Year: 2014
Type: dissertation
Abstract: The activator of mammalian target of rapamycin complex 1 (mTORC1), Ras homolog enriched in brain (Rheb), is a membrane-associated protein belonging to the Ras subfamily of small GTPases. Rheb's slow GTPase activity is stimulated by the GTPase activating protein (GAP) domain of tuberous sclerosis complex 1 and 2 (TSC1/2). Rheb hyperactivation, through its overexpression or loss of TSC1/2 GAP function, results in hyperactivated mTORC1 signaling culminating in tumourigenesis. The molecular details of Rheb GTP hydrolysis and the effect of membrane association on Rheb structure, dynamics and its GTPase function are currently not fully understood. The studies presented in this thesis focus on two key determinants of Rheb function i) the mechanism of Rheb GTP hydrolysis and ii) the structural and functional consequence of Rheb-bilayer membrane interaction. Through studies of fluorescent nucleotides, we revealed that the conserved G2-box residue Tyr35 auto-inhibits GTP hydrolysis in Rheb. We demonstrated that a non-canonical catalytic residue, Asp65, in the switch II region of Rheb, contributed more to the GTP hydrolysis rate than Gln64, which corresponds to the canonical Ras Gln61. This non-canonical auto-inhibited mechanism of GTP hydrolysis was required for optimal mTORC1 regulation. These structural insights were then used to guide the design of novel gain- and loss-of function mutants by substitutions of the ultra-conserved G3-box Gly63 of Rheb. Finally, using solution NMR spectroscopy, we monitored the Rheb GTPase cycle and characterized its nucleotide-dependent membrane orientations on nanodisc-based phospholipid bilayers. Rheb was shown to sample two orientations in which its C-terminal helix was semi-perpendicular or semi-parallel with respect to the bilayer plane. The semi-parallel orientation, where switch II residues critical for mTORC1 communication are accessible, was favored in the GTP bound conformation, suggesting that membrane-tethering modulates Rheb function. These structural insights into the catalytic machinery of Rheb and its membrane interface suggest new approaches to modulate these key determinants of Rheb function through small molecules towards development of therapeutic avenues for Rheb-mediated pathogenesis such as tuberous sclerosis or cancer.
Source: TSpace (University of Toronto)
Author: Mohammad T. Mazhab‐Jafari
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2765127138