Dataset Information

Phosphorylation Francisella

ABSTRACT: Francisella possesses a non-canonical T6SS that is essential for efficient phagosomal escape and access to the cytosol of infected macrophages. Using a global and site-specific phosphoproteomic analysis of Francisella we identified here a unique phosphorylation site on IglB, the TssC homologue and a key component of the T6SS contractile sheath. Phosphorylation of the sheath may constitute a previously unrecognized mechanism contributing to the dynamics of assembly-disassembly of the T6SS.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Escherichia Coli

SUBMITTER: Chiara guerrera

LAB HEAD: Chiara Guerrera

PROVIDER: PXD012507 | Pride | 2023-12-20

REPOSITORIES: Pride

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Publications

Critical Role of a Sheath Phosphorylation Site On the Assembly and Function of an Atypical Type VI Secretion System.

Ziveri Jason J Chhuon Cerina C Jamet Anne A Rytter Héloïse H Prigent Guénolé G Tros Fabiola F Barel Monique M Coureuil Mathieu M Lays Claire C Henry Thomas T Keep Nicholas H NH Guerrera Ida Chiara IC Charbit Alain A

Molecular & cellular proteomics : MCP 20191002 12

The bacterial pathogen <i>Francisella tularensis</i> possesses a noncanonical type VI secretion system (T6SS) that is required for phagosomal escape in infected macrophages. KCl stimulation has been previously used to trigger assembly and secretion of the T6SS in culture. By differential proteomics, we found here that the amounts of the T6SS proteins remained unchanged upon KCl stimulation, suggesting involvement of post-translational modifications in T6SS assembly. A phosphoproteomic analysis i ...[more]

PMID: 31578219

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Project description:Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by a non-coding CTG repeat expansion in the DMPK gene. This mutation generates a toxic CUG RNA that interferes with the RNA processing of target genes in multiple tissues. Despite debilitating neurological impairment, the pathophysiological cascade of molecular and cellular events in the central nervous system has been less extensively characterized than the molecular pathogenesis of muscle/cardiac dysfunction. Particularly, the contribution of different cell types to DM1 brain disease is not clearly understood. We first used transcriptomics to compare the impact of expanded CUG RNA on the transcriptome of primary neurons, astrocytes and oligodendrocytes derived from DMSXL mice, a transgenic model of DM1. RNA sequencing revealed more frequent expression and splicing changes in glia than neuronal cells. In particular, primary DMSXL oligodendrocytes showed the highest number of transcripts differentially expressed, while DMSXL astrocytes displayed the most severe splicing dysregulation. Interestingly, the expression and splicing defects of DMSXL glia recreated molecular signatures suggestive of impaired cell differentiation: while DMSXL oligodendrocytes failed to upregulate a subset of genes that are naturally activated during the oligodendroglia differentiation, a significant proportion of missplicing events in DMSXL oligodendrocytes and astrocytes increased the expression of RNA isoforms typical of precursor cell stages. Together these data suggest that expanded CUG RNA in glial cells affects preferentially differentiation-regulated molecular events. This hypothesis was corroborated by gene ontology (GO) analyses, which revealed an enrichment for biological processes and cellular components with critical roles during cell differentiation. Finally, we combined exon ontology with phosphoproteomics and cell imaging to explore the functional impact of CUG-associated spliceopathy on downstream protein metabolism. Changes in phosphorylation, protein isoform expression and intracellular localization in DMSXL astrocytes demonstrate the far-reaching impact of the DM1 repeat expansion on cell metabolism. Our multi-omics approaches provide insight into the mechanisms of CUG RNA toxicity in the CNS with cell type resolution, and support the priority for future research on non-neuronal mechanisms and proteomic changes in DM1 brain disease.

Project description:Poly(ADP-ribose) Polymerase 1 (PARP-1) is a nuclear enzyme that catalyse the transfer of ADP-ribose units from NAD+ to several target proteins involved in cellular stress responses. Using the cell line WRL68, we have previously shown that the activation of PARP-1 induced by oxidative stress after H2O2 treatment lead to depletion of cellular NAD+ and ATP, which promote cell death. In this work, we used LC-MS/MS based phosphoproteomics to show that the oxidative damage induced by H2O2 treatment modified the phosphorylation of several proteins in WRL68 cells, most of them related to transcription process. Interestingly, genetic or pharmacological inhibition of PARP-1 reduced the phosphorylation of YAP1, a transcriptional coactivator involved in cell survival, in WRL68 cells after H2O2 treatment. Inhibition of PARP-1 increased cell viability after oxidative treatment and diminished the degradation of YAP1; the elimination of YAP1 by gene silencing abrogated the protective effect of PARP-1 inhibition, indicating that YAP1 is important for the survival of WRL68 cells after oxidative damage. In the same way, the supplementation of NAD+, substrate of PARP-1, on WRL68 cells suffering oxidative damage reduced the phosphorylation of YAP1, suggesting that the loss of cellular NAD+ caused by PARP-1 activation after oxidative treatment is responsible for the phosphorylation of YAP1. Finally, PARP-1 silencing after oxidative treatment diminished the activation of AMPK, a master regulator of cell metabolism implicated in the restoration of energy balance during metabolic stress. Since NAD+ supplementation reduced the phosphorylation of some AMPK substrates in WRL68 cells after oxidative treatment, we hypothesize that the loss of cellular NAD+ after PARP-1 activation may induce a metabolic stress that activates AMPK. In summary, we showed a new crucial role of PARP-1 in the response to oxidative stress in WRL68 cells; PARP-1 activation reduces the cell viability by promoting the phosphorylation and degradation of YAP1, which is mediated by the energy stress caused by PARP-1 activation.

Dataset Information

Phosphorylation Francisella

Publications

Critical Role of a Sheath Phosphorylation Site On the Assembly and Function of an Atypical Type VI Secretion System.

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