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Global site-specific neddylation profiling reveals that NEDDylated cofilin regulates actin dynamics.

Nat Struct Mol Biol. 2020 Feb;27(2):210-220. doi: 10.1038/s41594-019-0370-3. Epub 2020 Feb 3.

Authors/Editors: Vogl AM, Phu L, Becerra R, Giusti SA, Verschueren E, Hinkle TB, Bordenave MD, Adrian M, Heidersbach A, Yankilevich P, Stefani FD, Wurst W, Hoogenraad CC, Kirkpatrick DS, Refojo D, Sheng M.
Publication Date: 2020

02_vogl

Abstract

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.

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