Group Leader: Professor Kelvin Cain
Summary of Research Interests
The MRC Toxicology Unit Protein Profiling Group is a project driven interactive protein profiling facility studying molecular mechanisms in toxicology, cancer and neurodegeneration. We have specialised in developing ‘quantitative proteomics’ and affinity targeting of important biological proteins and both soluble/insoluble and membrane complexes involved in cell death, neurodegeneration and cancer-related toxicity (e.g. the apoptosome and DISC complexes). Quantitative proteomics allows rapid identification and quantitation of proteins after separation of native protein(s)/complexes on SDS-PAGE gels, tryptic digestion and analysis by LC-MS/MS. Coupled with Scaffold bioinformatics software, ‘quantitative proteomics’ provides an essential technology platform for protein profiling of cells and tissue in a variety of toxicological, cancer and neurodegenerative scenarios (Fig.1). Emerging bioinformatics software such as to assess pathway interactions is also being used interpret quantitative proteomics data.
The protein profiling group collaborates with other members of the Toxicology Unit in a variety of projects. The projects vary with some being led by the Protein Profiling Groups to other projects where the group provides protein identities on a sample by sample basis. A number of projects involve joint collaboration with Unit programmes often by means of joint PhD studentships. Selected specific projects are listed below.
1. Cell death complexes-Identifying and characterising large multi-protein caspase-activating complexes such as the apoptosome which is involved is involved in mitochondrial-mediated (apoptosome) cell death. We are also in collaboration with Dr Marion MacFarlane (Receptor-Mediated and Cell Signalling in Cell Death) analysing the death-inducing signalling complex (DISC) which mediates both Fas and TRAIL receptor-induced cell death. Quantitative proteomics cannot only identify novel proteins in such complexes but can provide important stoichiometric information of the complex itself which can be used to understand how these multi-protein complexes signal for cell death or survival. This approach coupled with structural modelling studies has been used describe a new model structure for the DISC.
2. Protein profiling of leukemic plasma membrane and cell surface receptors- Chronic lymphocytic leukaemia and Mantle Cell Lymphoma (MCL) are aggressive B- cell malignancies which are not susceptible to current highly toxic chemotherapy. There is a great need to specifically target malignant specific cell membrane receptors and antigens. With Professor Martin Dyers group (Genomic Instability) we have used plasma membrane/lipid raft purification and ‘quantitative proteomics’, RT-PCR and immunoblotting to characterise MCL plasma membrane proteins. Major changes in the lipid raft signalling domains were identified along with a number of novel proteins, including a cation channel protein HVCN1. HVCN1 has since been shown to be an abundant protein in normal human peripheral B cells and modulates BCR signalling via reactive oxygen species (Fig. 2). New, unanticipated, BCR interacting proteins continue to be identified and our studies with MCL highlighted that quantitative proteomics has the ability to identify such changes. Thus, in HVCN1 deficient B-cells attenuated BCR signalling results in decreased activation of PI3K and AKT and impaired increases in glycolysis and oxidative phosphorylation (Fig. 2).
The voltage gated proton channel HVCN1 controls BCR signalling via BCR-dependent generation of reactive oxygen species.
3. Bionergetic and mitochondrial proteome studies. Tumors often rewire their metabolism (Warburg effect) to ensure constant supplies of ATP, reducing equivalents and intermediary metabolites for growth. Emerging evidence suggests that oxidative phosphorylation and aerobic glycolysis can cooperate to provide the energy for growth. Using a Seahorse Bioscience extra-cellular flux analyzer we can simultaneously analyse mitochondrial respiration and aerobic glycolysis in intact cells. Using this approach we identifed aberrant B-cell metabolism in HVCN1 knock out mice and altered neuronal bioenergetics in cerebral granule neurones isolated from thymidine kinase 2 KO mice. We have also investigated with Dr MacFarlanes group how switching metabolism from aerobic to oxidative phosphorylation can modify the response of cells to both intrinsic (TRAIL) and extrinsic cell death stimuli. Metabolic switching is controlled by interacting stress and cell-signalling pathways (Fig. 3). To study this we are analysing metabolism, and mitochondrial and glycolytic proteomes of primary and leukemic cell lines. We hope to identify changes in the mitochondrial proteome and associated proteins which can influence cellular metabolism, apoptotic cell death and response to cytotoxins. These combined approaches can be used to study aberrant B-cell signalling and energy metabolism in B-cell related diseases. For example, inappropriate signalling from the multi-protein B cell receptor (BCR) complex is a contributory factor to malignant B-cell survival in non-Hodgkin Lymphoma and new, unanticipated, BCR interacting proteins continue to be identified. Our studies with MCL and HVCN1 highlighted that quantitative proteomics has the ability to identify such changes.
Akt modulates cellular energy metabolism, cell survival and proliferation. Akt stimulates both aerobic glycolysis and oxidative phosphorylation by promoting metabolic coupling between these pathways through enhanced association between HK1/2, VDAC and mitochondria.
4. Other emerging projects involve collaborations with new groups who are concentrating on the role of protein translation in the cytotoxic response (Professor Anne Willis and Dr Martin Bushell). These studies will identify RNA binding proteins in controlling the response to toxic injury (Professor Anne Willis) and identification of potential kinase targets and other proteins involved in miR-RNA34c driven response (Dr Martin Bushell). Such studies and the above metabolic studies will involve mapping metabolic and cell–signalling pathways using pathway analysis (Fig. 4). Thus, we can correlate how changes in mitochondrial proteins can affect, aerobic glycolysis, levels of ATP and ROS production which can affect cell death and survival.
Mitochondria were purified from primary chronic lymphocytic and mantle cell leukemic cells and a Ramos cell lines, and then analysed by LC-MS/MS and individual proteins identified using Synapt G2 mass spectrometer. Quantitation used label-free spectral counting (Scaffold), and analysed by GeneGo Systems Biology and Pathway Analysis software. Data shows expression levels of various subunits of mitochondnal complex I.
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