PhD Projects

GABRIELA ALEXANDRU

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

A PhD studentship to identify novel substrates of p97/UBA-UBX protein complexes is available immediately in the group of Dr Gabriela Alexandru at the SCILLS Protein Ubiquitylation Unit.

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 degradation has been explored mainly in the context of endoplasmic reticulum-associated protein degradation, where p97 provides the driving force for protein retro-translocation from the ER into the cytosol [1]. We identified HIF1a as a novel substrate of p97 and its UBA-UBX cofactor, UBXD7 [2]. Our previous proteomic studies predict that HIF1a is just the tip of the iceberg and many other ‘soluble’, non-ERAD substrates of p97 await discovery.

The project will aim to identify specific substrates for each of the five UBA-UBX co-factors of p97 in human cells, using SILAC (stable isotope labelling by amino acids in cell culture) and the state-of-the-art mass spectrometry facility at SCILLS. Selected substrates will be further pursued using molecular/cell biology techniques.

HIF1α is so far the only non-ERAD substrate of p97 that has been linked to a particular UBA-UBX protein. The identification and characterization of other UBA-UBX protein – substrate pairs will not only expand our knowledge of p97 function and specificity, but will also allow a better understanding of the p97 mechanism of action towards substrates that do not require retro-translocation from the ER.

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.

Applications are now closed for 2010. New projects will be advertised for 2011 in due course.

ARNO ALPI

Ubiquitin-signaling in DNA damage responses

Undergraduate students with a strong scientific background in biochemistry and/or cell biology are invited for PhD Studentships to work with Dr Arno Alpi in the Protein Ubiquitylation Unit of the SCottish Institute for ceLL Signalling (SCILLS).

The laboratory aims to elucidate ubiquitin-signaling pathways that regulate cellular responses to DNA damage. In particular we are interested in the Fanconi anaemia ubiquitin-signaling pathway that is activated in the presence of DNA interstrand cross-links (ICLs). ICLs are highly toxic lesions that impede with DNA metabolism (e.g. DNA replication and transcription). If ICLs are not neatly repaired they cause genomic instability, a hallmark for cell transformation and carcinogenesis.

The goal of the project is to functionally dissect the FA ubiquitin-signaling pathway and its substrates to gain a better understanding of how this pathway protects cells from DNA damage.

The PhD project will initially focus on the identification of novel ubiquitin targets in the FA ubiquitin-signaling pathway. The student will be involved in the development of biochemical screens that utilize 2D-Dimensional Differential Gel Electrophoreses (2D-DIGE) or Stable Isotope Labeling with Amino acids in Cell culture (SILAC) proteomics. Somatic genetics and in vitro reconstitution assays will complement a set of techniques to study the molecular function of novel ubiquitin targets in the FA pathway.

Applications are now closed for 2010. New projects will be advertised for 2011 in due course.

PHILIP COHEN

The role of Pellino in regulating the innate immune system

Background
The major aim of Professor Cohen's research is to discover how the signaling pathways of the innate immune system are regulated during infection by bacteria and viruses, and to find out how they trigger the production of the correct amounts of inflammatory mediators, such as pro-inflammatory cytokines, chemokines and interferons. These substances play key roles in mounting the responses to combat these pathogens, but the uncontrolled production of inflammatory mediators causes chronic inflammatory and autoimmune diseases, such as rheumatoid arthritis, psoriasis, asthma, lupus and septic shock. It is therefore critical that the extent and duration of activation of these pathways is under very tight control.

