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Darerca Owen

G proteins and their effectors as therapeutic targets in invasion and metastasis

For the majority of cancers the acquisition of invasive and metastatic characteristics leads to incurable disease in the host. A major challenge, therefore, is the elucidation of the underlying molecular changes that lead to the unimpeded ability of tumour cells to invade neighbouring tissue and disperse to secondary sites and the development of therapies to arrest such a progression. Small G proteins, particularly members of the Rho family, are known regulators of the actin cytoskeleton and therefore control the morphology and motility of mammalian cells. It is not surprising therefore, that they are implicated with increasing frequency in the transition to invasive and metastatic forms of cancers. This implies the existence of a therapeutic avenue directed against these proteins and their downstream effectors.

Our work addresses the relationship between structure and function in small G proteins and their effector complexes. We have determined the structures (by NMR) of the G protein, Cdc42, in complex with the G protein binding domains of its effector proteins ACK and PAK. ACK is known to function in integrin mediated adhesion pathways, while PAK is involved both in tumourigenicity and adhesion, being particularly identified as active in breast cancer metastasis. Our structures facilitated the design of mutations that selectively inhibited the interaction of Cdc42 with its effectors. Thermodynamic analysis of these mutants led to the identification of 'hotspots' on the G protein surface that define areas which could be targeted by small molecules for therapeutic purposes (figure 1). We have also studied the interaction of PRK1, another kinase with a potential role in invasion, with the Rho and Rac GTPases. We have solved the structure of the HR1b repeat of PRK1 and have used a combination of NMR and mutagenesis to determine the residues important for the interaction between Rac and PRK1 (figure 2).

Recently, we have also extended our investigations to include the Ras family member, Ral. Preliminary data indicate that the control of exocytosis by Ral is involved in cell motility and therefore another possible target for anti-metastasis therapies. First and foremost we hope our work will lead to a more detailed understanding of the protein/protein interactions involved in cell motility. The data generated, however, could also provide regulatory, structural, thermodynamic and in some cases kinetic data to assist rational drug design.

Lab members:  George Tetley, Naomi Robertson, Ana Masara Ahmad-Mokhtar, Millie Fox, Cat Hurd

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Key recent publications:

Fenwick, R.B., Campbell, L.J., Rajasekar, K., Prasannan, S., Nietlispach, D., Camonis, J., Owen, D. and Mott, H.R. (2010)The RalB-RLIP76/RalBP1 complex reveals a novel mode of Ral-effector interaction. Structure 18: 985-995

Mott, H.R. and Owen, D. (2010) RLIP76 (RalBP1): The first piece of the structural puzzle. Small GTPases 1(3): 157-160

Hutchinson, C.L., Lowe, P.N., McLaughlin, S.H., Mott, H.R.and Owen, D. (2011) Mutational analysis reveals a single binding interface between RhoA and its effector, PRK1. Biochemistry 50(14): 2860-2869

Rajasekar, K.V., Campbell, L.J., Nietlispach, D., Owen, D. and Mott, H.R. (2013) The structure of the RLIP76 (RalBP1) RhoGAP domain-Ral binding domain dyad: fixed position of the domains leads to dual engagement of small G proteins at the membrane. Structure 21(12): 2131 - 2142

Hutchinson, C.L., Lowe, P.N., McLaughlin, S.H., Mott, H.R.and Owen, D.  (2013) Differential binding of RhoA, B and C to the PRK isoforms PRK1, 2 and 3: PRKs have highest affinity for RhoB. Biochemistry 52(45): 7999–8011

Mott, H.R. and Owen, D. (2014) Structure and Function of RLIP76 (RalBP1): an intersection point between Ras and Rho signalling. Biochemical Society Transactions 42(1): 52-58

Campbell, L., Peppa, M., Crabtree, M., Shafiq, A., McGough, N., Mott, H.R.and Owen, D. (2015) Thermodynamic mapping of effector protein interfaces with RalA and RalB.Biochemistry 54: 1380-1389

Mott, H.R and Owen, D. (2015) Ras superfamily Effector Complexes: What have we learned in two decades? Critical Reviews in Biochemistry and Molecular Biology 50: 85-133

Watson, J.R., Nietlispach, D., Fox, H., Gallop, J. Owen, D. and Mott, H.R. (2016) Investigation of the Interaction Between Cdc42 and its Effector TOCA1: Handover of Cdc42 to the Actin Regulator N-WASP is Facilitated by Differential Binding Affinities. J. Biol. Chem. 291: 13875–13879

Thomas, J.T., Cooper, J., Clayton, N.S., Wang, C., White, M.A., Abell, C., Owen, D. and Mott, H.R.  (2016) Inhibition of Ral GTPases using a stapled peptide approach. J. Biol. Chem. 291: 18310-18325

Watson, J.R., Owen, D. and Mott, H.R. (2016) Cdc42 in actin dynamics: An ordered pathway governed by complex equilibria and directional effector handover. Small GTPases 7:1-8