Structural and Functional Analysis of mTORB

mTOR is a serine/threonine protein kinase that has been shown to be a key player in \nthe regulation of cell growth and proliferation. Furthermore, mTOR forms the \ncatalytic core of two known mTOR complexes, mTORC1 and mTORC2. These \ncomplexes sense various intra and extracellular signals, and regulate cellular \nprocesses that are critical for cell growth and proliferation. However, when \nconventional mTOR signalling is deregulated, cellular homeostasis is disrupted, \nresulting in a wide range of human diseases such as diabetes, neurodegeneration and \ncancer. Due to its involvement in tumorigenesis, mTOR has attracted enormous \ninterest as a therapeutic target. Initially, the classical mTOR inhibitor rapamycin was \ntested as a potential treatment. However, when the compound was assessed in clinical \ntrials, it proved to be of limited efficacy. This led to the design of novel types of \ninhibitors, which are currently being evaluated. The results obtained with rapamycin \nclearly indicated that our understanding of the mTOR signalling pathway is far from \ncomplete. \nIn addition, mTOR is currently known to exist in two isoforms, which are \ngenerated by alternative splicing of the transcript. These are known as mTORα and \nmTORβ respectively. The mTORα protein was the first isoform discovered and is \n2,549 residues long. mTORβ is approximately one third of the length at 706 amino \nacids. Both proteins share identical C-terminal domains, but mTORβ lacks the Nterminal \nHEAT and FAT repeats that mTORα possesses. Work done in our lab has \nshown that mTORβ is capable of forming complexes with Raptor and Rictor, which \nare the key components of mTORC1 and mTORC2. Furthermore, overexpression of \nmTORβ transforms immortal cells and causes tumour formation in nude mice. It is thought that modulation of cell proliferation via the mTOR signalling pathway could \nbe achieved through mTORβ, which behaves as a protooncogene. Thus, mTORβ has \nthe potential to be used as a target for anti-cancer therapies. \nThe first chapter of my thesis consisted of comparative modelling of \nmTORβ’s C-terminal region from the FRB domain to the kinase domain. The model \nthat was generated could then be used to give us insight into potential mechanisms for \nthe inhibition of mTOR by either rapamycin or ATP-competitive inhibitors. \nThe second chapter examined the effects of two different mutations in \nmTOR’s kinase domain on its activity. A point mutation (S2215Y) and a deletion of \n12 amino acids (12del) were introduced into the kinase domain of mTORβ. Mutant \nproteins were expressed in HEK293 mammalian cells and the phosphorylation status \nof various mTOR substrates was assessed under different experimental conditions. \nThe final chapter of my thesis described how a TAP-tag fusion protein was \ncreated. This would have been used to search for novel mTORβ binding partners in \nmammalian cells had I chosen to complete my PhD studies.