kelly crotty dr. tory hagen lipoic acid induces nrf2 activation in an mtor- dependent manner
Post on 19-Dec-2015
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• Influences the expression of over 200
genes with the Antioxidant Response
Element (ARE)
• These genes produce detoxification
enzymes and antioxidant proteins
Nrf2: A Transcription Factor in the Detoxification System
• Levels of Nrf2 in the nucleus decline with age
• The detoxification system is less effective with age
Lipoic acid
Supplementation
Old
• Lipoic Acid (LA) increases levels of nuclear Nrf2
Lipoic Acid improves stress response
Nrf2 Synthesis Under Normal Conditions: Cap Dependent
Translation
mRNA
4E-BP
mTOR Raptor
P P P
Nrf2 Synthesis
IRES
eIF4E
Ribosome and
proteins
P P
• mTOR is a kinase that senses energy and
stress levels in the cell
• Raptor is an adapter that brings mTOR to
the substrate (4E-BP)
Nrf2 Synthesis Under Oxidative Stress: Cap Independent
Translation
mRNAeIF4E
4E-BP
Internal
Ribosomal
Entry Site
Ribosome and
proteins
Synthesis of Stress
Response Proteins
(Nrf2)Stress
PP
P
Hypothesis:Lipoic acid increases levels of phosphorylated mTOR complex, inducing cap-dependent production of Nrf2
Prediction:Cells treated with lipoic acid will have increased amounts of phosphorylated mTOR complex compared to a non-treated control.
Methods:
Perform SDS-PAGE to separate proteins by weight
Treat cells in vitro with lipoic acid
Measure levels of mTOR and Raptor by Western
Blot analysis
Blocking
Primary
Antibody
Secondary
Antibody
Development
mTOR activation is lost with LA treatment
mTOR
P-mTOR
Raptor P-Raptor
total mTOR in the cell
increases with LA treatment
Active mTOR disappears
with LA treatment
Total and active Raptor
disappear with LA treatment
No LA 4 hr. LA
No LA 4 hr. LA
No LA 4 hr. LA No LA 4 hr. LA
Raptor
mTOR
mTOR
P P
mTOR Complex is inactive
• Lipoic acid treatment increases total mTOR protein, but decreases mTOR complex activity
• This is counter to our hypothesis
eIF4E
4E-BP
mTOR Raptor
P P P
• LA decreases mTOR complex activity, so
we are not seeing cap-dependent
translation of Nrf2
Implications: Switch from Cap-Dependent to Cap-Independent Translation
Because lipoic acid does increase
translation of Nrf2, these data suggest cap-
independent translation of Nrf2.
Conclusion: Under lipoic acid treatment, cells inhibit global protein
synthesis and cap-independent translation is preferred
New Hypothesis: LA acts as a weak stress in the cell
• Hormesis: the favorable response of being exposed to a small stress regularly for an
extended period of time in order to have a larger capacity to respond to greater stresses
• LA treatment induces a stress response in the cell
• Old animals taking LA supplements respond to great stresses equally as well as young
animals
• LA is regimenting the cell to respond to stress
Next Step
• Do a time course to see when mTOR complex activity returns
• Probe for other proteins associated with cap-independent translation
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