Proteasome Activity Imaging and Profiling Characterizes Bacterial Effector Syringolin A
Proteasome Activity Imaging and Profiling Characterizes Bacterial Effector Syringolin A
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DOI：
10.1104/pp.110.163733
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发表时间：
2011-01-01
期刊：
影响因子：
7.4
通讯作者：
van der Hoorn, Renier A. L.
中科院分区：
文献类型：
--
作者：
Kolodziejek, Izabella;Misas-Villamil, Johana C.;van der Hoorn, Renier A. L.
丁香菌素 A (SylA) 是细菌病原体丁香假单胞菌丁香菌 (Pseudomonas syringae pv syringae) 产生的一种非核糖体环肽，可抑制真核蛋白酶体。蛋白酶体是一种多亚基蛋白水解复合物，存在于细胞核和细胞质中，包含具有不同催化活性的三个亚基：β1、β2 和β5。在这里，我们研究了 SylA 如何使用基于活性的分析和成像来靶向活细胞中的植物蛋白酶体。我们通过引入新的、更具选择性的探针并建立活拟南芥（Arabidopsis thaliana）细胞的无创成像程序，进一步开发了这项技术。这些研究表明，SylA 在体外和体内优先靶向植物蛋白酶体的 β2 和 β5。结构-活性分析表明，SylA 的二肽尾部有助于 β2 特异性，并鉴定出一种非反应性 SylA 衍生物，该衍生物被证明对成像实验至关重要。有趣的是，使用基于 epoxomicin 和 SylA 的探针进行的亚细胞成像显示，SylA 在植物细胞核中积累，表明 SylA 以核蛋白酶体为目标。此外，亚细胞分级分离研究表明，SylA 标记核和细胞质蛋白酶体。讨论了 SylA 对催化亚基和亚细胞区室的选择性，并通过晶体学数据解释了亚基选择性。
Syringolin A (SylA) is a nonribosomal cyclic peptide produced by the bacterial pathogen Pseudomonas syringae pv syringae that can inhibit the eukaryotic proteasome. The proteasome is a multisubunit proteolytic complex that resides in the nucleus and cytoplasm and contains three subunits with different catalytic activities: beta 1, beta 2, and beta 5. Here, we studied how SylA targets the plant proteasome in living cells using activity-based profiling and imaging. We further developed this technology by introducing new, more selective probes and establishing procedures of noninvasive imaging in living Arabidopsis (Arabidopsis thaliana) cells. These studies showed that SylA preferentially targets beta 2 and beta 5 of the plant proteasome in vitro and in vivo. Structure-activity analysis revealed that the dipeptide tail of SylA contributes to beta 2 specificity and identified a nonreactive SylA derivative that proved essential for imaging experiments. Interestingly, subcellular imaging with probes based on epoxomicin and SylA showed that SylA accumulates in the nucleus of the plant cell and suggests that SylA targets the nuclear proteasome. Furthermore, subcellular fractionation studies showed that SylA labels nuclear and cytoplasmic proteasomes. The selectivity of SylA for the catalytic subunits and subcellular compartments is discussed, and the subunit selectivity is explained by crystallographic data.