Dataset Information

A chemoproteomics approach to discover regulators of innate immune signaling

ABSTRACT: Viral dsRNA binds to Retinoic acid Inducible Gene I (RIG-I) Like Receptors (RLRs), promoting the production of Interferon (IFN). Interferon then stimulates the innate and adaptive immune system in an autocrine and paracrine manner. Outside of conical pathways, regulators of the interferon (IFN) activation/response system are poorly characterized. In this study, we used a discovery-biased approach to identify Kinases that are part of the interferon system. Differential changes in phosphorylation sites, in the context of dsRNA RIG-I stimulation, were identified with unbiased mass-spec biased phospho-proteomics. We then computationally identified several Kinases upregulated after RIG-I stimulation from phospho-proteomics data. A Chemoproteomics screen was then used to characterize the altered interferon response in the presence of Kinases inhibitors for the upregulated kinases. Combining unbiased phosphoproteomics with a chemoproteomics screen, we identified several potentially novel regulators of the Interferon system whose inhibition blocked the production of Interferon Stimulated Genes.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

SUBMITTER: Jeffrey Johnson

LAB HEAD: Jeffrey R. Johnson

PROVIDER: PXD041369 | Pride | 2023-06-02

REPOSITORIES: Pride

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A Chemical Proteomics Approach to Discover Regulators of Innate Immune Signaling.

Kurland Andrew P AP Bonaventure Boris B Johnson Jeffrey R JR

Viruses 20230503 5

Innate immune pathways are tightly regulated to balance an appropriate response to infectious agents and tolerable levels of inflammation. Dysregulation of innate immune pathways can lead to severe autoinflammatory disorders or susceptibility to infections. Here, we aimed to identify kinases in common cellular pathways that regulate innate immune pathways by combining small-scale kinase inhibitor screening with quantitative proteomics. We found that inhibitors of kinases ATM, ATR, AMPK, and PLK1 ...[more]

PMID: 37243198

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Project description:Beneficial effects of SIRT1 on healthspan are likely to be pleiotropic and may include effects on DNA methylation. We demonstrated recently that manipulating SIRT1 in human cells affected DNA methylation of a panel of test genes, and that genes with expression modified by dietary restriction corresponded with genes that underwent changes in DNA methylation during ageing. Here we tested the hypothesis that genes particularly susceptible to SIRT1-induced effects on DNA methylation across the genome map to genes for which DNA methylation changes during ageing. We increased or reduced SIRT1 expression in human intestinal (Caco-2) and vascular endothelial (HuVEC) cells by transient transfection with an expression construct or with siRNA, respectively. Effects on DNA methylation were measured by enriching for the methylated fraction then either sequencing (HuVEC) or hybridising to a human promoter microarray (Caco-2). Effects using these two different cell lines and techniques for analysis were remarkably consistent. Genes with a DNA methylation status affected by SIRT1 manipulation were enriched for those that undergo age-dependent changes in DNA methylation, thus supporting our hypothesis. Polycomb group protein target genes (PCGTs), which are suppressed by epigenetic mechanisms in stem cells and have been shown previously to correspond with loci particularly susceptible to age-related changes in DNA methylation, were over-represented within the set of genes showing altered DNA methylation in response to SIRT1 manipulation in both cell lines. We thus propose that effects of SIRT1 to extend healthspan include influences on the DNA methylation status of genes affected during ageing, in particular PCGTs. Analysis of methylation profiles of Caco-2 cells meeting the following conditions: SIRT1 overexpressed by transfection with pCMV6-ENTRY-SIRT1 (2 replicates), corresponding vector control (2 replicates), each of the siRNAs that target SIRT1 (1 & 2 replicates, respectively) and control siRNA (2 replicates).

Dataset Information

A chemoproteomics approach to discover regulators of innate immune signaling

Publications

A Chemical Proteomics Approach to Discover Regulators of Innate Immune Signaling.

Similar Datasets

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