Dataset Information

Ubiquitin-mediated modulation of necroptotic effector MLKL

ABSTRACT: We aimed to identify lysine (K) residues that are endogenously ubiquitylated upon TNF-induced necroptosis. To this end, we used a quantitative mass spectrometry-based approach. Briefly, MDFs were treated with TSZ or left untreated and protein lysates were digested with trypsin into peptides. Ubiquitylated peptides bearing the di-glycine (K-GG) remnant were enriched by immunoprecipitation with anti-K-ε-GG antibody. A total of three biological replicates were prepared and the 6 samples were labelled with Tandem Mass Tags (TMT). Samples were mixed and analysing by Liquid Chromatography-Tandem Mass Spectroscopy (LC-MS). Quantification was performed to determine the relative abundance of site-specific ubiquitylation events in each condition.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture, Fibroblast

SUBMITTER: James Wright

LAB HEAD: Jyoti Choudhary

PROVIDER: PXD018857 | Pride | 2021-04-13

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	LRG02TMT6plex-V5.msf	Msf
	LRG02TMT6plex-V5.pdResult	Other
	LRG02TMT6plex-V5_PeptideGroups.txt	Txt
	LRG02TMT6plex-V5_Proteins.txt	Txt
	Lumos50cm_LRG02TMT6plex-P01.raw	Raw

Items per page:

1 - 5 of 12

Publications

Ubiquitylation of MLKL at lysine 219 positively regulates necroptosis-induced tissue injury and pathogen clearance.

Garcia Laura Ramos LR Tenev Tencho T Newman Richard R Haich Rachel O RO Liccardi Gianmaria G John Sidonie Wicky SW Annibaldi Alessandro A Yu Lu L Pardo Mercedes M Young Samuel N SN Fitzgibbon Cheree C Fernando Winnie W Guppy Naomi N Kim Hyojin H Liang Lung-Yu LY Lucet Isabelle S IS Kueh Andrew A Roxanis Ioannis I Gazinska Patrycja P Sims Martin M Smyth Tomoko T Ward George G Bertin John J Beal Allison M AM Geddes Brad B Choudhary Jyoti S JS Murphy James M JM Aurelia Ball K K Upton Jason W JW Meier Pascal P

Nature communications 20210607 1

Necroptosis is a lytic, inflammatory form of cell death that not only contributes to pathogen clearance but can also lead to disease pathogenesis. Necroptosis is triggered by RIPK3-mediated phosphorylation of MLKL, which is thought to initiate MLKL oligomerisation, membrane translocation and membrane rupture, although the precise mechanism is incompletely understood. Here, we show that K63-linked ubiquitin chains are attached to MLKL during necroptosis and that ubiquitylation of MLKL at K219 sig ...[more]

PMID: 34099649

Similar Datasets

Project description:Oncolytic Reovirus type 3 Dearing (RT3D), is a naturally occurring double-stranded (ds) RNA virus that is under development as an oncolytic immunotherapy We used an unbiased high-throughput cytotoxicity screen of different targeted therapeutic agents with the aim of identifying potential drug-viral sensitizers to enhance RT3D tumour killing. Talazoparib, a clinical poly(ADP)-ribose polymerase 1 (PARP-1) inhibitor, was identified as a top hit and found to cause profound sensitisation to RT3D. This effect was not seen with other classes of oncolytic virus and was not mediated by enhanced viral replication or PARP inhibitor-related effects on the DNA damage response. RT3D interacts with retinoic acid-induced gene-1 (RIG-I) and activates PARP-1, with consequent PARylation of components of the extrinsic apoptosis pathway. Pharmacological and genetic inhibition of PARP-1 abrogates this PARylation and increases levels of extrinsic apoptosis, NF-kB signalling and pro-inflammatory cell death. Direct interaction between PARP-1 and RIG-I following RT3D/talazoparib treatment is a key factor in activating downstream signaling pathways that lead to IFN- and TNF-/TRAIL production which, in turn, amplify the therapeutic effect through positive feedback. Critically, it was possible to phenocopy the effect of RT3D through the use of non-viral ds-RNA therapy and RIG-I agonism. In in vivo studies, we demonstrated profound combinatorial efficacy of RT3D and talazoparib in human A375 melanoma in immunodeficient mice. More impressively, in immunocompetent mouse models of 4434 murine melanoma, we achieved 100% tumour control and protection from subsequent tumour rechallenge with the combination regimen. Correlative immunophenotyping confirmed significant innate and adaptive immune activation with the combination of RT3D and PARP inhibition. Taken together, these data provide a clear line of sight to clinical translation of combined regimens of PARP inhibition or ds-RNA agonism, with either viral or non-viral agents, in tumour types beyond the relatively narrow confines of current licensed indications for PARP inhibition.