Download Document

STP2 & GAPDH:
Partners in crime towards -Synuclein
Toxicity
By,
Arun George Paul
BIO 324
Acknowledgements
•
•
•
•
•
•
•
•
•
Dr. Shubhik K. DebBurman
Ruja Shrestha
Isaac Holmes
Chris Prater
Tulaza Vaidya
Sara Herrera
Katrina Brandies
Jessica Price
Saccharomyces cerevisiae
The Big Picture: Parkinson Disease
(PD)
•
Affects 4 million people world wide
•
A Progressive Neurodegerative Disorder
•
Major symptoms:
- Tremor at rest
- Slowness of movement
- Reduced facial expressions
- Balance problems
Pathology of PD
Death of Dopaminergic
Neurons
Substantia Lewy
nigra Bodies
Perves, Augustine, Fitzpatrick et al.Neuroscience, 2nd edition
Spillantini, Schmidt, Lee et al -Synuclein in lewy bodies. Nature 1997.
Diseased
Healthy
Molecular Theme of PD
?
-Synuclein
?
Mutant
-Synuclein
?
Aggregated
-Synuclein
Lewy Bodies
• A30P
• A53T
Over expression of
-Synuclein
-Synuclein
Aggregates
What is -Synuclein ?
What we know?
• Membrane Binding Protein
• Proposed to be a dopamine transport protein
What we do not know?
• Why misfolded in dopaminergic neurons
• Proteins Linked to Misfolding
The Willingham Study
Mildly Toxic
Over expression of
-Synuclein
One Among
86 Genetic Deletions…
I selected STP2
My Goal: How?
Willingham, Stephen, Tiago Fleming Outerio, Michael J. Devit, Susan L. Lindquist, and Paul Muchowski. 2003. Yeast Genes that Enhance the Toxicity of a Mutant Huntingtin Fragment or -synuclein. Science, 302, 1769.
Why STP2?
Over expression of
-Synuclein
&
Huntingtin
Mildly Toxic
Among 86…
Only STP2 Enhanced
Toxicity in Both Cases
What is STP2?
STP2 is required for amino acid intake
Amino Acid
Nucleus
STP2
de Boer M, Nielsen PS, Bebelman JP, Heerikhuizen H, Andersen HA, and Planta RJ. Stp1P, Stp2P, and Abfip are involved in regulation of expression of the amino acid transporter gene BAP3 of Saccharomyces cerevisiae. Nucleic Acids Res., 28, 974.
How is STP2 & -Synuclein Toxicity
Linked ?
STP2 deletion increases GAPDH Levels
Increased
GAPDH
STP2
Nucleus
Eckert-Boulet N, Nielsen PS, Friis C, dos Santos MM, Nielsen J, Kielland-Brandt MC, and Regenberg B. 2004. Transcriptional profiling of extracellular amino acid sensing in Saccharomyces cerevisiae and the role of stp2 and stp2. Yeast, 21, 635.
How is GAPDH important?
• GAPDH is a glycolytic protein
• GAPDH deletion rescues PD neurons
• GAPDH is localized with -Synuclein in Lewy
Bodies
• GAPDH3/Hsp35 is a heat shock protein
How is STP2 & -Synuclein Toxicity
Linked ?
My Hypothesis:
More
Cell
Death
Mildly
Toxic
Over expression of
-Synuclein
STP2
Increased
Enhanced by GAPDH
STP2 Deletion
How do I test my hypothesis?
Aim 1:
?
Over expression of
-Synuclein
Mildly Toxic
Enhanced by
STP2 Deletion
How do I test my hypothesis?
Aim 2:
?
Over expression of
-Synuclein
By
STP2 Deletion
-Synuclein
Aggregates
How do I test my hypothesis?
Aim 3:
STP2
?
Increased
GAPDH
How do I test my hypothesis?
Aim 4:
More
CellCell
Death
Reduced
Death
Cell Death
Over expression of
-Synuclein
STP2
?
