Department of Immunology, University of Washington, Seattle, WA 98195, USA.
Proc Natl Acad Sci U S A. 2010 Feb 16;107(7):2848-53. doi: 10.1073/pnas.0914821107. Epub 2010 Feb 1.
Gram-negative bacteria deliver a cadre of virulence factors directly into the cytoplasm of eukaryotic host cells to promote pathogenesis and/or commensalism. Recently, families of virulence proteins have been recognized that function as E3 Ubiquitin-ligases. How these bacterial ligases integrate into the ubiquitin (Ub) signaling pathways of the host and how they differ functionally from endogenous eukaryotic E3s is not known. Here we show that the bacterial E3 SspH2 from S. typhimurium selectively binds the human UbcH5 ~ Ub conjugate recognizing regions of both UbcH5 and Ub subunits. The surface of the E2 UbcH5 involved in this interaction differs substantially from that defined for other E2/E3 complexes involving eukaryotic E3-ligases. In vitro, SspH2 directs the synthesis of K48-linked poly-Ub chains, suggesting that cellular protein targets of SspH2-catalyzed Ub transfer are destined for proteasomal destruction. Unexpectedly, we found that intermediates in SspH2-directed reactions are activated poly-Ub chains directly tethered to the UbcH5 active site (UbcH5 ~ Ub(n)). Rapid generation of UbcH5 ~ Ub(n) may allow for bacterially directed modification of eukaryotic target proteins with a completed poly-Ub chain, efficiently tagging host targets for destruction.
革兰氏阴性菌将一系列毒力因子直接递送到真核宿主细胞的细胞质中，以促进发病机制和/或共生。最近，已经识别出一系列作为 E3 泛素连接酶发挥作用的毒力蛋白家族。这些细菌连接酶如何整合到宿主的泛素 (Ub) 信号通路中，以及它们在功能上与内源性真核 E3 有何不同，目前尚不清楚。在这里，我们表明来自 S. typhimurium 的细菌 E3 SspH2 选择性地结合人 UbcH5Ub 缀合物，识别 UbcH5 和 Ub 亚基的区域。涉及此相互作用的 E2 UbcH5 的表面与涉及真核 E3 连接酶的其他 E2/E3 复合物定义的表面有很大不同。在体外，SspH2 指导 K48 连接多 Ub 链的合成，这表明 SspH2 催化 Ub 转移的细胞蛋白靶标注定要被蛋白酶体破坏。出乎意料的是，我们发现 SspH2 定向反应的中间产物是直接连接到 UbcH5 活性位点的激活多 Ub 链（UbcH5Ub(n)）。UbcH5~Ub(n)的快速生成可能允许细菌对具有完整多 Ub 链的真核靶蛋白进行定向修饰，有效地标记宿主靶标进行破坏。
