Genome-wide association (GWA) studies have provided significant insight into the underlying genetic components of common human diseases such as type 2 diabetes (T2D). However, the translation of such genetic findings into biological and clinical insight remains a major challenge. One of the genes implicated in T2D pathogenesis and effects on related glycaemic and lipidaemic traits by the GWA approach is GCKR, encoding glucokinase regulatory protein (GKRP). GKRP inhibits the glycolytic enzyme glucokinase (GCK) in the liver, sequestering it in an inactive form in the nucleus. Together, GCK and GKRP exert a significant proportion of control of hepatic glycolytic flux, facilitating glucose uptake and disposal in the fed state and inhibiting glycolysis in the fasting state. A common nonsynonymous variant in GCKR, p.P446L, was identified by a combination of genetic and functional approaches as the likely causative variant underlying the GWA findings. The P446L variant protein has been shown to have diminished capacity to inhibit GCK relative to wild-type (WT) GKRP, resulting in enhanced hepatic glycolytic flux and activation of liver synthetic pathways, including those generating triglycerides. Genes such as GCKR harbouring common variants of discernible functional effect may also contain rare variants of large effect. Accordingly, I aimed to comprehensively identify and functionally characterise nonsynonymous variants across the allelic frequency spectrum in GCKR, both by applying and adapting existing kinetic techniques and by developing methodologies to assess the effects of variants on the subcellular localisation of GKRP and GCK. Additionally, I aimed to relate these findings back to clinically important phenotypes, and to further characterise the binding of GCK and GKRP by searching for novel small- molecule modulators of this interaction. Using fluorescent fusion proteins transiently transfected into HeLa cells, I showed that human GKRP localises to the nucleus and sequesters human GCK. I also demonstrated that the common P446L variant significantly reduces the ability of G KRP to localise to the nucleus and sequester GCK. I then investigated the cellular and kinetic characteristics of 18 additional nonsynonymous GCKR variants identified in the National Institutes of Health's ClinSeqTM cohort, determining that the majority of variants affected protein function, and that these effects could be divided into distinct sub-classes. Variants causing a significant loss of function were associated with increased lipid levels in the cohort. Finally, I developed robust assays capable of measuring the interaction of re comb in ant GKRP and GCK in a format suitable for quantitative high-throughput screening of up to 400,000 small-molecule compounds. While no compounds that specifically affected this interaction have been identified to date, the assays developed could be useful in future studies of GCK and GKRP. These data provide further insight into the critical regulatory role of GCKR in metabolism, the structure and function of the GKRP protein, and the potential pathogenic consequences of human variants within GCKR.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:589604 |
| Date | January 2012 |
| Creators | Rees, Matthew Geoffrey |
| Contributors | Collins, Francis ; Gloyn, Anna ; McCarthy, Mark |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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