Identification of a novel 29-linked polyubiquitin binding protein, Ufd3, using polyubiquitin chain analogues.
Identification of a novel 29-linked polyubiquitin binding protein, Ufd3, using polyubiquitin chain analogues.
复制标题
DOI：
10.1021/bi035626r
复制
发表时间：
2004-04
期刊：
影响因子：
2.9
通讯作者：
N. Russell;K. Wilkinson
中科院分区：
文献类型：
--
作者：
N. Russell;K. Wilkinson
赖氨酸 48 连接的多聚泛素链是人们最了解的多聚泛素形式，对于泛素-蛋白酶体系统的功能是必需的。然而，体内也存在其他形式的多聚泛素（例如 K29 和 K63 连接链）。关于这些连接的功能作用或与这些形式的多聚泛素特异性相互作用的蛋白质知之甚少。使用天然多聚泛素链来鉴定结合蛋白由于合成和稳定性的困难而变得复杂。在这里，我们报告了在亲和支持物上合成 29 连接多聚泛素链的不可水解类似物及其在识别结合 29 连接多聚泛蛋白链的蛋白质中的用途。 29 连接的 Ub4 树脂稳定且紧密结合重组人异肽酶 T (USP5)，这是一种已知可结合 29 连接的多聚泛素链的去泛素化酶。从酿酒酵母裂解物中鉴定出 29 个连接的多聚泛素类似物的两个高亲和力相互作用因子。它们被鉴定为 Ubp14（异肽酶 T 的酵母直系同源物）和 Ufd3（泛素融合降解途径的成员，功能未知）。纯化的重组 Ufd3 也与树脂结合，证实 Ufd3 是多聚泛素的新型结合伴侣。这些结果证明了使用多聚泛素类似物亲和支持物来鉴定特异性连接的多聚泛素链的新型结合配偶体的功效。这些蛋白质的鉴定将有助于更好地了解不同多聚泛素连接的生理相关性。
Lysine 48-linked polyubiquitin chains are the best understood form of polyubiquitin and are necessary for the function of the ubiquitin-proteasome system. However, other forms of polyubiquitin (e.g., K29- and K63-linked chains) are also present in vivo. Less is known about the functional roles of these linkages or the proteins specifically interacting with these forms of polyubiquitin. Use of native polyubiquitin chains to identify binding proteins is complicated by the difficulties of synthesis and stability. Here, we report the synthesis of a nonhydrolyzable analogue of 29-linked polyubiquitin chains on an affinity support and its use in identifying proteins that bind 29-linked polyubiquitin chains. The 29-linked Ub4 resin was stable and tightly bound recombinant human Isopeptidase T (USP5), a deubiquitinating enzyme known to bind the 29-linked polyubiquitin chains. Two high affinity interactors of the 29-linked polyubiquitin analogues were identified from Saccharomyces cerevisiae lysates. They were identified as Ubp14, the yeast ortholog of Isopeptidase T, and Ufd3, a member of the ubiquitin-fusion degradation pathway with unknown function. Purified recombinant Ufd3 bound to the resin as well, confirming that Ufd3 is a novel binding partner of polyubiquitin. These results demonstrate the efficacy of using polyubiquitin analogue affinity supports to identify novel binding partners of specifically linked polyubiquitin chains. Identification of these proteins will lead to a greater understanding of the physiological relevance of different polyubiquitin linkages.