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Identification of the NEDDylated proteome by Nedd8-specific K-GG mass spectrometry uncovers modulation of Actin dynamics by neddylated Cofilin

ABSTRACT: Neddylation is the post-translational protein modification that is most closely related to ubiquitination. The Ubiquitin-like protein NEDD8 is mostly studied for its role in activating the Cullin-RING E3 Ubiquitin ligases, however little is known about other NEDD8 targets. We developed serial NEDD8-Ubiquitin Substrate Profiling (sNUSP), a method that employs Nedd8-R74K knock-in cells allowing discrimination of endogenous NEDD8- and Ubiquitin-modification sites by mass spectrometry after Lys-C digestion and K-GG- peptide enrichment. Using sNUSP, we identified 607 neddylation sites dynamically regulated by the neddylation inhibitor MLN4924 and the de-neddylating enzyme NEDP1/SENP8. Among the candidates, we characterized lysine 112 (K112) 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 and site-specific neddylation of Cofilin at K112 regulates neurite outgrowth. These data show that Cofilin neddylation contributes to the regulation of neuronal Actin dynamics.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: erik verschueren Donald Kirkpatrick

PROVIDER: MSV000084683 | MassIVE | Thu Dec 12 20:59:00 GMT 2019

REPOSITORIES: MassIVE

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Project description:Post-translational control by ubiquitin regulates various aspects of cellular biology. This chemical modification on proteins presents itself in numerous iterations, from a single mono-ubiquitination event to chains of poly-ubiquitin. Among the various types of poly-ubiquitin constructed are untethered species that comprise a series of ubiquitin moieties linked to one another, but free from another protein. The current notion is that these unanchored poly-ubiquitin species are deleterious to the cell and are rapidly deconstructed. We recently examined the toxicity and utilization of untethered poly-ubiquitin in an intact organism by using the fruit fly, Drosophila melanogaster. We found that these ubiquitin species are largely innocuous to flies and that free poly-Ub can be controlled by being degraded by the proteasome or being conjugated onto another protein as a single unit. However, whether the fly is mounting an organismal defense against untethered chains was not explored in detail. Here, we conducted RNA-seq analyses to examine at the transcriptome level the impact of unanchored poly-Ub in the fly. We found ~90 transcripts whose expression is altered in the presence of different types of unanchored poly-ubiquitin. The set of genes identified was mostly devoid of ubiquitin-, proteasome- or autophagy-related components. The largest gene ontology category identified, housing ~15 of the altered genes, was proteolysis. The seeming absence of a large and multipronged response to unanchored ubiquitin chains in the fly supports the conclusion that these species need not be toxic in vivo and underscores the need to reexamine the role of free ubiquitin chains in the cell. We designed two types of poly-Ub chains with six Ub molecules in tandem. They cannot be cleaved by DUBs (there are no "GG" motifs linking the Ub molecules). Ub6-GG can be conjugated in toto onto other proteins; Ub6-Stop cannot. We drove ubiquitous expression using sqh-Gal4, and then performed RNA-Seq on adult flies to determine any cellular response to the presence of our unanchored Ub chains.

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Identification of the NEDDylated proteome by Nedd8-specific K-GG mass spectrometry uncovers modulation of Actin dynamics by neddylated Cofilin

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