<br>肿瘤抑制因子 PTEN 泛素化分析的半合成方法,Journal of the American Chemical Society

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肿瘤抑制因子 PTEN 泛素化分析的半合成方法
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2023-03-10 , DOI: 10.1021/jacs.2c13871
Reina Iwase _{1,

2} , Daniel R Dempsey _{1,

2,

3} , Samuel D Whedon _{1,

2} , Hanjie Jiang _{1,

2} , Brad A Palanski _{1,

2} , Bedphiny Deng _{4,

5} , Philip A Cole _{1,

2}

Affiliation

磷酸酶和张力蛋白同源物 (PTEN) 肿瘤抑制蛋白是一种 PIP3 脂质磷酸酶，会受到多方面的翻译后修饰。其中一种修饰是 Lys13 的单泛素化，它可能会改变其细胞定位，但也可能会影响其多种细胞功能。为了探索泛素对 PTEN 生化特性的调节影响及其与泛素连接酶和去泛素酶的相互作用，位点特异性和化学计量泛素化蛋白的生成可能是有益的。在这里，我们描述了一种半合成方法，该方法依赖于连续表达的蛋白质连接步骤，将泛素安装在近全长 PTEN 的 Lys13 模拟物上。这种方法允许在 PTEN 中同时安装 C 端修饰，从而有助于分析 N 端泛素化和 C 端磷酸化之间的相互作用。我们发现 PTEN 的 N 端泛素化抑制其酶功能，减少其与脂质囊泡的结合，通过 NEDD4-1 E3 连接酶调节其加工，并被去泛素酶 USP7 有效切割。我们的连接方法应该激发相关的努力来揭示复杂蛋白质泛素化的影响。

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Semisynthetic Approach to the Analysis of Tumor Suppressor PTEN Ubiquitination

Phosphatase and tensin homologue (PTEN) tumor suppressor protein is a PIP3 lipid phosphatase that is subject to multifaceted post-translational modifications. One such modification is the monoubiquitination of Lys13 that may alter its cellular localization but is also positioned in a manner that could influence several of its cellular functions. To explore the regulatory influence of ubiquitin on PTEN’s biochemical properties and its interaction with ubiquitin ligases and a deubiquitinase, the generation of a site-specifically and stoichiometrically ubiquitinated protein could be beneficial. Here, we describe a semisynthetic method that relies upon sequential expressed protein ligation steps to install ubiquitin at a Lys13 mimic in near full-length PTEN. This approach permits the concurrent installation of C-terminal modifications in PTEN, thereby facilitating an analysis of the interplay between N-terminal ubiquitination and C-terminal phosphorylation. We find that the N-terminal ubiquitination of PTEN inhibits its enzymatic function, reduces its binding to lipid vesicles, modulates its processing by NEDD4-1 E3 ligase, and is efficiently cleaved by the deubiquitinase, USP7. Our ligation approach should motivate related efforts for uncovering the effects of ubiquitination of complex proteins.

更新日期：2023-03-10

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