Team:IISER Bhopal/Model Module1.html
Smac Internalization Kinetics
R8 – Smac (n7) Internalization
Description
After the fusion peptide cleaves by metalloprotease, the part containing R8-Smac(n7) internalises the cancer cell to inhibit XIAP. The rate of internalisation of this protein plays an essential role in the apoptosis of the cancer cell. The rate of uptake determines how effectively the fusion peptide population can inhibit the XIAPs. In other words, if the protein internalises quickly and enters the cell in large quantities, the protein can bind with more XIAPs and stop them from inhibiting the caspase-cascade reaction.
Methods and Pathways
We have made a deterministic model using Michaelis Menten kinetics. We have assumed the protein R8-Smac(n7) present outside the cell forms an enzyme-substrate complex with the cell membrane. The complex then dissociates to give internalised R8-Smac(n7).
The pathway for this process can written as
R8-Smac(n7)outside + CM ⇋ R8-Smac(n7)-CM → R8-Smac(n7)inside + CM
Here the reactants are,
| Reactants | Description |
|---|---|
| R8-Smac(n7)outside | Fusion peptide present outside the cell, produced by bacteria in the tumour microenvironment. |
| CM | The cell membrane of cancer cells |
| R8-Smac(n7)-CM | Protein-Lipid Bilayer complex formed due to the protein-lipid interaction |
| R8-Smac(n7)inside | Internalised fusion peptide previously present outside the cell. |
Parameters for reactions
The reaction rate constants were estimated using experimental data from various papers discussing polyarginine to transport cargo via membrane translocation and experimented on HeLa cells.
| Reaction | Rate constant |
|---|---|
| R8-Smac(n7)outside + CM → R8-Smac(n7)-CM | 6.85e-7 (l/nmol*min) |
| R8-Smac(n7)-CM → R8-Smac(n7)outside + CM | 0.0850 (1/min) |
| R8-Smac(n7)-CM → R8-Smac(n7)inside + CM | 0.05 (1/min) |
Results and Conclusions
The model shows that the rate of internalisation is high initially and decreases as the reaction proceeds. Experimental data from various sources showed that the time taken for 90% internalisation cargo proteins attached with polyarginine was around 120+ minutes and the cargo protein size (150-250 amino acids) used is much bigger than Smac(n7)-R8 (only 16 amino acids). Since our fusion protein is small, we assumed that our protein will have a higher rate of internalisation. However, moderately big proteins tend to have a better rate of internalisation since they can make a bigger pore in the cell membrane. Considering these factors, we predicted that 90% of the protein will get internalised after 1.5 - 2 hours.