Endoplasmic Reticulum Protein Quality Control Is Determined by Cooperative Interactions between Hsp/c70 Protein and the CHIP E3 Ligase
Endoplasmic Reticulum Protein Quality Control Is Determined by Cooperative Interactions between Hsp/c70 Protein and the CHIP E3 Ligase
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DOI：
10.1074/jbc.m113.479345
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发表时间：
2013-10-25
影响因子：
4.8
通讯作者：
Skach, William R.
中科院分区：
文献类型：
--
作者：
Matsumura, Yoshihiro;Sakai, Juro;Skach, William R.
Hsp70相互作用蛋白(CHIP)E3连接酶的C端通过将HSP/C70(90)的C端(M/I)EEVD肽基序与其N端四肽重复(TPR)结构域结合，并通过其C端催化U盒促进错误折叠的客户蛋白的多泛素化，从而起到调节蛋白质质量的关键作用。使用CFTR作为模型客户端，我们最近表明，Hsc70-客户端结合周期的持续时间是稳定性的主要决定因素。然而，控制芯片募集到HSP/C70的分子特征，以及因此HSP/C70客户的命运，仍然未知。为了了解CHIP是如何识别HSP/C70的，我们利用了一个显性的负性突变体，在该突变体中，U-box结构域(P269A)中保守的脯氨酸的缺失会消除E3连接酶的活性。在无细胞重组的内质网相关降解系统中，P269A芯片以剂量依赖的方式抑制Hsc70依赖的CFTR泛素化和降解。最佳抑制需要TPR和U-box，表明两个结构域之间的协同作用。野生型和P269A突变体都没有改变Hsc70与CFTR的结合程度，也没有改变Hsc70-CFTR复合体的解离速率。然而，U-box突变刺激了芯片与Hsc70的结合，同时促进了芯片的寡聚。Hsc70客户端的存在也刺激了芯片与Hsc70结合，其中Hsc70客户端偏爱ADP结合状态。因此，HSP/C70(M/I)EEVD基序不是TPR结构域的简单锚。相反，芯片招募涉及其TPR和U-box结构域与HSP/C70的底物结合和C末端结构域之间的互换变构相互作用。
The C terminus of Hsp70 interacting protein (CHIP) E3 ligase functions as a key regulator of protein quality control by binding the C-terminal (M/I)EEVD peptide motif of Hsp/c70(90) with its N-terminal tetratricopeptide repeat (TPR) domain and facilitating polyubiquitination of misfolded client proteins via its C-terminal catalytic U-box. Using CFTR as a model client, we recently showed that the duration of the Hsc70-client binding cycle is a primary determinant of stability. However, molecular features that control CHIP recruitment to Hsp/c70, and hence the fate of the Hsp/c70 client, remain unknown. To understand how CHIP recognizes Hsp/c70, we utilized a dominant negative mutant in which loss of a conserved proline in the U-box domain (P269A) eliminates E3 ligaseactivity. In a cell-free reconstituted ER- associated degradation system, P269A CHIP inhibited Hsc70-dependent CFTR ubiquitination and degradation in a dose-dependent manner. Optimal inhibition required both the TPR and the U-box, indicating cooperativity between the two domains. Neither the wild type nor the P269A mutant changed the extent of Hsc70 association with CFTR nor the dissociation rate of the Hsc70-CFTR complex. However, the U-box mutation stimulated CHIP binding to Hsc70 while promoting CHIP oligomerization. CHIP binding to Hsc70 binding was also stimulated by the presence of an Hsc70 client with a preference for the ADP-bound state. Thus, the Hsp/c70 (M/I) EEVD motif is not a simple anchor for the TPR domain. Rather CHIP recruitment involves reciprocal allosteric interactions between its TPR and U-box domains and the substrate-binding and C-terminal domains of Hsp/c70.