Exploring the mechanism of CCT using small molecule inhibitors
Supervisor(s): Professors Keith Jones and Keith Willison
Section of Cancer Therapeutics (including the Cancer Research UK Centre for Cancer Therapeutics) and Section of Cell and Molecular Biology (including the Cancer Research UK Centre for Cell and Molecular Biology)
Teams: Medicinal Chemistry Three and Protein Folding and Assembly
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Summary
The eukaryotic chaperonin CCT is a megadalton protein nanomachine constructed from 8 individual subunit proteins (Fig. 1). It folds the key cellular scaffold (cytoskeletal) proteins actin and tubulin, and also the cell cycle specific regulators CDH1 and CDC20 to their native functional states. Depletion of CCT activity in human cancer cells causes complete and immediate cessation of cell division and also induces cell motility defects [1]. The cell proliferation component of CCT activity is positively regulated through growth factor dependent activation, via Ras-MAPK and PI3K-mTOR pathways, of the ribosomal kinases, RSK and S6K, which phosphorylate serine 260 in the substrate binding domain of the CCT beta/2 subunit [2]. CCT complex possesses an intrinsic ATPase activity which drives the folding function but what are the contributions of each of the 8 subunits to the global activity of the machine? It is well known that conformational changes in chaperonins are governed by allosteric signals that originate upon ATP binding/hydrolysis and occur at the intra- and inter-ring level [3]. All the chaperonins appear to have negative inter-ring co-operativity, but their behaviour with respect to intra-ring co-operativity is complex [3]. CCT has allosteric transitions that are sequential rather than concerted and we have developed a model in which ATP binding/hydrolysis occurs in a hierarchical manner, starting in CCT-alpha and ending in CCT zeta/epsilon region.
Figure 1
The initial aim of this project is to synthesise a small library of ATP-mimetics and probe the heterogeneous ATP binding pockets of the 8 CCT subunits. The compounds will first be tested in steady state ATPase assays [4] and an activity series assembled. Selected molecules would then be used in a chemical genetics screen against yeast CCT and ATP-site mutants in various of its subunits. Recent work in the Willison laboratory has led to an X-ray crystal structure for yeast CCT which is of sufficiently high resolution to guide the design of mutants. This will allow a level of understanding of this complex molecular machine which has not been achieved by any other approach. If time permits, the understanding gained of both the biology of CCT and the small molecules will be used to develop drug-like inhibitors of human CCT.
References
- Grantham, J., et al (2006) Substantial CCT activity is required for cell cycle progression and cytoskeletal organisation in mammalian cells. Exp. Cell. Res Vol 312, No 12, p2309-2324
- Abe, Y., et al. (2009) p90 ribosomal S6 kinase and p70 ribosomal S6 kinase link phosphorylation of the eukaryotic chaperonin containing TCP-1 to growth factor, insulin, and nutrient signalling. J.Biol.Chem Vol 284, No 22, p14939-14948
- Horovitz, A., and Willison, K.R. (2005) Allosteric regulation of chaperonins. Current Opinion in Structural Biology Vol 15, No 6, p646-651
- Shimon, L., et al (2008) ATP-induced allostery in the eukaryotic chaperonin CCT is abolished by the mutation G345D in CCT4 that renders yeast temperature-sensitive for growth. J.Mol. Biol Vol 377, No 2, p469-477
