Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, United States.
Neuroscience Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, United States.
Biochemistry. 2020 Jun 9;59(22):2078-2088. doi: 10.1021/acs.biochem.0c00084. Epub 2020 May 27.
The E3 ubiquitin ligase C-terminus of Hsc70 interacting protein (CHIP) plays a critical role in regulating the ubiquitin-dependent degradation of misfolded proteins. CHIP mediates the ubiquitination of the α-amino-terminus of substrates with the E2 Ube2w and facilitates the ubiquitination of lysine residues with the E2 UbcH5. While it is known that Ube2w directly interacts with the disordered regions at the N-terminus of its substrates, it is unclear how CHIP and UbcH5 mediate substrate lysine selection. Here, we have decoupled the contributions of the E2, UbcH5, and the E3, CHIP, in ubiquitin transfer. We show that UbcH5 selects substrate lysine residues independent of CHIP, and that CHIP participates in lysine selection by fine-tuning the subset of substrate lysines that are ubiquitinated. We also identify lysine 128 near the C-terminus of UbcH5 as a critical residue for the efficient ubiquitin transfer by UbcH5 in both the presence and absence of CHIP. Together, these data demonstrate an important role of the UbcH5/substrate interactions in mediating the efficient ubiquitin transfer by the CHIP/UbcH5 complex.
E3 泛素连接酶 C 端与 Hsc70 相互作用蛋白（CHIP）在调节错误折叠蛋白的泛素依赖性降解中起着关键作用。CHIP 介导 E2 Ube2w 对底物 α-氨基末端的泛素化，并促进 E2 UbcH5 对赖氨酸残基的泛素化。虽然已知 Ube2w 直接与底物 N 端的无规区域相互作用，但 CHIP 和 UbcH5 如何介导底物赖氨酸选择尚不清楚。在这里，我们已经将 E2、UbcH5 和 E3、CHIP 在泛素转移中的作用分离开来。我们表明，UbcH5 独立于 CHIP 选择底物赖氨酸残基，而 CHIP 通过微调被泛素化的底物赖氨酸亚基参与赖氨酸选择。我们还确定 UbcH5 的 C 端附近的赖氨酸 128 是 UbcH5 在有和没有 CHIP 的情况下有效进行泛素转移的关键残基。总之，这些数据表明 UbcH5/底物相互作用在介导 CHIP/UbcH5 复合物的有效泛素转移中起着重要作用。