Project
The signaling pathways of the innate immune system are activated by pathogen-derived molecules, termed Pathogen-Associated Molecular Patterns (PAMPs) when they bind to Toll-Like Receptors (TLRs) in the plasma and endosomal membranes of immune cells or to cytoplasmic receptors, such as  NOD1 and NOD2 (which bind bacterial peptidoglycans) or RIG-I and MDA5 (which bind viral RNAs).  The interaction of PAMPs with TLRs triggers the recruitment and activation of two protein kinases, termed IRAK1 and IRAK4, that are important in propagating downstream signaling but how they do this is poorly understood in molecular terms.  Activated IRAK1 is known to interact with the three related proteins, termed Pellino 1, 2 and 3, whose biological roles were unknown until recently.  Over the past two years we have discovered that the Pellinos are E3 ubiquitin ligases but only when they have been phosphorylated by IRAK1 or IRAK4 in vitro (Ordureau et al 2008, Biochem. J. 409, 43-52; Smith et al 2009, PNAS 106, 4584-4590). The Pellino isoforms attach lysine 63-linked polyubiquitin chains to other proteins that may include IRAK1, but how Pellino-mediated polyubiquitylation is involved in regulating the innate immune system is unknown.  To elucidate the roles of the Pellino isoforms in vivo we have now generated three different mouse lines in which the normal forms of Pellino 1, 2 and 3 are replaced by mutants in which the "RING-like" domain that carries the E3 ligase activity has been disabled. The aim of the proposed project is therefore to use cells from these "knock-in" mice to elucidate how the E3 ubiquitin ligase activities of Pellinos control the innate immune system.  The initial phase of the project will involve studying how the signaling pathways downstream of TLRs, NOD1/2 and RIG-I/MDA5 and the production of inflammatory mediators is altered in cells defective in Pellino  E3 ligase activity.  Further work will depend on the outcome of these studies, but is likely to focus on identifying the key cellular proteins that are ubiquitylated by Pellinos and whether the Pellinos are activated in vivo by other protein kinases distinct from  IRAK1 and IRAK4.

Applications are now closed for 2010. New projects will be advertised for 2011 in due course.

 

THIMO KURZ

The role of Cand1 in regulating the activation of Cullin-based E3 ubiqutin ligases

A PhD studentship to study the regulation of cullin-based E3 ubiquitin ligases by Cand1 is immediately available in the group of Dr Thimo Kurz at the University of Dundee in the SCILLS Protein Ubiquitylation Unit.

Cullin-Ring E3 Ligases (CRLs) are the most common type of ubiquitin ligases in the cell. These complexes are capable of ubiquitylating many different proteins due to their multi-subunit, modular composition. Tight regulation of CRLs is critical to ensure proper function of the complexes. One mode of regulation is based on the association of cullin core complexes with the ligase assembly inhibitor Cand1. Cand1 tightly binds to cullins and renders the ligase inactive by preventing the association of substrate-recognition modules.  To form a functional E3 ligase, Cand1 is released from the cullin, allowing the substrate adaptors to bind to the complex.

This PhD project will focus on the study of the Cullin-Cand1 interaction cycle. In particular, we would like to elucidate which signals regulate the binding of Cand1 to Cullins and what other proteins are involved in the process. Defects in cullin ligases and their activation have been implicated in many human diseases, including cancer, and the successful completion of this project will help us understand how this pathway contributes to disease development. The laboratory uses both, mammalian tissue culture cells and the budding yeast Saccharomyces cerevisiae as model systems. Initially, this project will start out in S. cerevisiae with the goal to later verify the obtained results in mammalian cells. The student will utilize a variety of techniques,  including genetics and biochemistry, in the course of this project.

Eligibility

Outstanding students from both the UK and overseas with a first degree in a relevant discipline are eligible to apply for this 3 year studentship.

Applications are now closed for 2010. New projects will be advertised for 2011 in due course.

 

PATRICK PEDRIOLI

Mass spectrometry based study of ubiquitin-like modifiers

Mass spectrometry (MS) allows for the rapid and efficient identification of many types of post-translational modifications (PTMs). However, due to, amongst other things, the complex fragmentation behavior of peptides conjugated to ubiquitin-like modifiers (UBLs), their study by MS remains challenging.

This PhD project is aimed at developing methodologies to enable the efficient, quantitative, and specific study of UBL-modified proteins by MS.
UBLs are key players in regulation of the emergent properties of a biological system and, as such, are involved in a number of diseases. This project will develop and provide highly demanded tools to study how the UBL modification status of the proteome changes in response to various stimuli as well as in normal vs. disease conditions.

The PhD position will be available with Dr Patrick Pedrioli starting at the beginning of 2010. The successful candidate will have a strong scientific background/interest in Biochemistry, Protein-chemistry, and Mass spectrometry. Familiarity with bioinformatic methodologies, as well as linux, C++ and PERL will be advantageous.

Applications are now closed for 2010. New projects will be advertised for 2011 in due course.