Identifying the substrate proteins of U-box E3s E4B and CHIP by orthogonal ubiquitin transfer.
Identifying the substrate proteins of U-box E3s E4B and CHIP by orthogonal ubiquitin transfer.
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通过正交泛素转移鉴定 U-box E3s E4B 和 CHIP 的底物蛋白
DOI：
10.1126/sciadv.1701393
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发表时间：
2018-01
期刊：
影响因子：
13.6
通讯作者：
Yin J
中科院分区：
文献类型：
--
作者：
Bhuripanyo K;Wang Y;Liu X;Zhou L;Liu R;Duong D;Zhao B;Bi Y;Zhou H;Chen G;Seyfried NT;Chazin WJ;Kiyokawa H;Yin J
通过噬菌体展示设计泛素转移级联可以有效地分析E3底物。E3泛素（UB）连接酶E4B和hsc70相互作用蛋白（CHIP）的羧基端使用一个共同的U-box基序将UB从E1和E2酶转移到它们的底物蛋白上，并调节多种细胞过程。为了分析它们在细胞中的泛素化目标，我们使用噬菌体展示来设计E2-E4B和E2-CHIP对，它们与天然UB转移级联没有交叉反应性。然后，我们使用工程化的E2-E3对构建“正交UB转移（OUT）”级联，以便突变的UB （xUB）可以被工程化的E4B或CHIP专门用于标记其底物蛋白。纯化xub结合蛋白，然后进行蛋白质组学分析，可以鉴定出人胚胎肾293细胞中数百种E4B和CHIP的潜在底物。激酶MAPK3（丝裂原活化蛋白激酶3）、甲基转移酶PRMT1（蛋白精氨酸n-甲基转移酶1）和磷酸酶PPP3CA（蛋白磷酸酶3催化亚基α）被确定为这两个E3s的共享底物。磷酸酶PGAM5（磷酸甘油酸突变酶5）和去泛素酶OTUB1（卵巢肿瘤结构域含泛素醛结合蛋白1）被证实为E4B底物，β-catenin和CDK4（细胞周期蛋白依赖性激酶4）被证实为CHIP底物。在OUT鉴定的CHIP-CDK4回路的基础上，我们揭示了CHIP信号CDK4在内质网应激下的降解。
Engineering the ubiquitin transfer cascades by phage display enables an efficient way to profile E3 substrates. E3 ubiquitin (UB) ligases E4B and carboxyl terminus of Hsc70-interacting protein (CHIP) use a common U-box motif to transfer UB from E1 and E2 enzymes to their substrate proteins and regulate diverse cellular processes. To profile their ubiquitination targets in the cell, we used phage display to engineer E2-E4B and E2-CHIP pairs that were free of cross-reactivity with the native UB transfer cascades. We then used the engineered E2-E3 pairs to construct “orthogonal UB transfer (OUT)” cascades so that a mutant UB (xUB) could be exclusively used by the engineered E4B or CHIP to label their substrate proteins. Purification of xUB-conjugated proteins followed by proteomics analysis enabled the identification of hundreds of potential substrates of E4B and CHIP in human embryonic kidney 293 cells. Kinase MAPK3 (mitogen-activated protein kinase 3), methyltransferase PRMT1 (protein arginine N-methyltransferase 1), and phosphatase PPP3CA (protein phosphatase 3 catalytic subunit alpha) were identified as the shared substrates of the two E3s. Phosphatase PGAM5 (phosphoglycerate mutase 5) and deubiquitinase OTUB1 (ovarian tumor domain containing ubiquitin aldehyde binding protein 1) were confirmed as E4B substrates, and β-catenin and CDK4 (cyclin-dependent kinase 4) were confirmed as CHIP substrates. On the basis of the CHIP-CDK4 circuit identified by OUT, we revealed that CHIP signals CDK4 degradation in response to endoplasmic reticulum stress.
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影响因子：
64.5
作者：
Koegl, M;Hoppe, T;Jentsch, S
通讯作者：
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影响因子：
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作者：
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DOI：
10.1073/pnas.220247197
发表时间：
2000-11-07
影响因子：
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作者：
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通讯作者：
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DOI：
10.1073/pnas.1621355114
发表时间：
2017-02-14
影响因子：
11.1
作者：
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通讯作者：
Mak, Tak W.