THIOREDOXIN REDOX STATE DETERMINATION

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THIOREDOXIN REDOX STATE DETERMINATION

• Hong Nguyen
• Mentor Dr. Gary Merrill

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Thioredoxin

• protein essential for life in mammals
• acts as an antioxidant to prevent oxidative
  stress
• acts as an electron donor in a variety of
  cellular reactions
• restores oxidatively inactivated enzymes to their
  reduced active state

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Thioredoxin exists in either the reduced or
oxidized form
Reduced Thioredoxin
Oxidized Thioredoxin
LEGEND SH thiol S-S disulfide
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Thioredoxin participates in dithiol-disulfide
exchange reactions
HS
HS
S
S
RNR oxidized
RNR reduced
H S
H S
S
S
reduced Trx
oxidized Trx
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Thioredoxin reductase thioredoxin
thioredoxin
ribonucleotide reductase
Trx
RNR
NADP
thioredoxin reductase
S
S
Trx
RNR
NADPH
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Hypothesis

• Thioredoxin reductase is a major determinant of
  thioredoxin redox state
• If true, then deletion of thioredoxin reductase
  should shift the redox state of thioredoxin from
  primarily reduced to primarily oxidized

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Protein electrophoretic mobility shift assay
(PEMSA)
SA-
SH
SA-
SA-
TRX red
TRX red
TRX red
TRX red
SA-
SH
SA-
SA-
SA-
SH
SA-
SA-
SA-
SH
SA-
SA-
SA-
SH
SA-
SA-
SA-
acetone wash
SH
SA-
SA-
DTT
IAM
IAA
Iodoacetamide
Iodoacetic acid
Dithiothreitol
SH
SA-
SA-
SA-
TRX ox
TRX ox
TRX ox
TRX ox
SH
SA-
SA-
SA-
SH
SA-
SA-
SA-
SH
SA-
SA-
SA-
S
S
SH
SM
S
S
SH
SM
LEGEND
A- negative charge
Thioredoxin
M neutral state
S
Disulfide
SH thiol
S
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Protein electrophoretic mobility shift assay
SH
SH
Trx red
Trx ox
SH
SH
SH
SH
SH
SH
S
SH
0
SH
S
-
-1
DTT
-2
urea PAGE
SH
-3
SH
SH
-4
TrxSS
SH
SH
SH
-5
IAA/IAM

-6
TrxSH
IAM
IAA
1
2
3
4
SM or SA-
SM
SA-
SM or SA-
SM
SA-
SM or SA-
SM
SA-
SM or SA-
SM
SA-
SM or SA-
SM
SA-
SM or SA-
SM
SA-
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Representative results
Wild Type
Mutant
1
2
3
4
5
200
201
37
229
230
Ladder
IAA only
IAM only
Trxox
mixed disulfide
Trxred
Trxox
mixed disulfide
Trxred
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Conclusion

• Thioredoxin reductase is not the major
  determinant of thioredoxin redox state
• Another pathway must be able to reduce
  thioredoxin

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Acknowledgement

• Dr. Gary Merrill
• Biochemistry Biophysics Lab
• Dr. Kevin Ahern
• Howard Hughes Medical Institute
• Undergraduate, Research, Innovation, Scholarship
  Creativity