The ubiquitin-like molecule NEDD8 controls several biological processes and is a promising target for therapeutic intervention. NEDDylation occurs through specific NEDD8 enzymes (canonical) or enzymes of the ubiquitin system (atypical). Identification of NEDD8 sites on substrates is critical for delineating the processes controlled by NEDDylation. By combining the use of the NEDD8 R74K mutant with anti-di-glycine (anti-diGly) antibodies, we identified 1,101 unique NEDDylation sites in 620 proteins. Bioinformatics analysis reveals that canonical and atypical NEDDylation have distinct proteomes; the spliceosome/mRNA surveillance/DNA replication and ribosome/proteasome, respectively. The data also reveal the formation of poly-NEDD8, hybrid NEDD8-ubiquitin, and NEDD8-SUMO-2 chains as potential molecular signals. In particular, NEDD8-SUMO-2 chains are induced upon proteotoxic stress (atypical) through NEDDylation of K11 in SUMO-2, and conjugates accumulate in previously described nucleolus-related inclusions. The study uncovers a diverse proteome for NEDDylation and is consistent with the concept of extensive cross-talk between ubiquitin and Ubls under proteotoxic stress conditions.

泛素样分子 NEDD8 控制多个生物过程，是治疗干预的有希望的靶点。NEDDylation 通过特定的 NEDD8 酶（规范）或泛素系统的酶（非典型）发生。识别基板上的 NEDD8 位点对于描述由 NEDDylation 控制的过程至关重要。通过将 NEDD8 R74K 突变体与抗二甘氨酸（抗二甘氨酸）抗体相结合，我们在 620 种蛋白质中鉴定了 1,101 个独特的 NEDDylation 位点。生物信息学分析表明，典型和非典型 NEDDylation 具有不同的蛋白质组；剪接体/mRNA 监视/DNA 复制和核糖体/蛋白酶体，分别。数据还揭示了作为潜在分子信号的多聚 NEDD8、混合 NEDD8-泛素和 NEDD8-SUMO-2 链的形成。特别是，NEDD8-SUMO-2 链通过 SUMO-2 中 K11 的 NEDD 化在蛋白毒性应激（非典型）下被诱导，并且缀合物在先前描述的核仁相关包涵体中积累。该研究揭示了 NEDDylation 的多种蛋白质组，并且与泛素和 Ubls 在蛋白质毒性应激条件下广泛串扰的概念一致。