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

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Structural Interaction of Thioredoxin (Trx) and
Thioredoxin-interacting Protein (Txnip)
Dr. Richard Lees Lab, BWH

• Haydeliz Martínez-Ruiz
• University of Puerto Rico at Mayagüez

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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

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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

."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.
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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.

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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.
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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

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Why crystallize Txnip?

• Understand the Complex
• (Txnip-Trx)
• Test Hypothesis
• Drug Development
• Diabetes
• Cancer

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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.
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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
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Protocol for Protein Purificationfor human-Txnip
HPLC Size Exchange Chromatography 7
Coomassie Blue Western Blot 9
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h-Txnip protein can be purified as a fusion
protein in native conditions
Western Blot
Coomasie
kDa 62 49
Fusion protein
?
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Thrombin cleaves the fusion protein creating
hTxnip and E coli. thioredoxin
Coomasie
Western Blot
kDa 62 47 15
Fusion
hTxnip
?
E. ColiTrx
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Size exclusion chromatography (TXNIP) after
thrombin cleavage
Txnip Aggregates
44kDa Txnip?
E.coli-Trx 17kDa
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Generation of Purified Recombinant Txnip
47kDa h-TXNIP
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Protocol for Protein PurificationTrx
Centrifugation (Supernatant) 3
Centrifugation (Pellet) And Sonication 2
LB Media with Bacteria (OD 0.6-1) 1
Wash Beads (Glutathione-Sepharose Beads and
Centrifuge 4
Thrombin to cleavage GST-Trx 5
Centrifuge and save the supernatant 6
Coomassie Blue Western Blot 8
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Trx protein can be purified as a fusion protein
in native conditions
GST26kDa Trx12kDa
Coomassie Blue Stain
32,35,73 Trx
Wild Type Trx
35STrx
73S Trx
GST-Trx (38kDa)
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Thrombin cleaves the fusion protein creating GST
and Trx
Coomassie after thrombin cleavage
32,35S,73S
35S, 73
Wild Type
73S
GST
Trx
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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

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DLS Results
GST-Trx
h-Txnip
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DLS Results (h-Txnip/GST-Trx interaction)
h-Txnip /GST-Wild type Trx
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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- Wild-type 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
GST-32S,35S,73S Trx 2.810 6.002 222.9kDa 3.281
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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 3.954 105.4 181.9 46.50
h-Txnip /GST-35S,73 Trx 3.954 79.76 94.84 35.14
h-Txnip /GST- 73S Trx 3.954 92.49 134.1 36.80
h-Txnip /GST-32S,35S,73S Trx 3.954 95.91 146.0 41.81
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Conclusion

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

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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

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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