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Identification of RNF111 substrates by integrative ubiquitome and proteome analysis

ABSTRACT: In the present work, we sought to identify exhaustively the endogenous substrates ubiquitinated and degraded by the E3 ubiquitin ligase RNF111 in presence of TGF-β signaling by performing label free quantitative proteomics after enrichment of ubiquitinated proteins (ubiquitome) in parental U2OS cell line compared to U2OS CRISPR engineered clones expressing a truncated form of RNF111 devoid of C-terminal Ring domain. We compare two methods of enrichment for ubiquitinated proteins prior to mass spectrometry proteomics analysis, the diGly remnant peptide immunoprecipitation with a K-e-GG antibody and a novel approach using protein immunoprecipitation with an ubiquitin pan (Pan UB) nanobody that recognizes all ubiquitin chains and mono-ubiquitination. These ubiquitomes were compared to the corresponding proteome to identify proteins ubiquitinated and degraded by RNF111 upon TGF-β signaling pathway.

INSTRUMENT(S): Q Exactive HF-X, Orbitrap Fusion, Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Osteosarcoma Cell Line

SUBMITTER: Victor Laigle

LAB HEAD: Damarys Loew

PROVIDER: PXD025890 | Pride | 2021-11-17

REPOSITORIES: Pride

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Project description:Huntington’s disease is caused by a polyglutamine repeat expansion at the N-terminus of the huntingtin protein which affects the function and folding of the protein, and results in intracellular protein aggregates. Here, we examined whether this mutation leads to altered ubiquitination of huntingtin and other proteins in both soluble and insoluble fractions of brain lysates of the Q175 knock-in Huntington disease mouse model and the Q20 wild-type mouse model. Ubiquitination sites are detected by identification of Gly-Gly (diGly) remnant motifs that remain on modified lysine residues after digestion. We identified K6, K9, K132, K804 and K837 as endogenous ubiquitination sites of soluble huntingtin, with wild-type huntingtin being mainly ubiquitinated at K132, K804 and K837. Mutant huntingtin protein levels were strongly reduced in the soluble fraction while K6 and K9 were mainly ubiquitinated. In the insoluble fraction increased levels of huntingtin K6 and K9 diGly sites were observed for mutant huntingtin as compared to wild type. Besides huntingtin, proteins with various roles, including membrane organization, transport, mRNA processing, gene transcription, translation, catabolic processes and oxidative phosphorylation, were differently expressed or ubiquitinated in wild-type and mutant huntingtin brain tissues. Correlating protein and diGly site fold-changes in the soluble fraction revealed that diGly site abundances of the majority of the proteins were not related to protein fold-changes, indicating that these proteins were differentially ubiquitinated in the Q175 mice. In contrast, both the fold-change of the protein level and diGly site level were increased for several proteins in the insoluble fraction, including ubiquitin, ubiquilin-2, sequestosome-1/p62 and myo5a. Our data sheds light on putative novel proteins involved in different cellular processes as well as their ubiquitination status in Huntington’s disease, which forms the basis for further mechanistic studies to understand the role of differential ubiquitination of huntingtin as well as ubiquitin-regulated processes in Huntington’s disease.

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Identification of RNF111 substrates by integrative ubiquitome and proteome analysis

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