Dataset Information

SILAC diGlycine proteomics in C. elegans to identify ULP-3 targets upon DNA damage.

ABSTRACT: We found a conserved role for the deNEDDylating enzyme NEDP1 (ULP-3 in C. elegans) in the DNA damage induced apoptosis. To gain mechanistic insights into the role of ULP-3 in the IR-induced apoptosis, we devised an unbiased proteomics approach to discover potential NEDD8 targets for ULP-3 upon DNA damage. Identification of diglycine (diGly) remnants left on lysine residues upon trypsin digestion of proteins by mass spectrometry demonstrates their modification with ubiquitin, NEDD8 or ISG15 (Kessler, 2013; Ordureau et al., 2015). We hypothesised that changes in the diGly signature upon deletion of the de-NEDDylating enzyme ULP-3, should specifically indicate changes in the NEDD8 modification repertoire. We combined Stable Isotope Labelling with Amino acid in nematodes (SILAC) (Larance et al., 2011) with the use of antibodies that recognise the diGly remnant on modified peptides (Kessler, 2013; Ordureau et al., 2015; Xu et al., 2010). Wild type and ulp-3 deleted worms were labelled with Light (K0R0) and Heavy (K8R10) isotopes respectively. To specifically address the effect of ULP-3 on the NEDD8 proteome upon DNA damage, both sets of worms were exposed to IR. Synchronised worms were grown on nitrogen free nematode growth medium (NGM-N) as described previously (Larance et al., 2011) seeded with SLE1 bacteria labelled with Light (K0R0) and Heavy (K8R10) Isotopes (Eurisotope). Larvae stage 1 synchronised worms were grown until late adult stage and synchronised again by bleaching before growing them on 9cm NGM-N plates seeded with SILAC bacteria (SLE1). Approximately 10 plates of 4000 worms each were used to grow synchronised and labelled worms until late L4-young adult stage that were irradiated with 90Gy of IR using a Blood-Xrad apparatus. Labelled worms proteins were extracted in 50mM Hepes-NaOH pH8.0, Urea 9M using a bead beater and zirconium 0.7mm beads. Extracts were mixed in 1:1 ratio before immunoprecipitation with the anti-diGly antibody followed by MS/MS, performed by Cell Signaling Technology.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Nematodes (ncbitaxon:333870)

SUBMITTER: Dimitris Xirodimas

PROVIDER: MSV000084214 | MassIVE | Mon Aug 19 07:32:00 BST 2019

REPOSITORIES: MassIVE

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Publications

The Balance between Mono- and NEDD8-Chains Controlled by NEDP1 upon DNA Damage Is a Regulatory Module of the HSP70 ATPase Activity.

Bailly Aymeric P AP Perrin Aurelien A Serrano-Macia Marina M Maghames Chantal C Leidecker Orsolya O Trauchessec Helene H Martinez-Chantar M L ML Gartner Anton A Xirodimas Dimitris P DP

Cell reports 20191001 1

Ubiquitin and ubiquitin-like chains are finely balanced by conjugating and de-conjugating enzymes. Alterations in this balance trigger the response to stress conditions and are often observed in pathologies. How such changes are detected is not well understood. We identify the HSP70 chaperone as a sensor of changes in the balance between mono- and poly-NEDDylation. Upon DNA damage, the induction of the de-NEDDylating enzyme NEDP1 restricts the formation of NEDD8 chains, mainly through lysines K1 ...[more]

PMID: 31577950

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