Browsing by Author "Gleaton, Jeremy"

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    Insertion of a Synthetic Switch Into Insulin Provides Metabolite-Dependent Regulation of Hormone-Receptor Activation
    (Endocrine Society, 2021-05-03) Chen, Yen-Shan; Gleaton, Jeremy; Yang, Yanwu; Dhayalan, Balamurugan; Phillips, Nelson B.; Liu, Yule; Broadwater, Laurie; Jarosinski, Mark; Chatterjee, Deepak; Lawrence, Michael C.; Hattier, Thomas; Michael, Dodson M.; Weiss, Michael Aaron; Biochemistry and Molecular Biology, School of Medicine
    Insulin signaling requires conformational change: whereas the free hormone and its receptor each adopt autoinhibited conformations, their binding leads to large-scale structural reorganization. To test the coupling between insulin’s “opening” and receptor activation, we inserted an artificial ligand-dependent switch into insulin. Ligand binding disrupts an internal tether designed to stabilize the hormone’s native closed and inactive conformation, thereby enabling productive receptor engagement. This scheme exploited a diol sensor (meta-fluoro-phenylboronic acid at GlyA1) and internal diol (3,4-dihydroxybenzoate at LysB28). The sensor recognizes monosaccharides (fructose > glucose). Studies of insulin signaling in human hepatoma-derived cells (HepG2) demonstrated fructose-dependent receptor autophosphorylation leading to appropriate downstream signaling events, including a specific kinase cascade and metabolic gene regulation (gluconeogenesis and lipogenesis). Addition of glucose (an isomeric ligand with negligible sensor affinity) did not activate the receptor. Similarly, metabolite-regulated signaling was not observed in control studies of (i) an unmodified insulin analog or (ii) an analog containing a diol sensor in the absence of internal tethering. Although as expected CD-detected secondary structure was unaffected by ligand binding, heteronuclear NMR studies revealed subtle local and nonlocal monosaccharide-dependent changes in structure. Insertion of a synthetic switch into insulin has thus demonstrated coupling between hinge-opening and holoreceptor signaling. In addition to this basic finding, our results provide proof of principle for a mechanism-based metabolite-responsive insulin. In particular, replacement of the present fructose sensor by an analogous glucose sensor may enable translational development of a “smart” insulin analog designed to mitigate risk of hypoglycemia in the treatment of diabetes mellitus.
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    Insertion of a synthetic switch into insulin provides metabolite-dependent regulation of hormone–receptor activation
    (National Academy of Sciences, 2021) Chen, Yen-Shan; Gleaton, Jeremy; Yang, Yanwu; Dhayalan, Balamurugan; Phillips, Nelson B.; Liu, Yule; Broadwater, Laurie; Jarosinski, Mark A.; Chatterjee, Deepak; Lawrence, Michael C.; Hattier, Thomas; Michael, M. Dodson; Weiss, Michael A.; Biochemistry and Molecular Biology, School of Medicine
    Ligand-dependent conformational switches are ubiquitous in biological macromolecules, from allosteric proteins to RNA riboswitches. Molecular design of artificial switches provides a general strategy to test relationships between macromolecular structure and function. The present study exploited recent structures of complexes between an ancestral signaling protein (insulin) and the ectodomain of its cellular receptor to insert a metabolite-regulated switch into the hormone. Whereas binding of ligands often stabilizes structure, this design envisioned metabolite-dependent “opening” of a closed, inactive insulin conformation. Assessment of hormone-directed receptor autophosphorylation and a downstream signaling cascade in liver-derived cells demonstrated that binding of metabolite (a monosaccharide) enabled hormonal signaling. These results suggest a mechanism-based strategy to design “smart” glucose-responsive analogs to more safely treat insulin-dependent diabetes mellitus.