Molecular Analysis of Thyroid Hormone Receptor Beta and Peroxisome Proliferator-Activated Receptor Gamma Action
The nuclear receptor superfamily comprises a group of ligand-activated transcription factors that regulate the expression of target genes. They play a central role in diverse physiological pathways, and are therefore extremely important in the aetiology of various human disorders and as pharmaceutical therapeutic targets. This thesis describes molecular analyses of the thyroid hormone receptor (TR) and the peroxisome proliferator-activated receptor gamma (PPARγ), in disorders of thyroid hormone and insulin action respectively. The syndrome of Resistance to Thyroid Hormone (RTH), characterized by reduced tissue responsiveness to circulating thyroid hormones, is associated with diverse mutations in the ligand-binding domain of the thyroid hormone β receptor, localizing to three clusters around the hormone binding cavity. The first part of this thesis describes three novel RTH mutations (S314C, S314F, S314Y), due to different amino acid substitutions in the same codon, occurring in six separate families. Characterization of these mutant receptors showed marked differences in their functional impairment. In the second part of the thesis I report detailed functional studies of natural and synthetic receptor agonists with loss-of-function PPARγ mutants (P467L; V290M), previously identified in patients with severe insulin resistance, type 2 diabetes mellitus and hypertension. Both PPARγ mutants act as dominant negative inhibitors of wild type receptor (WT) action because of their failure to fully dissociate from corepressors. My results provide evidence that tyrosine-based rather than thiazolidinedione PPARγ agonists, may represent a more rational therapeutic approach to restoring mutant receptor function and ameliorating insulin resistance in our patients. Then, in an unrelated kindred a different, digenic mechanism of insulin resistance, with a combination of loss-of-function mutations in PPARγ and PPP1R3 (muscle-specific subunit of protein-phosphatase 1 mediating glycogen synthesis) is described. Functional characterisation of these mutant proteins provides unique insights into the complex interplay between this nuclear receptor and a second metabolic signalling pathway. Finally, three novel heterozygous mutations, in the ligand and DNA binding domains of PPARγ, identified in three unrelated subjects with partial lipodystrophy, severe insulin resistance, dyslipidaemia and hypertension are described. Their functional characterization suggests that they inhibit WT action via a novel, non DNA-binding interference mechanism.