Dataset Information

SILAC proteomics on MG132-induced nuclear aggregates

ABSTRACT: H1299 cells were labelled either with light (Lys0/Arg0) or heavy (Lys8/Arg10) amino acids and were either unstressed (light), stressed (heavy) with MG132 (15hrs) or stressed with MG132 and allowed to recover for 8hrs (light). Equal number of unstressed and MG132 stressed cells or MG132 stressed cells and cells after 8hrs recovery were mixed before isolation of nuclear inclusions (N-Lauroylsarcosine insoluble inclusions). Isolated inclusions were solubilised in 5% SDS before trypsin digestion (10g of sample) in columns using the universal proteomics sample preparation kit ProtiFi according to manufacturer’s instructions before eluted peptides were analysed by mass-spectrometry. Quantitation of data from 3 independent experiments.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Emmanuelle Mouton Barbosa

LAB HEAD: Odile Schiltz

PROVIDER: PXD054637 | Pride | 2025-05-07

REPOSITORIES: Pride

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Publications

A nuclear protein quality control system for elimination of nucleolus-related inclusions.

Brunello Lorène L Polanowska Jolanta J Le Tareau Léo L Maghames Chantal C Georget Virginie V Guette Charlotte C Chaoui Karima K Balor Stéphanie S O'Donohue Marie-Françoise MF Bousquet Marie-Pierre MP Gleizes Pierre-Emmanuel PE Xirodimas Dimitris P DP

The EMBO journal 20241217 3

The identification of pathways that control elimination of protein inclusions is essential to understand the cellular response to proteotoxicity, particularly in the nuclear compartment, for which our knowledge is limited. We report that stress-induced nuclear inclusions related to the nucleolus are eliminated upon stress alleviation during the recovery period. This process is independent of autophagy/lysosome and CRM1-mediated nuclear export pathways, but strictly depends on the ubiquitin-activ ...[more]

PMID: 39690241

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Project description:Background: The combination of Proteasome inhibitor with Glucocorticoid is one of the most promising antileukemic treatments. Both agents act through pluripotent signal mediators. The two types of agents inhibit key signals that are considered crucial to their effects on malignant cells. Methodology/Principal Findings: Combined use of the two reagents on the lymphoblastic leukemia cell line CCRF-CEM has a range of effects on the viability that depend on the dose and the time used. Even though both reagents are capable of enhancing cell death on the CEM cell line, no combinatorial increase in cell death is detectable, until after the first 120 hours of treatment. In contrast, there are a number of combinatorial effects on the cell cycle phase distribution of treated cells, which indicates a potential for mutual signal disruption between glucocorticoid and proteasome inhibitor at multiple levels. Microarray analysis indicates that Prednisolone and MG132 elicit highly divergent, early-response molecular signatures on this cell line. We assayed levels of the antiapoptotic protein Mcl-1 as a potential model of late, downstream target regulation by both glucocorticoid and proteasome. Synchronized use of Prednisolone with MG132 results in temporary stabilization of Mcl-1 in CEM cells. Stabilization is not the result of a common mechanism of action on the genome, as Prednisolone and MG132 elicit nonreduntant molecular signatures, and it occurs also when the cells are treated at different timepoints with either agent. Conclusions/Significance: Our results show that this glucocorticoid-resistant ALL lymphoblast line is highly sensitive to proteasome inhibitor, and suggest that the proteasome inhibitor and the glucocorticoid regulate different direct target genes. This difference in immediate targets, when the two agents are applied in combination, may lead to negative or positive interference with mechanisms regulating viability of the leukemic lymphoblast. Signal interference between glucocorticoid Prednisolone and the proteasome inhibitor MG132 is expected to occur at more than one level, resulting in complex effects on intracellular signal transduction pathways. The net result depends on the development of individual downstream effects and interactions between key signal mediators. Elucidation of the conditions of interference between glucocorticoid and proteasome targets on leukemic cell fate is expected to improve effects of applied treatment combinations. Experimental setups consisted of the three following samples obtained after 4 h treatment: control, 10nM prednisolone, 1uM prednisolone, 10uM prednisolone, 100M-NM-<M prednisolone, 700M-NM-<M prednisolone, 200nM MG132, 2M-NM-<M MG132 and 20M-NM-<M MG132. Untreated cells were used as reference.

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