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Browsing by Author "Whittaker, Jonathan"
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Item “Register-shift” insulin analogs uncover constraints of proteotoxicity in protein evolution(Elsevier, 2020-03-06) Rege, Nischay K.; Liu, Ming; Dhayalan, Balamurugan; Chen, Yen-Shan; Smith, Nicholas A.; Rahimi, Leili; Sun, Jinhong; Guo, Huan; Yang, Yanwu; Haataja, Leena; Phillips, Nelson F. B.; Whittaker, Jonathan; Smith, Brian J.; Arvan, Peter; Ismail-Beigi, Faramarz; Weiss, Michael A.; Biochemistry and Molecular Biology, School of MedicineGlobular protein sequences encode not only functional structures (the native state) but also protein foldability, i.e. a conformational search that is both efficient and robustly minimizes misfolding. Studies of mutations associated with toxic misfolding have yielded insights into molecular determinants of protein foldability. Of particular interest are residues that are conserved yet dispensable in the native state. Here, we exploited the mutant proinsulin syndrome (a major cause of permanent neonatal-onset diabetes mellitus) to investigate whether toxic misfolding poses an evolutionary constraint. Our experiments focused on an invariant aromatic motif (PheB24-PheB25-TyrB26) with complementary roles in native self-assembly and receptor binding. A novel class of mutations provided evidence that insulin can bind to the insulin receptor (IR) in two different modes, distinguished by a "register shift" in this motif, as visualized by molecular dynamics (MD) simulations. Register-shift variants are active but defective in cellular foldability and exquisitely susceptible to fibrillation in vitro Indeed, expression of the corresponding proinsulin variant induced endoplasmic reticulum stress, a general feature of the mutant proinsulin syndrome. Although not present among vertebrate insulin and insulin-like sequences, a prototypical variant ([GlyB24]insulin) was as potent as WT insulin in a rat model of diabetes. Although in MD simulations the shifted register of receptor engagement is compatible with the structure and allosteric reorganization of the IR-signaling complex, our results suggest that this binding mode is associated with toxic misfolding and so is disallowed in evolution. The implicit threat of proteotoxicity limits sequence variation among vertebrate insulins and insulin-like growth factors.Item An ultra-stable single-chain insulin analog resists thermal inactivation and exhibits biological signaling duration equivalent to the native protein(American Society for Biochemistry and Molecular Biology, 2018-01-05) Glidden, Michael D.; Aldabbagh, Khadijah; Phillips, Nelson B.; Carr, Kelley; Chen, Yen-Shan; Whittaker, Jonathan; Phillips, Manijeh; Wickramasinghe, Nalinda P.; Rege, Nischay; Swain, Mamuni; Peng, Yi; Yang, Yanwu; Lawrence, Michael C.; Yee, Vivien C.; Ismail-Beigi, Faramarz; Weiss, Michael A.; Biochemistry and Molecular Biology, School of MedicineThermal degradation of insulin complicates its delivery and use. Previous efforts to engineer ultra-stable analogs were confounded by prolonged cellular signaling in vivo, of unclear safety and complicating mealtime therapy. We therefore sought an ultra-stable analog whose potency and duration of action on intravenous bolus injection in diabetic rats are indistinguishable from wild-type (WT) insulin. Here, we describe the structure, function, and stability of such an analog, a 57-residue single-chain insulin (SCI) with multiple acidic substitutions. Cell-based studies revealed native-like signaling properties with negligible mitogenic activity. Its crystal structure, determined as a novel zinc-free hexamer at 2.8 Å, revealed a native insulin fold with incomplete or absent electron density in the C domain; complementary NMR studies are described in the accompanying article. The stability of the analog (ΔGU 5.0(±0.1) kcal/mol at 25 °C) was greater than that of WT insulin (3.3(±0.1) kcal/mol). On gentle agitation, the SCI retained full activity for >140 days at 45 °C and >48 h at 75 °C. These findings indicate that marked resistance to thermal inactivation in vitro is compatible with native duration of activity in vivo Further, whereas WT insulin forms large and heterogeneous aggregates above the standard 0.6 mm pharmaceutical strength, perturbing the pharmacokinetic properties of concentrated formulations, dynamic light scattering, and size-exclusion chromatography revealed only limited SCI self-assembly and aggregation in the concentration range 1-7 mm Such a combination of favorable biophysical and biological properties suggests that SCIs could provide a global therapeutic platform without a cold chain.