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

Tumor Cells Cells grow uncontrollably

Tumor Cells Cells grow uncontrollably Benign or Malignant

Tumor Cells Cells grow uncontrollably Benign or Malignant Malignant=Cancerous

Growth of Tumor Cells More nutrients = More growth

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

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

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

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

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?

Barton et al 2010 If Ang1 present→ Tie2 Dimerization

If Ang1 present→ Tie2 Dimerization = Angiogenesis

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

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

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

Tie2 Tie1 Barton et al 2010

Tie2 Tie1

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

Experiment 1)Find Amino Acids to mutate

Experiment 1)Find Amino Acids to mutate → Pymol

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

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

Experiment→ QuickChange Site-Directed Mutagenesis

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

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

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

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

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

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

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

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

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

Outcome Successful: Tie1/Tie2 stay together

Outcome Successful: Tie1/Tie2 stay together Attraction strong enough

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

Possible Issues Electrostatic Interaction→ Not strong bond

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

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

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

Questions?

Further Explanation of FRET Tie 2 Tie 1 Tie 2 Tie 1

Pymol Further Explanation

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.

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. https://doi.org/10.1016/j.molcel.2010.02.007 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), 7205–7210. https://doi.org/10.1073/pnas.1216890110 Adair, T. H., & Montani, J.-P. (2010). Overview of Angiogenesis. Retrieved from https://www.ncbi.nlm.nih.gov/books/NBK53238/ 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), 33571–33585. https://doi.org/10.18632/oncotarget.16827 QuickChange Site-Directed Mutagenesis, http://kirschner.med.harvard.edu/files/protocols/Stratagene_quickchangepdf.pdf FRET image, http://www.rowland.harvard.edu/labs/bacteria/images/fret2.jpg