Ubiquitin C-terminal electrophiles are activity-based probes for identification and mechanistic study of ubiquitin conjugating machinery.
Ubiquitin C-terminal electrophiles are activity-based probes for identification and mechanistic study of ubiquitin conjugating machinery.
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DOI：
10.1021/cb9000348
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发表时间：
2009-04-17
影响因子：
4
通讯作者：
Ploegh, Hidde L.
中科院分区：
文献类型：
--
作者：
Love, Kerry Routenberg;Pandya, Renuka K.;Spooner, Eric;Ploegh, Hidde L.
泛素（Ub）和泛素样修饰剂（Ubl）对蛋白质的修饰需要激活（E1）、缀合（E2）和连接（E3）酶的作用，是特异性破坏蛋白质的关键步骤。去泛素化酶（DUB）使被Ub/Ubls修饰的底物去缀合，并在细胞内回收Ub。基于与具有已知功能的蛋白质的序列同源性的基因组挖掘已将许多酶分配给该途径，而无需确认缀合或DUB活性。功能依赖性方法仍然是最有用的快速鉴定或评估的生物活性的表达蛋白质从细胞。基于活性的蛋白质分析（ABPP）使用化学探针，这些探针是活性位点定向的，用于复杂混合物中蛋白质活性的分类。在这里，我们表明，设计和使用一套扩展的基于UB的亲电探针，使我们能够恢复和识别成员的泛素-蛋白酶体系统中的每一类酶，包括E3连接酶和DUBs与以前未经验证的活动。我们表明，表位标记的Ub-亲电探针可用作E3连接酶鉴定的活性为基础的探针，通过体外标记和纯化的酶的活性研究，确定从复杂的混合物中的细胞裂解物。此外，我们的探针与E3 Ub连接酶ARF-BP 1的HECT结构域的反应性表明，多个半胱氨酸可能在E2结合位点附近，并且能够将Ub转移到自身或底物蛋白。
Protein modification by ubiquitin (Ub) and ubiquitin-like modifiers (Ubl) requires the action of activating (E1), conjugating (E2), and ligating (E3) enzymes and is a key step in the specific destruction of proteins. Deubiquitinating enzymes (DUBs) deconjugate substrates modified with Ub/Ubls and recycle Ub inside the cell. Genome mining based on sequence homology to proteins with known function has assigned many enzymes to this pathway without confirmation of either conjugating or DUB activity. Function-dependent methodologies are still the most useful for rapid identification or assessment of biological activity of expressed proteins from cells. Activity-based protein profiling (ABPP) uses chemical probes that are active-site directed for the classification of protein activities in complex mixtures. Here we show that the design and use of an expanded set of Ub-based electrophilic probes allowed us to recover and identify members of each enzyme class in the ubiquitin-proteasome system, including E3 ligases and DUBs with previously unverified activity. We show that epitope-tagged Ub-electrophilic probes can be used as activity-based probes for E3 ligase identification by in vitro labeling and activity studies of purified enzymes identified from complex mixtures in cell lysate. Furthermore, the reactivity of our probe with the HECT domain of the E3 Ub ligase ARF-BP1 suggests that multiple cysteines may be in the vicinity of the E2-binding site and are capable of the transfer of Ub to self or to a substrate protein.
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影响因子：
--
作者：
Lim, Kah-Leong;Tan, Jeanne M. M.
通讯作者：
Tan, Jeanne M. M.
影响因子：
--
作者：
Davies JE;Sarkar S;Rubinsztein DC
通讯作者：
Rubinsztein DC
影响因子：
3.6
作者：
Artavanis-Tsakonas, Katerina;Misaghi, Shahram;Ploegh, Hidde L.
通讯作者：
Ploegh, Hidde L.
影响因子：
5.8
作者：
Larkin, M. A.;Blackshields, G.;Higgins, D. G.
通讯作者：
Higgins, D. G.
影响因子：
3.7
作者：
Catic A;Misaghi S;Korbel GA;Ploegh HL
通讯作者：
Ploegh HL