Fu, C; (2013) Structural and Functional Analysis of mTORB. Masters thesis , UCL (University College London).

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Abstract

mTOR is a serine/threonine protein kinase that has been shown to be a key player in the regulation of cell growth and proliferation. Furthermore, mTOR forms the catalytic core of two known mTOR complexes, mTORC1 and mTORC2. These complexes sense various intra and extracellular signals, and regulate cellular processes that are critical for cell growth and proliferation. However, when conventional mTOR signalling is deregulated, cellular homeostasis is disrupted, resulting in a wide range of human diseases such as diabetes, neurodegeneration and cancer. Due to its involvement in tumorigenesis, mTOR has attracted enormous interest as a therapeutic target. Initially, the classical mTOR inhibitor rapamycin was tested as a potential treatment. However, when the compound was assessed in clinical trials, it proved to be of limited efficacy. This led to the design of novel types of inhibitors, which are currently being evaluated. The results obtained with rapamycin clearly indicated that our understanding of the mTOR signalling pathway is far from complete. In addition, mTOR is currently known to exist in two isoforms, which are generated by alternative splicing of the transcript. These are known as mTORα and mTORβ respectively. The mTORα protein was the first isoform discovered and is 2,549 residues long. mTORβ is approximately one third of the length at 706 amino acids. Both proteins share identical C-terminal domains, but mTORβ lacks the Nterminal HEAT and FAT repeats that mTORα possesses. Work done in our lab has shown that mTORβ is capable of forming complexes with Raptor and Rictor, which are the key components of mTORC1 and mTORC2. Furthermore, overexpression of mTORβ transforms immortal cells and causes tumour formation in nude mice. It is thought that modulation of cell proliferation via the mTOR signalling pathway could be achieved through mTORβ, which behaves as a protooncogene. Thus, mTORβ has the potential to be used as a target for anti-cancer therapies. The first chapter of my thesis consisted of comparative modelling of mTORβ’s C-terminal region from the FRB domain to the kinase domain. The model that was generated could then be used to give us insight into potential mechanisms for the inhibition of mTOR by either rapamycin or ATP-competitive inhibitors. The second chapter examined the effects of two different mutations in mTOR’s kinase domain on its activity. A point mutation (S2215Y) and a deletion of 12 amino acids (12del) were introduced into the kinase domain of mTORβ. Mutant proteins were expressed in HEK293 mammalian cells and the phosphorylation status of various mTOR substrates was assessed under different experimental conditions. The final chapter of my thesis described how a TAP-tag fusion protein was created. This would have been used to search for novel mTORβ binding partners in mammalian cells had I chosen to complete my PhD studies.

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