDa 62 47 15 hTxnip E. Coli Trx Fusion ? Western Blot

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

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

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

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 h-Txnip GST-Trx

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 Protein Expected Radius (nm) Mass Spectra Radius (nm) Molecular Weight Radius Equivalent Polydispersion (nm) h-Txnip3.085127.9286.0MDa66.69 GST- Wild- type Trx 2.8105.487180.8kDa3.021 GST-35S,73 Trx 2.8106.855304.3kDa3.938 GST- 73S Trx2.8107.473372.4kDa3.548 GST- 32S,35S,73S Trx 2.8106.002222.9kDa3.281

22. DLS Results h-Txnip with Mutated GST-Trx Table 2. DLS Results for Mass Spectra for each complex Protein Expected Radius (nm) Mass Spectra Radius (nm) Molecular Weight Radius Equivalent (MDa) Polydispersion (nm) h-Txnip /GST- Wild-type Trx 3.954105.4181.946.50 h-Txnip /GST- 35S,73 Trx 3.95479.7694.8435.14 h-Txnip /GST- 73S Trx 3.95492.49134.136.80 h-Txnip /GST- 32S,35S,73S Trx 3.95495.91146.041.81

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