structural interaction of thioredoxin (trx) and thioredoxin- interacting protein (txnip) haydeliz...
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![Page 1: Structural Interaction of Thioredoxin (Trx) and Thioredoxin- interacting Protein (Txnip) Haydeliz Martínez-Ruiz University of Puerto Rico at Mayagüez Dr](https://reader035.vdocuments.mx/reader035/viewer/2022062300/56649d545503460f94a30ff3/html5/thumbnails/1.jpg)
Structural Interaction of Structural Interaction of Thioredoxin (Trx) and Thioredoxin-Thioredoxin (Trx) and Thioredoxin-
interacting Protein (Txnip)interacting Protein (Txnip)
Haydeliz Martínez-RuizHaydeliz Martínez-Ruiz
University of Puerto Rico at MayagüezUniversity of Puerto Rico at Mayagüez
Dr. Richard Lee’s Lab, BWH
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OutlineOutline
Thioredoxin and Txnip are important in Thioredoxin and Txnip are important in regulating both redox and glucose regulating both redox and glucose metabolismmetabolism
Structural information about Txnip is not Structural information about Txnip is not knownknown
Generation of pure recombinant Txnip Generation of pure recombinant Txnip protein for crystallography studiesprotein for crystallography studies
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Thioredoxin (Trx) 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
C62C69
."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)Thioredoxin-interacting protein(Txnip)
Inhibits thioredoxin reducing activity.Inhibits thioredoxin reducing activity. Binding to thioredoxin requires two cysteines, Binding to thioredoxin requires two cysteines,
suggesting an interaction through a disulfide bondsuggesting an interaction through a disulfide bond Regulates glucose metabolism in humans.Regulates glucose metabolism in humans.
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Trx - Txnip mechanismTrx - 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 projectAims of the project
Define the structure of Txnip and the Define the structure of Txnip and the mechanism of the Txnip-Trx interaction by mechanism of the Txnip-Trx interaction by crystallizationcrystallization
Test methods to produce pure protein and Test methods to produce pure protein and eliminate aggregation of Txnipeliminate aggregation of Txnip
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Why crystallize Txnip?Why crystallize Txnip?
Understand the Complex Understand the Complex (Txnip-Trx)(Txnip-Trx)
Test HypothesisTest HypothesisDrug DevelopmentDrug Development
– DiabetesDiabetes– CancerCancer
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Could incubating Thioredoxin with Could incubating Thioredoxin with Txnip reduce aggregation?Txnip reduce aggregation?
Hypotesis: Hypotesis: Incubating Incubating Txnip with GST-Txnip with GST-Thioredoxin would Thioredoxin would prevent disaggregationprevent disaggregation
Mutated Trxs: Mutated Trxs: 35S,73; 73S; 35S,73; 73S; 32S,35S,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.
![Page 9: Structural Interaction of Thioredoxin (Trx) and Thioredoxin- interacting Protein (Txnip) Haydeliz Martínez-Ruiz University of Puerto Rico at Mayagüez Dr](https://reader035.vdocuments.mx/reader035/viewer/2022062300/56649d545503460f94a30ff3/html5/thumbnails/9.jpg)
Protocol for Protein PurificationProtocol for Protein Purificationfor human-Txnipfor human-Txnip
Dialysis 44
CentrifugationAnd Sonication 22
Column Purification(Ni-NTA) 33
TB Media with Bacteria(OD 0.6-1) 11
Concentrate (Polyethylene Glycol) 55
Thrombin to cleavage 66
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Protocol for Protein PurificationProtocol for Protein Purificationfor human-Txnipfor human-Txnip
HPLCSize Exchange Chromatography 7
Coomassie BlueWestern Blot 9
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h-Txnip protein can be purified as a fusion protein h-Txnip protein can be purified as a fusion protein in native conditionsin native conditions
CoomasieWestern
Blot
kDa
62
49
Fusion protein
?
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Thrombin cleaves the fusion protein Thrombin cleaves the fusion protein creating hTxnip and E coli. thioredoxincreating hTxnip and E coli. thioredoxin
hTxnipE. coli Trx His
thrombin
15 kDa 47 kDa
62 kDa
TxnipTxnip Coomasie
kDa
62
47
15
hTxnip
E. ColiTrx
Fusion
?
Western Blot
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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 PurificationProtocol for Protein PurificationTrxTrx
Wash Beads (Glutathione-Sepharose Beads and Centrifuge 44
Centrifugation (Pellet)And Sonication 22
Centrifugation (Supernatant) 33
LB Media with Bacteria(OD 0.6-1) 11
Thrombin to cleavage GST-Trx 55
Centrifuge and save the supernatant 66
Coomassie BlueWestern Blot 88
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Trx protein can be purified as a fusion Trx protein can be purified as a fusion protein in native conditionsprotein in native conditions
GST=26kDa
Trx=12kDa CoomassieCoomassie Blue StainBlue Stain
GST-Trx (38kDa)
32,35,73 Trx
35STrx
73S Trx Wild Type Trx
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Thrombin cleaves the fusion protein Thrombin cleaves the fusion protein creating GST and Trxcreating GST and Trx
26kDa
GST Trx
12kDa
38kDa
thrombin
TrxTrx
Coomassie after thrombin cleavage
Trx
GST
32,35S,73S
35S, 73
73S Wild Type
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Dynamic Light Scattering StudiesDynamic Light Scattering Studies(DLS)(DLS)
Allows to know if there is aggregation by mass Allows to know if there is aggregation by mass spectraspectra
GST-Trx, h-Txnip, Mutated GST-Trx, GST-Trx and GST-Trx, h-Txnip, Mutated GST-Trx, GST-Trx and h-Txnip complexh-Txnip complex
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DLS ResultsDLS Results
h-Txnip
GST-Trx
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DLS Results (h-Txnip/GST-Trx DLS Results (h-Txnip/GST-Trx interaction)interaction)
h-Txnip /GST-Wild type Trx
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DLS Results DLS Results Table 1. DLS Results for Mass Spectra for each protein
ProteinExpected
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
Trx2.810 6.002 222.9kDa 3.281
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DLS Results h-Txnip with DLS Results h-Txnip with Mutated GST-TrxMutated GST-Trx
Table 2. DLS Results for Mass Spectra for each complex
ProteinExpected
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
Trx3.954 95.91 146.0 41.81
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ConclusionConclusion
Successfully purified recombinant Successfully purified recombinant Txnip proteinTxnip protein
Incubating Txnip with GST-Incubating Txnip with GST-Thioredoxin did not prevent Thioredoxin did not prevent disaggregationdisaggregation
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RecommendationsRecommendations
Concentrate GST-Trx without loosing Concentrate GST-Trx without loosing protein during Size Exclusionprotein during Size Exclusion
Eliminating GST from the Trx by Size Eliminating GST from the Trx by Size Exclusion ChromatographyExclusion Chromatography
Try DLS with Trx-Txnip complexTry DLS with Trx-Txnip complex
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AcknowledgementsAcknowledgements Dr. Richard Lee (P.I.)Dr. Richard Lee (P.I.) Dr. Jun Yoshioka (Mentor)Dr. Jun Yoshioka (Mentor) Dr. Parth PatwariDr. Parth Patwari Dr. Alexander SigalovDr. Alexander Sigalov Dr. Zarixia ZavalaDr. Zarixia Zavala Dr. Eric SchreiterDr. Eric Schreiter Dr. Bruce BirrenDr. Bruce Birren Shawna YoungShawna Young Dr. Neal LernerDr. Neal Lerner BROAD InstituteBROAD Institute