Lawson, Alexander James Frank (2010)
Ph.D. thesis, University of Birmingham.
11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is a key enzyme in the conversion of cortisone to the functional glucocorticoid hormone cortisol. This activation has been implicated in several human disorders, notably the metabolic syndrome where 11β-HSD1 has been identified as a novel target for potential therapeutic drugs. Crystal structures have revealed the presence of a pronounced hydrophobic surface patch lying on two helices at the C-terminus. The physiological significance of this region has been attributed to facilitating substrate access by allowing interactions with the endoplasmic reticulum membrane. In order to initiate investigations into the potential interaction between 11β-HSD1 and membrane, hydrophobic residues in the C-terminal helices of human and guinea pig 11β-HSD1 were mutated to glutamic acid. One of these mutants, F278E, displayed greatly increased yields of soluble protein on expression in E.coli together with an increase in both monodispersity and activity. No change in structure was observed when compared to the previously reported wild-type structure. Human F278E 11β-HSD1 enzyme was then used as a background to construct a hybrid dimer system to analyse novel heterozygous mutations in the HSD11B1 gene (R137C and K187N) which are thought to give rise to cortisone reductase deficiency (CRD). Expression of the heterodimers in E.coli revealed that the negative effects of both mutations can extend to the normally-active WT partner, leading to a marked suppression of 11β-HSD1 activity, which could account for the phenotype observed in patients presenting with CRD. Finally, a comparison of human WT and F278E 11β-HSD1 enzymes was used to probe the membrane association of 11β-HSD1. A combination of western blot analysis and fluorescence quenching experiments revealed that the binding of 11β-HSD1 to membrane is via both hydrophobic and electrostatic interactions.
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