DNA Damage Regulates Translation through β-TRCP Targeting of CReP.
DNA Damage Regulates Translation through β-TRCP Targeting of CReP.
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DOI：
10.1371/journal.pgen.1005292
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发表时间：
2015-06
期刊：
影响因子：
4.5
通讯作者：
Toczyski DP
中科院分区：
文献类型：
--
作者：
Loveless TB;Topacio BR;Vashisht AA;Galaang S;Ulrich KM;Young BD;Wohlschlegel JA;Toczyski DP
Skp 1-Cul 1-F盒复合物（SCF）与许多F盒蛋白中的任何一种相关联，其充当底物结合衔接子。人F盒蛋白βTRCP指导泛素与多种底物蛋白的结合，导致底物被蛋白酶体破坏。为了鉴定βTRCP底物，我们采用了最近开发的技术，称为连接酶捕获，其中泛素连接酶与泛素结合结构域融合以“捕获”泛素化底物。我们检查的候选底物中有88%是真正的底物，包括12个先前验证的底物，11个新底物和3个假阳性。一种βTRCP底物CReP是蛋白磷酸酶1（PP 1）特异性亚基，靶向翻译起始因子eIF 2 α，以促进去除应激诱导的抑制性磷酸化并增加帽依赖性翻译。我们发现CReP被βTRCP靶向降解DNA损伤。使用一个稳定的CReP等位基因，我们表明，CReP的耗尽是DNA损伤后完全诱导eIF 2 α磷酸化所必需的，并且有助于在细胞从DNA损伤中恢复时保持低水平的翻译。大约有600个人类基因编码的酶作为泛素连接酶，促进小蛋白泛素转移到数千个底物蛋白;“标签”与泛素通常促进底物的蛋白酶体的降解。在本文中，我们采用了一种称为连接酶捕获的技术用于哺乳动物细胞。连接酶捕获是一种用于确定泛素连接酶靶向哪些底物的高度准确的方法。在这里，我们用它来确定新的人类细胞周期调节剂βTRCP的底物。我们的筛选确实非常准确，因为我们能够验证我们通过质谱鉴定的88%的候选底物。这些新底物中有一些是不稳定的蛋白质，通过抑制βTRCP或βTRCP作为其一部分的整类泛素连接酶而稳定。然而，其他的似乎是稳定的或冗余的靶向底物，这是更难以确定与目前的技术。这表明连接酶捕获将能够可靠地鉴定人泛素连接酶的新底物。此外，新的βTRCP底物之一CReP在DNA损伤时特异性地被耗尽，并且CReP的耗尽有助于DNA损伤时翻译机制的失活。
The Skp1-Cul1-F box complex (SCF) associates with any one of a number of F box proteins, which serve as substrate binding adaptors. The human F box protein βTRCP directs the conjugation of ubiquitin to a variety of substrate proteins, leading to the destruction of the substrate by the proteasome. To identify βTRCP substrates, we employed a recently-developed technique, called Ligase Trapping, wherein a ubiquitin ligase is fused to a ubiquitin-binding domain to “trap” ubiquitinated substrates. 88% of the candidate substrates that we examined were bona fide substrates, comprising twelve previously validated substrates, eleven new substrates and three false positives. One βTRCP substrate, CReP, is a Protein Phosphatase 1 (PP1) specificity subunit that targets the translation initiation factor eIF2α to promote the removal of a stress-induced inhibitory phosphorylation and increase cap-dependent translation. We found that CReP is targeted by βTRCP for degradation upon DNA damage. Using a stable CReP allele, we show that depletion of CReP is required for the full induction of eIF2α phosphorylation upon DNA damage, and contributes to keeping the levels of translation low as cells recover from DNA damage. Approximately 600 human genes encode enzymes that act as ubiquitin ligases, which facilitate the transfer of the small protein ubiquitin to thousands of substrate proteins; “tagging” with ubiquitin often promotes the degradation of the substrate by the proteasome. In this paper, we adapt a technique called Ligase Trapping for use in mammalian cells. Ligase Trapping is a highly accurate method for determining which substrates are targeted by a ubiquitin ligase. Here we use it to identify new substrates of the human cell cycle regulator βTRCP. Our screen was indeed highly accurate, as we were able to validate 88% of the candidate substrates we identified by mass spectrometry. Some of these new substrates were unstable proteins that were stabilized by inhibition of βTRCP, or of the entire class of ubiquitin ligases of which βTRCP is a part. However, others appear to be stable or redundantly-targeted substrates, which have been more difficult to identify with current techniques. This suggests that Ligase Trapping will be able to reliably identify new substrates of human ubiquitin ligases. Further, one of the new βTRCP substrates, CReP, is specifically depleted upon DNA damage, and depletion of CReP contributes to inactivation of the translational machinery upon DNA damage.
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影响因子：
21.3
作者：
Benanti, Jennifer A.;Cheung, Stephanie K.;Toczyski, David P.
通讯作者：
Toczyski, David P.
影响因子：
4.8
作者：
D'Annibale, Sara;Kim, Jihoon;Guardavaccaro, Daniele
通讯作者：
Guardavaccaro, Daniele
影响因子：
10.5
作者：
Jin, JP;Shirogane, T;Harper, JW
通讯作者：
Harper, JW
影响因子：
16
作者：
Harding, HP;Novoa, I;Ron, D
通讯作者：
Ron, D
影响因子：
9.2
作者：
Deng, J;Harding, HP;Sonenberg, N
通讯作者：
Sonenberg, N