Substitution of an Internal Disulfide Bridge with a Diselenide Enhances both Foldability and Stability of Human Insulin.
Substitution of an Internal Disulfide Bridge with a Diselenide Enhances both Foldability and Stability of Human Insulin.
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用二硒化物取代内部二硫桥可增强人胰岛素的折叠性和稳定性。
DOI：
10.1002/chem.201900892
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发表时间：
2019
期刊：
影响因子：
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通讯作者：
Metanis,Norman
中科院分区：
文献类型：
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作者：
Weil-Ktorza,Orit;Rege,Nischay;Lansky,Shifra;Shalev,DeborahE;Shoham,Gil;Weiss,MichaelA;Metanis,Norman
胰岛素类似物是现代糖尿病治疗的主要药物，也是蛋白质工程在分子药理学中的应用。尽管单个A和B链的化学合成在20世纪60年代早期完成，但由于竞争性二硫键配对和聚集，它们组合形成天然胰岛素仍然效率低下。为了克服这些限制，我们设想了一种替代方法：用硒代半胱氨酸（Sec，U）成对取代半胱氨酸残基。为此，CysA 6和CysA 11（形成内部链内A6-A11二硫键）分别被Sec取代。通过固相肽合成制备A链[C6 U，C11 U]变体;而生物合成人胰岛素的亚硫酸盐解提供野生型B链-二-S-磺酸盐。这些位点上硒原子的存在显著提高了链结合的速率和保真度，从而解决了化学胰岛素合成中的一个长期挑战。Se-胰岛素类似物对凝集素纯化的胰岛素受体的亲和力与WT-胰岛素的亲和力不可区分。值得注意的是，类似物在25 °C下的热力学稳定性（根据胍变性研究推断）得到增强（ΔΔGu ≥ 0.8 kcal mol−1）。 根据这种增强的稳定性，Se-胰岛素类似物的还原性解折叠和对Glu-C蛋白酶酶裂解的抗性比WT-胰岛素慢四倍。2D-NMR和X射线晶体学研究证明了天然的三维结构，其中二硒桥被容纳在疏水核中，没有空间冲突。
Insulin analogues, mainstays in the modern treatment of diabetes mellitus, exemplify the utility of protein engineering in molecular pharmacology. Whereas chemical syntheses of the individual A and B chains were accomplished in the early 1960s, their combination to form native insulin remains inefficient because of competing disulfide pairing and aggregation. To overcome these limitations, we envisioned an alternative approach: pairwise substitution of cysteine residues with selenocysteine (Sec, U). To this end, CysA6and CysA11(which form the internal intrachain A6–A11 disulfide bridge) were each replaced with Sec. The A chain[C6U, C11U] variant was prepared by solid‐phase peptide synthesis; while sulfitolysis of biosynthetic human insulin provided wild‐type B chain‐di‐S‐sulfonate. The presence of selenium atoms at these sites markedly enhanced the rate and fidelity of chain combination, thus solving a long‐standing challenge in chemical insulin synthesis. The affinity of the Se‐insulin analogue for the lectin‐purified insulin receptor was indistinguishable from that of WT‐insulin. Remarkably, the thermodynamic stability of the analogue at 25 °C, as inferred from guanidine denaturation studies, was augmented (ΔΔGu≈0.8 kcal mol−1). In accordance with such enhanced stability, reductive unfolding of the Se‐insulin analogue and resistance to enzymatic cleavage by Glu‐C protease occurred four times more slowly than that of WT‐insulin. 2D‐NMR and X‐ray crystallographic studies demonstrated a native‐like three‐dimensional structure in which the diselenide bridge was accommodated in the hydrophobic core without steric clash.