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, the first drug that targets the Ub system to be developed, in treating Multiple Myeloma 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 team that we have set 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 a 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 informatics infrastructure required for this screening platform has been established in the College of Life Sciences at Dundee in which the Protein Ubiquitylation Unit is embedded. We have carried out a wide range of screens using either unmodified cells or cells that have been engineered to deliver a quantifiable output from biological pathways that have important links to disease pathology. A variety of different screening formats can be employed measuring cell viability, transcriptional reporter readouts or high content imaging. siRNA screening is particularly suited to the analysis of "synthetic lethality".
An excellent example of this approach is the exquisite sensitivity of BRCA1/2 mutant cells to inhibition of PARP. Searching for such synthetic lethal interactions with a known pharmacological agent can now be accomplished using siRNA screening. Cells treated with siRNA are exposed to a sub-lethal dose of a pharmacological agent and after an appropriate time cell viability assays are performed. siRNAs that sensitise the cells to the action of the drug are candidate synthetic lethal interactors of the drug. This information will identify pathways that function redundantly with the target of the drug and may define additional targets for pharmacological intervention or define genetic populations that may benefit most from use of the drug. siRNA screens can be carried out on cells which have been engineered to measure a transcriptional output that relates to a particular disease. Thus activation (or repression) of a programme of gene expression can be easily measured using a transcription factor dependent luciferase reporter that has been stably integrated into the genome of an appropriate cell. siRNAs which result in reduced transcriptional output from these transcription factors would represent excellent targets for therapeutic intervention in these diseases. While these cell lines would represent the basis for the cell based siRNA screening effort, they would also be an extremely useful resource for future testing of chemical entities directed against particular Ub and Ubl pathway components. In our hands a highly robust and flexible screening modality is the use of high content imaging using either InCell2000 or Operetta automated microscopes. If high quality antibodies to the target of interest are available then the levels, or subcellular localisation, of a particular protein can be followed during the course of a physiological response. The siRNA screen would thus identify those siRNAs that interfere with such a response. This approach is particularly suited to cells that have been engineered to express a protein of interest fused to a GFP tag that can be followed without recourse to antibody staining.
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 components, 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.