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STP2 & GAPDH: Partners in crime towards  -Synuclein Toxicity

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.

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STP2 & GAPDH: Partners in crime towards  -Synuclein Toxicity

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  1. STP2 & GAPDH:Partners in crime towards -Synuclein Toxicity By, Arun George Paul BIO 324

  2. Acknowledgements • Dr. Shubhik K. DebBurman • Ruja Shrestha • Isaac Holmes • Chris Prater • Tulaza Vaidya • Sara Herrera • Katrina Brandies • Jessica Price • Saccharomyces cerevisiae

  3. 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

  4. Death of Dopaminergic Neurons Lewy Bodies Diseased Healthy Pathology of PD Substantia nigra Perves, Augustine, Fitzpatrick et al.Neuroscience, 2nd edition Spillantini, Schmidt, Lee et al -Synuclein in lewy bodies. Nature 1997.

  5. ? ? ? -Synuclein Mutant -Synuclein Aggregated -Synuclein Lewy Bodies • A30P • A53T -Synuclein Aggregates Over expression of -Synuclein Molecular Theme of PD

  6. 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

  7. One Among 86 Genetic Deletions… I selected STP2 Mildly Toxic Over expression of -Synuclein My Goal: How? The Willingham Study 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.

  8. Over expression of -Synuclein & Huntingtin Mildly Toxic Among 86… Only STP2 Enhanced Toxicity in Both Cases Why STP2?

  9. Amino Acid What is STP2? STP2 is required for amino acid intake 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.

  10. Increased GAPDH How is STP2 & -Synuclein Toxicity Linked ? STP2 deletion increases GAPDH Levels Nucleus STP2 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.

  11. 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

  12. More Cell Death Increased GAPDH Enhanced by STP2 Deletion Mildly Toxic Over expression of -Synuclein STP2 How is STP2 & -Synuclein Toxicity Linked ? My Hypothesis:

  13. How do I test my hypothesis? Aim 1: ? Mildly Toxic Enhanced by STP2 Deletion Over expression of -Synuclein

  14. How do I test my hypothesis? Aim 2: ? -Synuclein Aggregates By STP2 Deletion Over expression of -Synuclein

  15. STP2 Increased GAPDH How do I test my hypothesis? Aim 3: ?

  16. More Cell Death Cell Death Reduced Cell Death Over expression of -Synuclein Increased GAPDH STP2 How do I test my hypothesis? Aim 4: ?

  17. -Syn WT A30P A53T A30P/A53T +STP2 +STP2 -Syn Control: pYES2 Over expresses -Synuclein -Syn -STP2 -STP2 Our Pet… Saccharomyces cerevisiae or Baker’s Yeast

  18. Testing Aim 1 by Spotting Assay STP2 Prediction 1 Over expression of -Synuclein + pYES2 - + ? WT Enhanced by STP2 Deletion - + A30P - + Mildly Toxic A53T - + A30P/ A53T -

  19. A30P/ A53T pYES2 WT A30P A53T Ladder + + + + + - - - - - >150 kDA STP2 62 KDa Model Blot Testing Aim 2: By Immunoblotting Prediction 2 STP2 Deletion -Synuclein

  20. Potential Aggregate -Synuclein’s Membrane Localization Testing Aim 2: By GFP Microscopy Prediction 2 STP2 Deletion -Synuclein

  21. Testing Aim 2: By GFP Microscopy Prediction 2 STP2 Deletion -Synuclein STP2 Deletion and Non-Deletion Showed the Following Phenotypes A30P/ A53T pYES2 A53T WT A30P Peripheral Cytoplasmic Peripheral Cytoplasmic

  22. Potential -Synuclein Aggregates Testing Aim 2: By GFP Microscopy Prediction 2 STP2 Deletion -Synuclein STP2 Deletion and Non-Deletion Showed the Following Phenotypes A53T A53T WT WT

  23. A30P/ A53T pYES2 WT A30P A53T Ladder STP2 + + + + + - - - - - STP2 Increased GAPDH 43.5 kDA 43.5 KDa Model Blot Testing Aim 3: By Immunoblotting Prediction 3

  24. Testing Aim 4 by Spotting Assay STP2 Prediction 4 + pYES2 GAPDH - + WT - ? + A30P - + Reduced Cell Death A53T - + A30P/ A53T -

  25. More Cell Death Increased GAPDH Enhanced by STP2 Deletion Mildly Toxic Over expression of -Synuclein STP2 My Hypothesis:

  26. More Cell Death Increased GAPDH Mildly Toxic Over expression of -Synuclein What did we see? STP2

  27. Negative Results are as important as positive results in the pursuit of science

  28. p426 -Syn -Syn -Syn By Willingham et. al., (2003) pYES2 By Us The Act of Troubleshooting: What did we do different?

  29. The Act of Troubleshooting: What did we do different? Protein-Quality Control Systems Protein-Quality Control Systems Misfolded Proteins Misfolded Proteins Misfolded Proteins Protein-Quality Control Systems

  30. -Syn Action of Protein-Quality Control Systems! Mildly Toxic Not Enough -Synuclein? pYES2 -Synuclein Aggregates Over expression of -Synuclein The Act of Troubleshooting: What did we do different?

  31. -Syn -Syn -Syn M4054 Increased GAPDH By Eckert-Boulet et. al., (2004) By Us 4741 The Act of Troubleshooting: What did we do different? STP2

  32. Increased GAPDH The Act of Troubleshooting: What did we do different? STP2 • They used a DNA Microarray • GAPDH signal might have been a false positive

  33. Non- 4741/pYES2 -Synuclein System 4741/pYES2 -Synuclein System Mildly Toxic No Cell Death Over expression of -Synuclein Not Enhanced by STP2 Deletion through GAPDH Enhanced by STP2 Deletion through GAPDH Our Proposal

  34. 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

  35. Questions?

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