Polyubiquitin-Photoactivatable Crosslinking Reagents for Mapping Ubiquitin Interactome Identify Rpn1 as a Proteasome Ubiquitin-Associating Subunit.
Polyubiquitin-Photoactivatable Crosslinking Reagents for Mapping Ubiquitin Interactome Identify Rpn1 as a Proteasome Ubiquitin-Associating Subunit.
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DOI：
10.1016/j.chembiol.2017.02.013
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发表时间：
2017-04-20
影响因子：
8.6
通讯作者：
Glickman MH
中科院分区：
文献类型：
--
作者：
Chojnacki M;Mansour W;Hameed DS;Singh RK;El Oualid F;Rosenzweig R;Nakasone MA;Yu Z;Glaser F;Kay LE;Fushman D;Ovaa H;Glickman MH
泛素 (Ub) 信号传导是由小蛋白 Ub 和多泛素链与一系列细胞蛋白靶点共价连接控制的一组不同过程。最有记录的 Ub 信号通路是将多聚泛素蛋白传递至 26S 蛋白酶体进行降解的信号通路。然而，由于这些相互作用的瞬时性和疏水性以及缺乏研究它们的工具，对该过程中涉及的分子相互作用的研究受到了阻碍。在这里，我们开发了 Ub-phototrap (UbPT)，一种含有光活化交联侧链的合成 Ub 变体。酶促聚合成指定长度和连接类型的链提供了一组试剂，从而将 Rpn1 鉴定为第三个蛋白酶体泛素相关亚基，协调底物穿梭机的对接、底物的卸载和多聚泛素结合物的锚定。我们的工作展示了 UbPT 的价值，我们期望其未来的用途将有助于定义和研究泛素相互作用组。多聚泛素光捕获 (polyUbPT) 是一组新型的链特异性诱导光交联探针，其应用能够通过泛素的疏水性斑块捕获瞬时伴侣。 PolyUbPT 从完整的蛋白酶体复合物中捕获 Rpn1。 Rpn1 作为蛋白酶体亚基加入 Rpn10 和 Rpn13，对 PolyUb 和 Ub 样结构域具有亲和力。
Ubiquitin (Ub) signaling is a diverse group of processes controlled by covalent attachment of small protein Ub and the polyUb chains to a range of cellular protein targets. Best documented Ub signaling pathway is the one that delivers polyUb-proteins to the 26S proteasome for degradation. However, studies of molecular interactions involved in this process have been hampered by the transient and hydrophobic nature of these interactions and the lack of tools to study them. Here, we develop Ub-phototrap (UbPT), a synthetic Ub variant containing a photoactivatable crosslinking side chain. Enzymatic polymerization into chains of defined lengths and linkage types provided a set of reagents that led to identification of Rpn1 as a third proteasome ubiquitin-associating subunit that coordinates docking of substrate shuttles, unloading of substrates, and anchoring of polyUb-conjugates. Our work demonstrates the value of UbPT and we expect that its future uses will help define and investigate the ubiquitin interactome. Application of polyubiquitin-phototrap (polyUbPT), a novel set of chain-specific inducible photo-crosslinking probes, enables trapping of transient partners through the hydrophobic patch of ubiquitin. PolyUbPT captured Rpn1 from intact proteasome complexes. Rpn1 joins Rpn10 and Rpn13 as proteasome subunits with affinity for polyUb and Ub-like domains.
影响因子：
0.9
作者：
Chen T;Zhang D;Matiuhin Y;Glickman M;Fushman D
通讯作者：
Fushman D