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From Proteopedia
An ultra-stable single-chain insulin analog resists thermal inactivation and exhibits biological signaling duration equivalent to the native protein
Structural highlights
DiseaseINS_HUMAN Defects in INS are the cause of familial hyperproinsulinemia (FHPRI) [MIM:176730.[1] [2] [3] [4] Defects in INS are a cause of diabetes mellitus insulin-dependent type 2 (IDDM2) [MIM:125852. IDDM2 is a multifactorial disorder of glucose homeostasis that is characterized by susceptibility to ketoacidosis in the absence of insulin therapy. Clinical fetaures are polydipsia, polyphagia and polyuria which result from hyperglycemia-induced osmotic diuresis and secondary thirst. These derangements result in long-term complications that affect the eyes, kidneys, nerves, and blood vessels.[5] Defects in INS are a cause of diabetes mellitus permanent neonatal (PNDM) [MIM:606176. PNDM is a rare form of diabetes distinct from childhood-onset autoimmune diabetes mellitus type 1. It is characterized by insulin-requiring hyperglycemia that is diagnosed within the first months of life. Permanent neonatal diabetes requires lifelong therapy.[6] [7] Defects in INS are a cause of maturity-onset diabetes of the young type 10 (MODY10) [MIM:613370. MODY10 is a form of diabetes that is characterized by an autosomal dominant mode of inheritance, onset in childhood or early adulthood (usually before 25 years of age), a primary defect in insulin secretion and frequent insulin-independence at the beginning of the disease.[8] [9] [10] FunctionINS_HUMAN Insulin decreases blood glucose concentration. It increases cell permeability to monosaccharides, amino acids and fatty acids. It accelerates glycolysis, the pentose phosphate cycle, and glycogen synthesis in liver. Publication Abstract from PubMedThermal degradation of insulin complicates its delivery and use. Previous efforts to engineer ultra-stable analogs were confounded by prolonged cellular signaling in vivo, complicating mealtime therapy and of unclear safety. 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 A, revealed a native insulin fold with incomplete or absent electron density in the C domain; complementary NMR studies are described in a companion article. The stability of the analog (DeltaGu 5.0(+/-0.1) kcal/mol at 25 degrees 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 degrees C and >48 hours at 75 degrees C. 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. These findings indicate that marked resistance to thermal inactivation in vitro is compatible with native duration of activity in vivo. Such a combination of favorable biophysical and biological properties suggests that SCIs could provide a global therapeutic platform without a cold chain. An ultra-stable single-chain insulin analog resists thermal inactivation and exhibits biological signaling duration equivalent to the native protein.,Glidden MD, Aldabbagh K, Phillips NB, Carr K, Chen YS, Whittaker J, Phillips M, Wickramasinghe NP, Rege N, Swain M, Peng Y, Yang Y, Lawrence MC, Yee VC, Ismail-Beigi F, Weiss MA J Biol Chem. 2017 Nov 7. pii: jbc.M117.808626. doi: 10.1074/jbc.M117.808626. PMID:29114035[11] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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