AP-SWATH Reveals Direct Involvement of VCP/p97 in Integrated Stress Response Signaling Through Facilitating CReP/PPP1R15B Degradation
AP-SWATH Reveals Direct Involvement of VCP/p97 in Integrated Stress Response Signaling Through Facilitating CReP/PPP1R15B Degradation
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DOI：
10.1074/mcp.ra117.000471
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发表时间：
2018-07-01
影响因子：
7
通讯作者：
Meyer, Hemmo
中科院分区：
文献类型：
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作者：
Huelsmann, Julia;Kravic, Bojana;Meyer, Hemmo
泛素介导的AAA-ATP酶VCP/p97在多种细胞应激反应途径中促进受损或错误折叠蛋白的降解。因此，解决其与伴侣和底物蛋白相互作用的复杂性并了解其与应激信号传导的联系是一个重大挑战。在这里，我们使用亲和纯化SWATH质谱法（AP-SWATH）来鉴定与底物捕获突变体p97-E578 Q特异性相互作用的蛋白质。AP-SWATH鉴定了从丰富的p97辅因子到途径特异性伴侣和单个连接酶（如被捕获在p97-E578 Q复合物中的RNF 185和MUL 1）的大检测范围内的差异相互作用。此外，我们确定了各种底物蛋白和候选物，包括PP 1调节剂CReP/PPP 1 R15 B，其使eIF 2去磷酸化，从而抵消应激激酶引起的翻译衰减。我们提供的证据表明，p97与其Ufd 1-Npl 4衔接子确保了未受干扰的细胞中快速的组成性周转和CReP的平衡水平。此外，我们表明，p97介导的降解，连同减少CReP合成，是必不可少的及时应激诱导的CReP水平的降低，因此，强大的eIF 2磷酸化，以加强应激反应。因此，我们的研究结果表明，p97不仅有利于批量降解的错误折叠的蛋白质在压力下，但也直接调制的综合应激反应的信号水平。
The ubiquitin-directed AAA-ATPase VCP/p97 facilitates degradation of damaged or misfolded proteins in diverse cellular stress response pathways. Resolving the complexity of its interactions with partner and substrate proteins and understanding its links to stress signaling is therefore a major challenge. Here, we used affinity-purification SWATH mass spectrometry (AP-SWATH) to identify proteins that specifically interact with the substrate-trapping mutant, p97-E578Q. AP-SWATH identified differential interactions over a large detection range from abundant p97 cofactors to pathway-specific partners and individual ligases such as RNF185 and MUL1 that were trapped in p97-E578Q complexes. In addition, we identified various substrate proteins and candidates including the PP1 regulator CReP/PPP1R15B that dephosphorylates eIF2 and thus counteracts attenuation of translation by stress-kinases. We provide evidence that p97 with its Ufd1-Npl4 adapter ensures rapid constitutive turnover and balanced levels of CReP in unperturbed cells. Moreover, we show that p97-mediated degradation, together with a reduction in CReP synthesis, is essential for timely stress-induced reduction of CReP levels and, consequently, for robust eIF2 phosphorylation to enforce the stress response. Thus, our results demonstrate that p97 not only facilitates bulk degradation of misfolded proteins upon stress, but also directly modulates the integrated stress response at the level of signaling.