| Popis: |
GTP-cyclohydrolase-1 (GCH1) catalyses the rate-limiting step in the biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor for enzymes including aromatic amino acid hydroxylases and nitric oxide synthases. Strategies that increase vascular BH4 biosynthesis represent a promising therapeutic approach for the treatment of endothelial dysfunction. GCH1 is subject to feedback and feed-forward regulation by BH4 and L-phenylalanine (L-phe) respectively, via an allosteric protein interaction with GCH1 feedback regulatory protein (GFRP). The aim of this thesis was to investigate the GCH1-GFRP interaction using recombinant proteins, and to validate the functional significance of the interaction in the vasculature using animal models. Human GCH1 and GFRP proteins were recombinantly expressed. Studies compared the activity and protein interactions of native GCH1 with a truncated mutant. A kinetic GCH1 activity assay was modified to enable a high-throughput screen of a fragment library. GCH1-GFRP interactions were assessed using surface plasmon resonance (SPR). Finally, in-vivo and ex-vivo functional studies in rodents investigated the effects of L-phe on vascular function and BH4 levels. Studies using recombinant proteins revealed the activity of truncated GCH1 exceeds that of native GCH1. A high-throughput screen successfully identified four compounds that modulate GCH1 activity. Biophysical analysis (SPR) demonstrated that both native and truncated GCH1 bind to GFRP in the absence of natural ligands. The kinetics and binding rate constants have been reported for the first time. In functional studies, oral L-phe supplementation in rodents led to a rise of BH4 levels within aortic tissue, and reversed vascular dysfunction observed in vessels obtained from spontaneously hypertensive rats. This thesis demonstrates that modulation of the GCH1-GFRP interactions represents a novel therapeutic target to regulate endogenous BH4 levels. SPR data suggests that GCH1 and GFRP are constitutively bound in-vivo and indicate that the N-terminal region of GCH1 may directly interact with GFRP and modulate basal enzyme activity. The functional effects of L-phe on vascular BH4 levels and function, validate the potential of the GCH1-GFRP pathway as a therapeutic target for cardiovascular disease. |
