Synthesis of calcineurin-resistant derivatives of FK506 and selection of compensatory receptors
Synthesis of calcineurin-resistant derivatives of FK506 and selection of compensatory receptors
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DOI：
10.1016/s1074-5521(02)00085-6
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发表时间：
2002-01-01
影响因子：
--
通讯作者：
Schreiber, SL
中科院分区：
文献类型：
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作者：
Clemons, PA;Gladstone, BG;Schreiber, SL
我们利用烯烃复分解法合成了FK506的C40衍生物，并测定了它们与FKBP12络合后抑制钙调磷酸酶活性的能力。我们鉴定了含有钙调磷酸酶A和B多肽片段的模块化二聚化结构域（CABS）。在哺乳动物细胞和酵母或哺乳动物三杂交实验中，这些cab对FK506均有反应。利用化学遗传选择，我们鉴定出补偿突变型cab，这些cab对抗钙调磷酸酶FK506衍生物的反应浓度远低于仅含有野生型钙调磷酸酶序列的cab的反应阈值。这些试剂提供了与现有二聚体系统正交的小分子蛋白质组合，并可与现有系统一起使用，以增加诱导接近实验的复杂性。这种“凹凸孔”策略的新用途保护靶细胞免受内源性钙调磷酸酶抑制引起的并发症。
We used olefin metathesis to synthesize C40 derivatives of FK506 and measured their ability, when complexed to FKBP12, to inhibit calcineurin's phosphatase activity. We identified modular dimerization domains (CABS) containing segments of the calcineurin A and B polypeptides. These CABs respond to FK506 both when overexpressed in mammalian cells and in yeast or mammalian three-hybrid assays. Using chemical genetic selection, we identified compensatory mutant CABs that respond to a calcineurin-resistant FK506 derivative at concentrations well below the response threshold for CABS containing only wild-type calcineurin sequence. These reagents provide a small molecule protein combination orthogonal to existing dimerizer systems and may be used with existing systems to increase the complexity of induced-proximity experiments. This new use of the "bump-hole" strategy protects target cells from complications arising from the inhibition of endogenous calcineurin.