Recognition of nonproline N -terminal residues by the Pro/N-degron pathway
Recognition of nonproline N -terminal residues by the Pro/N-degron pathway
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DOI：
10.1073/pnas.2007085117
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发表时间：
2020-06-23
影响因子：
11.1
通讯作者：
Min, Jinrong
中科院分区：
文献类型：
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作者：
Dong, Cheng;Chen, Shun-Jia;Min, Jinrong
真核N-降解子途径是一种蛋白质降解系统，其唯一的特征是能够识别含有N-末端(NT)降解信号的蛋白质，称为N-降解子，并通过26S蛋白酶体或自噬来靶向降解这些蛋白质。GID4是GID泛素连接酶的一个亚基，是Pro/N-degron途径的主要识别成分。在存在特定下游序列基序的情况下，GID4通过其NT-Pro残基或位置2上的Pro靶向蛋白质。在这里，我们发现人GID4还可以识别除Pro以外的疏水性NT-残基。一个例子是带有NT-ILE的序列NT-IGLW。NT-IGLW与野生型人GID4结合的Kd为16mM，而其他相同的NT-Pro序列PGLW与GID4结合更紧密，Kd为1.9mM。尽管NT-IGLW与NT-PGLW的GID4亲和力不同，但我们发现酿酒酵母GID4介导的Pro/N-degron途径可以靶向携带NT-IGLW的蛋白进行快速降解。我们解决了人GID4与含有NT-Ile或NT-Val的多肽结合的晶体结构。我们还改变了人GID4的特异性残基，并测定了产生的突变体GID4对NT-IGLW和NT-PGLW的亲和力，从而确定了特定GID4残基对GID4介导的NT-Pro与非Pro的NT-残基识别的相对贡献。这些和相关的结果促进了对Pro/N-degron通路靶向的理解，并极大地扩大了GID泛素连接酶在人和酵母细胞中的底物识别范围。
Eukaryotic N-degron pathways are proteolytic systems whose uni- fying feature is their ability to recognize proteins containing N -terminal (Nt) degradation signals called N-degrons, and to target these proteins for degradation by the 26S proteasome or auto- phagy. GID4, a subunit of the GID ubiquitin ligase, is the main rec- ognition component of the proline (Pro)/N-degron pathway. GID4 targets proteins through their Nt-Pro residue or a Pro at position 2, in the presence of specific downstream sequence motifs. Here we show that human GID4 can also recognize hydrophobic Nt-residues other than Pro. One example is the sequence Nt-IGLW, bearing Nt-Ile. Nt-IGLW binds to wild -type human GID4 with a K d of 16 mu M, whereas the otherwise identical Nt-Pro -bearing sequence PGLW binds to GID4 more tightly, with a K d of 1.9 mu M. Despite this differ- ence in affinities of GID4 for Nt-IGLW vs. Nt-PGLW, we found that the GID4-mediated Pro/N-degron pathway of the yeast Saccharo- myces cerevisiae can target an Nt-IGLW -bearing protein for rapid degradation. We solved crystal structures of human GID4 bound to a peptide bearing Nt-Ile or Nt-Val. We also altered specific residues of human GID4 and measured the affinities of resulting mutant GID4s for Nt-IGLW and Nt-PGLW, thereby determining relative con- tributions of specific GID4 residues to the GID4-mediated recogni- tion of Nt-Pro vs. Nt-residues other than Pro. These and related results advance the understanding of targeting by the Pro/N-degron pathway and greatly expand the substrate recognition range of the GID ubiquitin ligase in both human and yeast cells.