1. Sarah Otih Mentor: Dr. William Barton
Tie1 and Tie2 Dimerization and the Possible Inhibition of Angiogenesis in Tumor endothelial cells.
Sarah Otih
Mentor: Dr. William Barton

2. Tumor Cells
Cells grow uncontrollably

3. Tumor Cells
Cells grow uncontrollably
Benign or Malignant

4. Tumor Cells Cells grow uncontrollably Benign or Malignant
Malignant=Cancerous

5. Growth of Tumor Cells
More nutrients = More growth

6. Growth of Tumors More nutrients = More growth
More growth = More Blood Supply

7. Growth of Tumors More nutrients = More growth
More growth = More Blood Supply
Stop Blood Supply→ Stop Growth

9. How to cut off this Blood Supply?
How new blood vessels form from preexisting vessels
Tie Receptors
Location→ Cellular surface

10. Proposal Question:
If angiogenesis is permanently stopped, could this result in cellular death?

11. Proposal Question:
If angiogenesis is permanently stopped, could this result in cellular death?
Could a constant physical interaction between Tie1 and Tie2 result in the hault of angiogenesis in targeted endothelial cells?

12. Barton et al 2010
If Ang1 present→
Tie2 Dimerization

13. If Ang1 present→ Tie2 Dimerization=
Angiogenesis

14. If NO Ang1→ Tie1 & Tie2 bound = NO Angiogenesis
Barton et al 2010

15. How is Tie1/Tie2 bound? → Positive Charge Surface= Tie1
Barton et al.(2010)
→ Positive Charge Surface= Tie1

16. How is Tie1/Tie2 bound? → Positive Charge Surface= Tie1
Barton et al.(2010)
→ Positive Charge Surface= Tie1
→ Negative Charge Surface= Tie2

17. Tie Tie1
Barton et al 2010

18. Tie Tie1

19. Keep Tie1/Tie2 bound=NO angiogenesis
How?
Mutation of Tie1 and Tie2 surfaces→
Increase charge density

20. Experiment
1)Find Amino Acids to mutate

21. Experiment
1)Find Amino Acids to mutate
→ Pymol

22. Experiment 1)Find Amino Acids to mutate → Pymol
→ Tie1-increase positive charge

23. Experiment 1)Find Amino Acids to mutate → Pymol
→ Tie1-increase positive charge
→ Tie2-increase negative charge

24. Experiment→ QuickChange Site-Directed Mutagenesis

26. Cell Line→ Step 3→Control
Remove normal Endothelial cells from animal

27. Cell Line→ Step 3→Control
Remove normal Endothelial cells from animal
Add Media

28. Cell Line→ Step 3→ Control
Remove normal Endothelial cells from animal
Add Media
Cell growth

29. Cell Line→ Step 3→ Mutated
Remove Endothelial cells from animal

30. Cell Line→ Step 3→ Mutated
Remove Endothelial cells from animal
Incorporate Mutated Plasmid into Media

31. Cell Line→ Step 3→ Mutated
Remove Endothelial cells from animal
Incorporate Mutated Plasmid into Media
Mutated Cell Growth

32. Experiment
4)Measure attraction-FRET(Fluorescence Resonance Energy Transfer)
→ Distance before mutation vs Distance after mutation

33. Experiment 3)Measure attraction-FRET(Quantitative)
→ Distance before mutation-Distance after mutation
→ FRET efficiency can only decrease

34. Experiment 3)Measure attraction-FRET(Quantitative)
→ Distance before mutation-Distance after mutation
→ FRET efficiency can only decrease
→ Introduce Ang1

35. Outcome
Successful: Tie1/Tie2 stay together

36. Outcome
Successful: Tie1/Tie2 stay together
Attraction strong enough

37. Outcome Successful: Tie1/Tie2 stay together Attraction strong enough
Unsuccessful: Tie1/Tie2 dissociate when Ang1 present

38. Possible Issues
Electrostatic Interaction→ Not strong bond

39. Possible Issues Electrostatic Interaction→ Not strong bond
Avoid Hydrophobic Areas when mutating

40. Possible Issues Electrostatic Interaction→ Not strong bond
Avoid Hydrophobic Areas when mutating
→ Protein Folding
→ Protein Function

41. The big picture... Success → Base for tumor treatment
→ Technological Advancements-targeting specific cells

42. Questions?

43. Further Explanation of FRET
Tie 2
Tie 1
Tie 2
Tie 1

44. Pymol Further Explanation

45. How do could we ensure that this Tie1/Tie2 dimerization would take place in only cancer cells?
No current cancer therapies/technologies
Once a technology is created, this Tie1/Tie2 dimerization could be a potential target.
Bivalent antibodies
In vivo, bivalent antibodies could potentially attach to both Tie1 and Tie2 and from there be inserted into targeted cells.

46. References
Seegar, T. C. M., Eller, B., Tzvetkova-Robev, D., Kolev, M. V, Henderson, S. C., Nikolov, D. B., & Barton, W. A. (2010). Article Tie1-Tie2 Interactions Mediate Functional Differences between Angiopoietin Ligands.
Yu, X., Seegar, T. C. M., Dalton, A. C., Tzvetkova-Robev, D., Goldgur, Y., Rajashankar, K. R.,
Barton, W. A. (2013). Structural basis for angiopoietin-1-mediated signaling initiation. Proceedings of the National Academy of Sciences of the United States of America, 110(18), 7205–
Adair, T. H., & Montani, J.-P. (2010). Overview of Angiogenesis. Retrieved from
Shlamkovich, T., Aharon, L., Barton, W. A., Papo, N., Shlamkovich, T., Aharon, L., … Papo, N. (2017). Utilizing combinatorial engineering to develop Tie2 targeting antagonistic angiopoetin-2 ligands as candidates for anti-angiogenesis therapy. Oncotarget, 8(20), 33571–
QuickChange Site-Directed Mutagenesis,
FRET image,