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Engineered Human Cells: STOP HIV



Problem & Idea
Project
Results
Team


Idea
Basic of our idea is to connect dimerization of two human transmembrane receptors CD4 and CCR5 or CXCR4 with apoptotic pathway. We discussed several ways how to do this on the end we decided to use the split ubiquitin assay as fundament for our new signaling pathway. Split ubiquitin assay is described on right.

Split ubiquitin assay

This assay is usually used for studying membrane protein interactions. Ubiquitin can be expressed as N-terminal (Nub) and C-terminal (Cub) half, which retain affinity for each other, and assemble into functional split ubiquitin. If another protein (reporter) is fused with C-terminal half of ubiquitin (Cub) it is cleaved off by so called cellular ubiquitin specific protease immediately after the assemble of Nub and Cub. Point mutation in the N-terminal half (NubG) completely abolishes the affinity of both halves for each other and fusion protein Cub-reporter is not recognized by ubiquitin specific protease. But fusion of NubG and Cub-reporter with interacting membrane proteins returns ability of both halves to assemble. Complex is later recognized by ubiquitin specific protease and reporter protein is cleaved off. Usually transcription factor is used instead of reporter protein fused to Cub, which than induces the promoter and starts transcription of reporter gene.
Split ubiquitin(low)


Solution
For the purpose of our project we fused CD4 transmembrane receptor with Cub, CCR5 with NubG and also CXCR4 with NubG (so our pathway is induced with HIV-2 viruses too). Basic idea of our project is that dimerization of receptors caused by HIV enables assembly of NubG and Cub, complex is later recognized by ubiquitin specific protease and efector protein is released from the CD4-Cub fusion protein. But virus causes dimerization of just few receptors, so we could not use direct efector (for instance caspase or DNAse), because in such case the signal would be too weak and probably would not activate apoptosis pathway. We decided to use very specific T7 RNA polymerase for fusion with Cub. This is polymerase from the T7 bacteriophage which translates only genes under the specific T7 bacteriophage promoter. Promoter T7 is very strong and is not recognized by any other cellular RNA polymerase. Under this promoter we inserted death efector caspase 3 gene which is part of the apoptosis pathway and causes controlled cell death. We also fused T7 RNA polymerase with nuclear localization signal (NLS) on C-termial end. To summarize, when dimerization of receptors occurs (HIV infection), T7 RNA polymerase is released from the membrane than translocated into the nucleus (because of NLS signal), where it transcribes efector genes under the T7 promoter. Apoptosis starts. We also designed construct T7 polymerase under the T7 promoter for self amplification of the signal. In later phases of experiment we also added specific HIV protease cleavage site between Cub and T7 polymerase. Therefore presence of virus protease also causes T7 polymerase translocation and apoptosis pathway occurs.




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