Molecular Analysis of Thyroid Hormone Receptor Beta and Peroxisome Proliferator-Activated Receptor Gamma Action
Abstract
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.