siRNA Screening Facility

The great majority of biological processes are controlled by the addition or removal of ubiquitin (Ub) and ubiquitin-like proteins (Ubl) from key regulatory molecules. Thus the level of regulatory molecules can be rapidly reduced by Ub-mediated proteasomal degradation, or protein activities can be altered by modification with atypical Ub chains or with Ubls, such as SUMO and NEDD8. The efficacy of the proteasome inhibitor Velcade in Multiple Myeloma, the first drug that targets the Ub system to be developed,  suggests that more substrate-specific inhibitors of proteasomal degradation may have increased efficacy and reduced side effects. However, identification of the most suitable targets for the development of new drugs requires a detailed knowledge of the molecules involved in protein modification and degradation by the Ub system. The siRNA screening facility that we are setting up is aimed at obtaining this information. Our first objective will therefore be to identify new targets in the Ub and Ubl protein systems that play key roles in biological pathways linked to disease processes, such as inflammation and cancer, followed by delineation of the pathways in which the identified molecules participate.

1) Identification of new targets in the Ub and Ubl protein systems that play key roles in biological pathways linked to disease

To identify disease related pathways, RNA interference will be used to switch off genes selectively and analyse how this affects particular biological processes. This technique has become an important research tool, and the Nobel Prize for Medicine was awarded to the scientists who discovered it in 2006. We will use an siRNA library directed against mRNAs encoding all known and suspected components of the Ub and Ubl conjugation and deconjugation systems in humans. The robotic and informatic infrastructure required for this screening platform has already been established in the College of Life Sciences at Dundee in which the Protein Ubiquitylation Unit is embedded. siRNA screens will be carried out on cells which have been engineered to deliver a quantifiable output from biological pathways that have important links to disease pathology. In many cases the final output from these pathways is activation of a programme of gene expression and this can easily be measured using a transcription factor-dependent luciferase reporter that has been stably integrated into the genome of an appropriate cell. Thus the first step will be to engineer cell lines expressing luciferase reporters responsive to various external stimuli, including pathogens.  These cell lines will not only be used for the cell based siRNA screening effort, but also for future testing of chemical entities directed against particular components of the Ub and Ubl pathways. An alternative strategy will be to fuse a particular protein of interest to a fluorescent protein and use the total fluorescent output as a measure of protein stability. This approach will be used with siRNA technology to identify genes that influence protein stability. The use of engineered fluorescent proteins is also amenable to high-throughput microscopy, where siRNAs will be used to identify genes that influence the transit of cells through the cell cycle.

2) Delineation of the pathways in which the identified molecules participate

Once components of the Ub system have been identified that modulate the activity of particular transcription factors, we will then identify the pathway(s) in which each molecule is(are) involved and the precise molecular target(s) of each factor. This will be accomplished by purification of protein complexes containing the protein of interest and identification of the components of the complexes using the state of the art mass spectrometry facility in Dundee.