PhD Projects

GABRIELA ALEXANDRU

Identification of Novel p97 Substrates through Exploration of the UBA-UBX Protein Interactome

A PhD studentship to identify novel substrates of p97/UBA-UBX protein complexes is available in the group of Dr Gabriela Alexandru at the SCILLS Protein Ubiquitylation Unit, University of Dundee.
p97 and its UBA-UBX cofactors interact with ubiquitylated proteins and facilitate their degradation by the proteasome. The role of p97 in regulating ubiquitin-dependent protein degradation has been explored mainly in the context of endoplasmic reticulum-associated degradation, where p97 provides the driving force for protein retro-translocation from the ER into the cytosol [1]. We identified HIF1 alpha as a novel substrate of p97 and its UBA-UBX cofactor UBXD7 [2]. Our previous proteomic studies suggest that HIF1 alpha is just the tip of the iceberg and many other ‘soluble’, non-ERAD substrates of p97 remain to be discovered.
This project aims to identify specific substrates for each of the five UBA-UBX cofactors of p97 in human cells, using SILAC (stable isotope labelling by amino acids in cell culture) and the state of the art SCILLS mass spectrometry facility. Selected substrates will be further analysed using molecular/cell biology techniques.
Up to now, only a couple of soluble substrates of p97 have been linked to a particular UBA-UBX protein [2, 3]. The identification and characterization of multiple UBA-UBX protein – substrate pairs will reveal what distinguishes each of these cofactors and will expand our knowledge of p97 function and specificity.

References
1. Ye, Y., Meyer, H.H., and Rapoport, T.A. (2001). The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol. Nature 414, 652-656.
2. Alexandru, G., Graumann, J., Smith, G.T., Kolawa, N.J., Fang, R., and Deshaies, R.J. (2008). UBXD7 binds multiple ubiquitin ligases and implicates p97 in HIF1alpha turnover. Cell 134, 804-816.
3. Zhang, L., Zhou, F., van Laar, T., Zhang, J., van Dam, H., and Ten Dijke, P. (2011). Fas-associated factor 1 antagonizes Wnt signaling by promoting beta-catenin degradation. Mol Biol Cell 22, 1617-1624.

ARNO ALPI

Characterization of a novel modulator of hypersensitivity to the anticancer drug cisplatin

Background
Cisplatin and other derivatives of platinum-based agents are commonly used in chemotherapy to treat a wide variety of solid tumors. Cisplatin is a potent genotoxin that interferes initially with proliferation of tumor cells but ultimately induces apoptosis of tumor cells. Despite the successful application of this drug for over 30 years, side effects, in particular myelosuppression and neurotoxicity, remain the limiting factor for its therapeutic efficacy. Furthermore, efficient therapy is frequently challenged by the problem that tumor cells are either intrinsically resistant or acquire resistance to platinum drugs. To further advance the therapeutic utilities of platinum-based drugs, it will be crucial to obtain a complete understanding of the molecular mechanism(s) underlying the cellular response to this type of drugs.

Aim
The project aims to understand better the cellular response(s) to the anticancer drug cisplatin, with the goal of identifying and characterizing modulators that enhance cells’ sensitivity to cisplatin. We designed and performed a small interference RNA synthetic lethality screen and identified a novel, not yet characterized, membrane associated E3 RING ubiquitin ligase. The project intends to study this ubiquitin-signaling component in a novel response to cisplatin and how it might impinge on DNA repair and apoptotic response pathways. The project will exploit biochemical (e.g. mass spectroscopic analysis of ubiquitin modifications) as well as genetic techniques.

PHILIP COHEN

Background
The major goal of my research is to discover how the signaling pathways that control the innate immune system are regulated during infection by bacteria and viruses and to work out how they trigger the production of inflammatory mediators and interferons. These substances are crucial for defence against infection, but their overproduction causes chronic inflammatory and autoimmune diseases, such as rheumatoid arthritis, psoriasis, lupus, sepsis and some leukaemias. Identifying the protein kinases and components of the ubiquitin system involved in regulating this process, finding out how they are controlled and how they exert their effects is therefore critical to evaluate their potential as drug targets for the treatment of inflammatory and autoimmune diseases.

Identifying the roles of Pellino isoforms in regulating innate immunity
Pellino was originally identified in the fruit fly Drosophila melanogaster as a protein that interacts with Pelle, the orthologue of interleukin-receptor associated kinase 1 (IRAK1) in the fly. Mammalian cells express three isoforms of Pellino, Pellino 1, 2 and 3. We established that all these isoforms are phosphorylated by IRAK1 and IRAK4 in vitro and that this transforms them from inactive to active E3 ubiquitin ligases [1, 2]. More recently, we made the surprising finding that the IKK-related kinases (TBK1 and IKKε), and not the IRAKs, phosphorylate and activate Pellino 1 in macrophages stimulated by synthetic (Poly(I:C), a mimetic of viral double-stranded RNA which activates Toll-Like Receptor 3 (TLR3),  and by the TLR4 ligand bacterial lipopolysaccharide (LPS) [3].  We also found that by activating the transcription factor Interferon Regulatory Factor 3 (IRF3) the IKK-related kinases stimulate transcription of the gene encoding Pellino 1, greatly increasing the level of expression of the Pellino 1 protein [3]. We generated a knock-in mouse in which the normal form of Pellino 1 has been replaced by an E3 ligase-deficient mutant and studies with macrophages from these mice have demonstrated a key role for Pellino 1 in the immune systems that cannot be replaced by the other isoforms Pellino 2 and Pellino 3 (unpublished work). The aim of the project is therefore to discover how Pellino 2 and Pellino 3 are regulated, to identify their physiological substrate(s) and to understand their roles in regulating the innate immune system. To this end, we are generating antibodies that recognize these proteins and have recently generated further knock-in mice in which the normal forms of Pellino 2 and Pellino 3 have been replaced by E3 ligase defective mutants. The project will also exploit novel techniques that we have developed for capturing Lys63-linked polyubiquitylated proteins.

References
1. Ordureau, A., Smith, H., Windheim, M., Peggie, M., Carrick, E., Morrice, N. and Cohen, P. (2008). The IRAK-catalysed activation of the E3 ligase function of Pellino isoforms induces the Lys63-linked polyubiquitination of IRAK1. Biochem. J. 409, 43-52.
2. Smith, H., Peggie, M., Campbell, D.G., Vandermoere, F., Carrick, E. and Cohen, P. (2009) Identification of the phosphorylation sites on the E3 ubiquitin ligase Pellino that are critical for activation by IRAK1 and IRAK4. Proc. Natl. Acad. Sci. USA 106, 4584-4590.
3. Smith, H. L., Liu, X. Y., Dai, L., Goh, E. T., Chan, A. T., Xi, J., Seh, C. C., Qureshi, I. A., Lescar, J., Ruedl, C., Gourlay, R., Morton, S., Hough, J., McIver, E. G., Cohen, P. and Cheung, P. C. (2011). The role of TBK1 and IKKε in the expression and activation of Pellino 1. Biochem. J. 434, 537-548.