Ljubljana/splitubiquitinassay

Split Ubiquitin Assay

Yeast two-hybrid system is widely used to detect the interactions between proteins, e.g. to determine the interactome. For integral membrane proteins most systems don't work and one of the few succesfull techniques is the split ubiquitin system. This assay, first described by Johnsson and Varshavsky (Johnsson and Varshavsky, 1994) is usually used for studying yeast 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. The assembling takes place only if both parts are brought together, thus applying an additional step, which can be controlled during the experiment, thus preventing spontaneous activation of the system. Another protein (e.g. reporter or effector) can be linked onto the Cub half without of major influence on ubiquitin assembly. The formed complex Nub-Cub-reporter is recognized by the ubiquitin specific protease and the reporter protein is cleaved off. Usually a transcription factor is used instead of the reporter protein, which then induces the promoter and starts transcription of a reporter gene. The system was developed to work in yeast cells, but it hasn't been shown before to work in mammalian membrane proteins.

HIV envelope consists of lipids and viral glycoproteins gp120 and gp41, which are crucial for binding of HIV to the host cell membrane and for entering into the cell. Inserted into the lipid bilayer are also other glycoproteins that guarantee firmness and protective function of the viral envelope. Gp120 binds to receptors (CD4) on the host cell surface, but additional co-receptors like chemokine receptors (CCR5, CXCR4) are also required for successful entry of HIV. Mutations in co-receptor genes can cause immunity – if HIV cannot enter host cells, HIV infection is prevented, and AIDS cannot develop.

The characteristic retroviral enzyme is reverse transcriptase, which transcripts viral RNA into DNA. Only DNA can integrate into host cell genome – this is the crucial step in expressing viral proteins that are needed for assembly of new viral particles. Viral gag and gag/pol genes are expressed as polyprotein; until this polyprotein is cut into functional units, it exerts no biological function. Polyprotein clipping is done by HIV protease. The resulting polyprotein fragments represent functional enzymes and structural proteins.

Transcription of viral RNA into DNA and processing of the viral polyprotein are two most important steps in HIV replication cycle. These are thus obvious targets for HIV therapeutics. Inhibitors of reverse transcriptase and HIV protease are currently used to treat acute HIV infection.

Current Disease Treatment

One of the first AIDS therapeutics were nucleotide or nucleoside analogues (NRTI – nucleoside-analogue reverse transcriptase inhibitors) – pseudosubstrates, that are during reverse transcription integrated into viral DNA instead of nucleosides and thus block the transcription. These drugs were superseded by non-nucleoside inhibitors (NNRTI) that could inhibit reverse transcriptase by binding into the alosteric site of the enzyme. The third type of drugs is a family of HIV protease inhibitors. In most cases specific inhibitors are very similar to protease substrates - the only difference is that because they cannot be cut, they block the active site by remaining bound into it.

Weakness of all these therapeutics is that they are very sensitive to HIV mutations – HIV can easily mutate and thus become drug resistant. A combination of drugs is used to minimize HIV's potential to develop resistance to each individual drug in the mixture. Some of the drugs induce mutations that have negative effect on the virulence and such drugs can be used in spite of developed resistance.

We still do not have a cure for AIDS that would be insensitive to HIV mutations. Our project presents new ways of potential AIDS therapeutics. Our approaches can be considered independent of HIV mutations. We have set up a few of biological ambushes; if HIV manages to avoid them, we presume that it would not be able to infect the cell anyway.

Classes of Antiretroviral Drugs

Antiretroviral drugs are mostly inhibitors of different stages in HIV life cycle. They are targeted at different enzymes or events that are typical for HIV infection – entry of the virus into the cell, reverse transcription, polyprotein cleavage... and are divided into seven main classes (REFERENCA!):

Development

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