Development of a Novel NMR-based Rheb GTPase Assay and Molecular Characterization of TSC2 GAP Activity.

摘要

The biological activity of the tuberous sclerosis 1/2 complex (TSC1/TSC2), key molecules in the etiology of tuberous sclerosis, is sensitive to a number of homeostatic signals within the cell, including cellular energy levels, availability of oxygen and nutrients and growth factor stimulation. Mechanistically, TSC1 appears to protect TSC2 from proteolytic degradation, whereas TSC2 possesses GTPase activating protein (GAP) activity towards a small GTPase from the Ras superfamily named Rheb. We hypothesized that nuclear magnetic resonance (NMR) could be used to monitor Rheb s guanine nucleotide- dependent Rheb s structural changes, in real time, to provide a dynamic readout of its intrinsic and stimulated GTPase activity. Based on these principles we developed a real-time NMR-based GTPase assay which allows unprecedented insight into the molecular mechanism of action of GTPases and implemented it to characterize the molecular mechanism of GTP catalysis by Rheb and the impact of the TSC2 GAP activity on this process. Further, we characterized a series of TSC2 GAP domain mutants found in patients with tuberous sclerosis and determined the molecular mechanism of action of the TSC2 GAP activity on Rheb.