Department of Neuroscience, Genentech, South San Francisco, CA, USA.
Department of Microchemistry, Proteomics and Lipidomics, Genentech, South San Francisco, CA, USA.
Nat Struct Mol Biol. 2020 Feb;27(2):210-220. doi: 10.1038/s41594-019-0370-3. Epub 2020 Feb 3.
Neddylation is the post-translational protein modification most closely related to ubiquitination. Whereas the ubiquitin-like protein NEDD8 is well studied for its role in activating cullin-RING E3 ubiquitin ligases, little is known about other substrates. We developed serial NEDD8-ubiquitin substrate profiling (sNUSP), a method that employs NEDD8 R74K knock-in HEK293 cells, allowing discrimination of endogenous NEDD8- and ubiquitin-modification sites by MS after Lys-C digestion and K-εGG-peptide enrichment. Using sNUSP, we identified 607 neddylation sites dynamically regulated by the neddylation inhibitor MLN4924 and the de-neddylating enzyme NEDP1, implying that many non-cullin proteins are neddylated. Among the candidates, we characterized lysine 112 of the actin regulator cofilin as a novel neddylation event. Global inhibition of neddylation in developing neurons leads to cytoskeletal defects, altered actin dynamics and neurite growth impairments, whereas site-specific neddylation of cofilin at K112 regulates neurite outgrowth, suggesting that cofilin neddylation contributes to the regulation of neuronal actin organization.
类泛素化是与泛素化最密切相关的蛋白质翻译后修饰。虽然 NEDD8 类泛素蛋白在激活 Cullin-RING E3 泛素连接酶方面的作用已得到充分研究，但对其他底物知之甚少。我们开发了串联 NEDD8-泛素底物谱分析（sNUSP），这是一种利用 NEDD8 R74K 敲入 HEK293 细胞的方法，在 Lys-C 消化和 K-εGG-肽富集后，通过 MS 可以区分内源性 NEDD8 和泛素修饰位点。使用 sNUSP，我们鉴定了 607 个动态受 neddylation 抑制剂 MLN4924 和去 neddylation 酶 NEDP1 调节的 neddylation 位点，这意味着许多非 Cullin 蛋白都被 neddylation 修饰。在候选蛋白中，我们将肌动蛋白调节剂丝切蛋白的赖氨酸 112 确定为新的 neddylation 事件。在发育中的神经元中，全局抑制 neddylation 会导致细胞骨架缺陷、肌动蛋白动力学改变和神经突生长受损，而丝切蛋白 K112 的特异性 neddylation 调节神经突生长，表明丝切蛋白 neddylation 有助于调节神经元肌动蛋白组织。
