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Dr. Richard Lee’s Lab, BWH Structural Interaction of Thioredoxin (Trx) and Thioredoxininteracting Protein (Txnip) Haydeliz Martínez-Ruiz University of Puerto Rico at Mayagüez 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 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 C73 C32 C35 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. 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. 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. 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 Why crystallize Txnip? Understand the Complex (Txnip-Trx) Test Hypothesis Drug Development –Diabetes –Cancer Could incubating Thioredoxin with Txnip reduce aggregation? Hypotesis: Incubating Txnip with GSTThioredoxin would prevent disaggregation Mutated Trxs: 35S,73; 73S; 32S,35S,73S Protocol for Protein Purification for human-Txnip TB Media with Bacteria (OD 0.6-1) 1 Centrifugation And Sonication 2 Column Purification (Ni-NTA) 3 Thrombin to cleavage 6 Concentrate (Polyethylene Glycol) 5 Dialysis 4 Protocol for Protein Purification for human-Txnip HPLC Size Exchange Chromatography 7 Coomassie Blue Western Blot 9 h-Txnip protein can be purified as a fusion protein in native conditions Coomasie kDa Fusion protein ? 62 49 Western Blot Thrombin cleaves the fusion protein creating hTxnip and E coli. thioredoxin Coomasie Txnip E. coli Trx His hTxnip kDa 62 thrombin 15 kDa Western Blot Fusion hTxnip 47 47 kDa ? 62 kDa 15 E. Coli Trx Size exclusion chromatography (TXNIP) after thrombin cleavage Txnip Aggregates 44kDa Txnip? E.coli-Trx 17kDa Generation of Purified Recombinant Txnip 47kDa h-TXNIP Protocol for Protein Purification Trx LB Media with Bacteria (OD 0.6-1) 1 Thrombin to cleavage GST-Trx 5 Centrifuge and save the supernatant 6 Centrifugation (Pellet) And Sonication 2 Centrifugation (Supernatant) 3 Wash Beads (Glutathione-Sepharose Beads and Centrifuge 4 Coomassie Blue Western Blot 8 Trx protein can be purified as a fusion protein in native conditions Coomassie Blue Stain 32,35, 73 Trx 35STrx 73S Trx GST-Trx (38kDa) Wild Type Trx GST=26kDa Trx=12kDa Thrombin cleaves the fusion protein creating GST and Trx Coomassie after thrombin cleavage Trx GST 26kDa Trx 32,35S, 73S GST thrombin 12kDa 38kDa Trx 35S, 73 73S Wild Type 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 DLS Results GST-Trx h-Txnip DLS Results (h-Txnip/GST-Trx interaction) h-Txnip /GST-Wild type Trx 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-Txnip 3.085 127.9 286.0MDa 66.69 GST- Wildtype Trx 2.810 5.487 180.8kDa 3.021 GST-35S,73 Trx 2.810 6.855 304.3kDa 3.938 GST- 73S Trx 2.810 7.473 372.4kDa 3.548 GST32S,35S,73S Trx 2.810 6.002 222.9kDa 3.281 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) h-Txnip /GSTWild-type Trx 3.954 105.4 181.9 46.50 h-Txnip /GST35S,73 Trx 3.954 79.76 94.84 35.14 h-Txnip /GST73S Trx 3.954 92.49 134.1 36.80 h-Txnip /GST32S,35S,73S Trx 3.954 95.91 146.0 41.81 Polydispersion (nm) Conclusion Successfully purified recombinant Txnip protein Incubating Txnip with GSTThioredoxin did not prevent disaggregation 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 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