Increased
GAPDH
Our Pet…
Saccharomyces cerevisiae or Baker’s Yeast
-Syn
+STP2
-Syn
Control: pYES2
WT
+STP2A30P
A53T Over expresses
A30P/A53T -Synuclein
-Syn
-STP2
-STP2
Testing Aim 1 by Spotting Assay
Prediction 1
STP2
Over expression of
-Synuclein
+
+
+
?
Enhanced by
STP2 Deletion
Mildly Toxic
pYES2
WT
A30P
A53T
A30P/
A53T
+
+
-
Testing Aim 2: By Immunoblotting
Prediction 2
STP2 Deletion
+
A53T
+
+
+
-
-
+
A30P
-
Ladder
WT
-
pYES2
A30P/
A53T
-
-Synuclein
STP2
>150 kDA
62 KDa
Model Blot
Testing Aim 2: By GFP Microscopy
Prediction 2
-Synuclein
STP2 Deletion
-Synuclein’s
Potential
Membrane
Aggregate
Localization
Testing Aim 2: By GFP Microscopy
Prediction 2
-Synuclein
STP2 Deletion
STP2 Deletion and Non-Deletion Showed the Following Phenotypes
pYES2
WT
A30P
A53T
A30P/
A53T
Peripheral Cytoplasmic Peripheral Cytoplasmic
Testing Aim 2: By GFP Microscopy
Prediction 2
-Synuclein
STP2 Deletion
STP2 Deletion and Non-Deletion Showed the Following Phenotypes
WT
A53T
WT
A53T
Potential -Synuclein Aggregates
Testing Aim 3: By Immunoblotting
Prediction 3
Increased
QuickTime™
Model Blotand a
A30P/
A53T
+
A53T
+
+
-
+
A30P
-
-
+
WT
-
pYES2
GAPDH
-
Ladder
STP2
STP2
43.5 KDa
43.5 kDA
Testing Aim 4 by Spotting Assay
STP2
Prediction 4
GAPDH
?
Reduced Cell Death
pYES2
WT
A30P
A53T
A30P/
A53T
+
+
+
+
+
-
My Hypothesis:
More
CellToxic
Death
Mildly
Over expression of
-Synuclein
STP2
Increased
Enhanced by GAPDH
STP2 Deletion
What did we see?
More
Cell
Death
Mildly
Toxic
Over expression of
-Synuclein
STP2
Increased
GAPDH
Negative Results are as important
as positive results in the pursuit
of science
The Act of Troubleshooting:
What did we do different?
-Syn
p426
-Syn
By Willingham et. al., (2003)
-Syn
pYES2
By Us
The Act of Troubleshooting:
What did we do different?
The Act of Troubleshooting:
What did we do different?
Mildly Toxic
Over expression of
-Synuclein
-Synuclein
Aggregates
Action of
Protein-Quality
Control Systems!
-Syn
pYES2
Not Enough
-Synuclein?
The Act of Troubleshooting:
What did we do different?
STP2
-Syn
Increased
GAPDH
-Syn
M4054
By Eckert-Boulet
et. al., (2004)
-Syn
By Us
4741
The Act of Troubleshooting:
What did we do different?
STP2
Increased
GAPDH
• They used a DNA Microarray
• GAPDH signal might have been a false
positive
Our Proposal
Non4741/pYES2
-Synuclein
System
4741/pYES2
-Synuclein
System
Mildly
No
Cell Toxic
Death
Over expression of
-Synuclein
Not
Enhanced
Enhanced
byby
STP2
STP2 Deletion
Deletion through
through GAPDH
GAPDH
Future Research
• Applying New Techniques for Detecting Toxicity:
FUN Racker Assay
• Testing our hypothesis on Non-4741/pYES2 Synuclein Systems
• Investigating the potential aggregates we saw
• Exploring the link of GAPDH and -Synuclein in
yeast
Questions?