Silva Gustavo M, Finley Daniel, Vogel Christine
Center for Genomics and Systems Biology, New York University, New York, New York, USA.
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.
Nat Struct Mol Biol. 2015 Feb;22(2):116-23. doi: 10.1038/nsmb.2955. Epub 2015 Jan 26.
Ubiquitination is a post-translational modification that signals multiple processes, including protein degradation, trafficking and DNA repair. Polyubiquitin accumulates globally during the oxidative stress response, and this has been mainly attributed to increased ubiquitin conjugation and perturbations in protein degradation. Here we show that the unconventional Lys63 (K63)-linked polyubiquitin accumulates in the yeast Saccharomyces cerevisiae in a highly sensitive and regulated manner as a result of exposure to peroxides. We demonstrate that hydrogen peroxide inhibits the deubiquitinating enzyme Ubp2, leading to accumulation of K63 conjugates assembled by the Rad6 ubiquitin conjugase and the Bre1 ubiquitin ligase. Using linkage-specific isolation methods and stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics, we identified >100 new K63-polyubiquitinated targets, which were substantially enriched in ribosomal proteins. Finally, we demonstrate that impairment of K63 ubiquitination during oxidative stress affects polysome stability and protein expression, rendering cells more sensitive to stress, and thereby reveal a new redox-regulatory role for this modification.
泛素化是一种翻译后修饰，它标志着包括蛋白质降解、运输和DNA修复在内的多个过程。在氧化应激反应期间，多聚泛素在整体上会积累，而这主要归因于泛素缀合增加以及蛋白质降解受到干扰。在此，我们表明，由于暴露于过氧化物，非常规的赖氨酸63（K63）连接的多聚泛素会以高度敏感且受调控的方式在酿酒酵母中积累。我们证明，过氧化氢会抑制去泛素化酶Ubp2，导致由Rad6泛素缀合酶和Bre1泛素连接酶组装的K63缀合物积累。使用基于细胞培养中氨基酸的连接特异性分离方法和稳定同位素标记（SILAC）定量蛋白质组学，我们鉴定出了100多个新的K63多聚泛素化靶点，这些靶点在核糖体蛋白中显著富集。最后，我们证明氧化应激期间K63泛素化的受损会影响多核糖体稳定性和蛋白质表达，使细胞对应激更敏感，从而揭示了这种修饰的一种新的氧化还原调节作用。
