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Biochemical and biophysical investigations of the interaction between human glucokinase and pro-apoptotic BAD.

ABSTRACT: The glycolytic enzyme glucokinase (GCK) and the pro-apoptotic protein BAD reportedly reside within a five-membered complex that localizes to the mitochondria of mammalian hepatocytes and pancreatic ?-cells. Photochemical crosslinking studies using a synthetic analog of BAD's BH3 domain and in vitro transcription/translation experiments support a direct interaction between BAD and GCK. To investigate the biochemical and biophysical consequences of the BAD:GCK interaction, we developed a method for the production of recombinant human BAD. Consistent with published reports, recombinant BAD displays high affinity for Bcl-xL (KD = 7 nM), and phosphorylation of BAD at S118, within the BH3 domain, abolishes this interaction. Unexpectedly, we do not detect association of recombinant, full-length BAD with recombinant human pancreatic GCK over a range of protein concentrations using various biochemical methods including size-exclusion chromatography, chemical cross-linking, analytical ultracentrifugation, and isothermal titration calorimetry. Furthermore, fluorescence polarization assays and isothermal titration calorimetry detect no direct interaction between GCK and BAD BH3 peptides. Kinetic characterization of GCK in the presence of high concentrations of recombinant BAD show modest (<15%) increases in GCK activity, observable only at glucose concentrations well below the K0.5 value. GCK activity is unaffected by BAD BH3 peptides. These results raise questions as to the mechanism of action of stapled peptide analogs modeled after the BAD BH3 domain, which reportedly enhance the Vmax value of GCK and stimulate insulin release in BAD-deficient islets. Based on our results, we postulate that the BAD:GCK interaction, and any resultant regulatory effect(s) upon GCK activity, requires the participation of additional members of the mitochondrial complex.

SUBMITTER: Rexford A

PROVIDER: S-EPMC5300155 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Biochemical and biophysical investigations of the interaction between human glucokinase and pro-apoptotic BAD.

Rexford Alix A Zorio Diego A R DA Miller Brian G BG

PloS one 20170209 2

The glycolytic enzyme glucokinase (GCK) and the pro-apoptotic protein BAD reportedly reside within a five-membered complex that localizes to the mitochondria of mammalian hepatocytes and pancreatic β-cells. Photochemical crosslinking studies using a synthetic analog of BAD's BH3 domain and in vitro transcription/translation experiments support a direct interaction between BAD and GCK. To investigate the biochemical and biophysical consequences of the BAD:GCK interaction, we developed a method fo ...[more]

PMID: 28182770

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Project description:Recent genetic and clinical evidence has implicated glucokinase regulatory protein (GKRP) in the pathogenesis of type 2 diabetes and related traits. The primary role of GKRP is to bind and inhibit hepatic glucokinase (GCK), a critically important protein in human health and disease that exerts a significant degree of control over glucose metabolism. As activation of GCK has been associated with improved glucose tolerance, perturbation of the GCK-GKRP interaction represents a potential therapeutic target for pharmacological modulation. Recent structural and kinetic advances are beginning to provide insight into the interaction of these two proteins. However, tools to comprehensively assess the GCK-GKRP interaction, particularly in the context of small molecules, would be a valuable resource. We therefore developed three robust and miniaturized assays for assessing the interaction between recombinant human GCK and GKRP: an HTRF assay, a diaphorase-coupled assay, and a luciferase-coupled assay. The assays are complementary, featuring distinct mechanisms of detection (luminescence, fluorescence, FRET). Two assays rely on GCK enzyme activity modulation by GKRP while the FRET-based assay measures the GCK-GKRP protein-protein interaction independent of GCK enzymatic substrates and activity. All three assays are scalable to low volumes in 1536-well plate format, with robust Z' factors (>0.7). Finally, as GKRP sequesters GCK in the hepatocyte nucleus at low glucose concentrations, we explored cellular models of GCK localization and translocation. Previous findings from freshly isolated rat hepatocytes were confirmed in cryopreserved rat hepatocytes, and we further extended this study to cryopreserved human hepatocytes. Consistent with previous reports, there were several key differences between the rat and human systems, with our results suggesting that human hepatocytes can be used to interrogate GCK translocation in response to small molecules. The assay panel developed here should help direct future investigation of the GCK-GKRP interaction in these or other physiologically relevant human systems.

Dataset Information

Biochemical and biophysical investigations of the interaction between human glucokinase and pro-apoptotic BAD.

Publications

Biochemical and biophysical investigations of the interaction between human glucokinase and pro-apoptotic BAD.

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