Determining the Effects of Neddylation on Cullin-RING Ligase-Dependent Protein Ubiquitination.
Determining the Effects of Neddylation on Cullin-RING Ligase-Dependent Protein Ubiquitination.
复制标题
DOI：
10.1002/cpz1.401
复制
发表时间：
2022-03
期刊：
影响因子：
--
通讯作者：
Liu X
中科院分区：
文献类型：
--
作者：
Wang K;Liu X
Cullin‐RING连接酶（CRL）是泛素（Ub）E3连接酶中最大的家族，在多种细胞过程中发挥着重要作用，其活性受到多种机制的严格调控。Neddylation，即Ub样蛋白NEDD 8与靶蛋白（如cullin）的结合，代表了CRL的关键调控机制。对一些CRL的生化和结构研究表明，cullin neddylation改变了CRL的构象，并激活了CRL依赖的蛋白泛素化。在这里，使用CUL 2-RING连接酶（CRL 2）作为一个例子，我们描述了我们的方案，用于制备重组CUL 2与或不与NEDD 8缀合，这是进一步用于定量确定neddylation对CRL 2依赖性蛋白泛素化的影响在体外。版权所有© 2022作者。由Wiley Periodicals LLC出版。 基本方案1：CUL 2·RBX 1在大肠杆菌中的表达和纯化 支持协议：在FPLC系统上用另外的层析进一步纯化CUL 2·RBX 1基本方案2：重构cullin neddylation用于体外定量泛素化测定
As the largest family of ubiquitin (Ub) E3 ligases, cullin‐RING ligases (CRLs) play crucial roles in various cellular processes, and their activities are tightly regulated by orchestrated mechanisms. Neddylation, the conjugation of a Ub‐like protein NEDD8 to a target protein such as the cullin, represents a key regulatory mechanism for CRLs. Biochemical and structural studies of a few CRLs have revealed that cullin neddylation alters the CRL conformation and activates CRL‐dependent protein ubiquitination. Here, using CUL2‐RING ligase (CRL2) as an example, we describe our protocols for the preparation of recombinant CUL2 with or without NEDD8 conjugation, which is further used to quantitatively determine the effect of neddylation on CRL2‐dependent protein ubiquitination in vitro. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression and purification of CUL2•RBX1 from Escherichia coli Support Protocol: Further purification of CUL2•RBX1 with additional chromatography on an FPLC system Basic Protocol 2: Reconstitution of cullin neddylation for quantitative ubiquitination assay in vitro