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

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

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.

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.

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.

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

Why crystallize Txnip?Why crystallize Txnip?

Understand the Complex Understand the Complex (Txnip-Trx)(Txnip-Trx)

Test HypothesisTest HypothesisDrug DevelopmentDrug Development

– DiabetesDiabetes– CancerCancer

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.

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

Protocol for Protein PurificationProtocol for Protein Purificationfor human-Txnipfor human-Txnip

HPLCSize Exchange Chromatography 7

Coomassie BlueWestern Blot 9

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

?

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

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 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

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

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

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

DLS ResultsDLS Results

h-Txnip

GST-Trx

DLS Results (h-Txnip/GST-Trx DLS Results (h-Txnip/GST-Trx interaction)interaction)

h-Txnip /GST-Wild type Trx

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

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

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

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

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