Histone Deacetylase 3 Associates With Key Regulators of NFκB Signaling in Lung Epithelial Cells Under Stimulation With LPS.
Introduction:Histone Deacetylase 3 (HDAC3) is a Class I member of the histone deacetylase family that is recruited to nuclear enhancers. HDAC3 is located within the cell nucleus, where it constitutes the sole endogenous HDAC interacting with the nuclear-receptor corepressor complex containing NCOR and SMRT (NCOR2). As such, HDAC3 has a unique role in modulating the transcriptional activity of nuclear receptors. We have previously demonstrated its involvement in the inflammatory response in Lipopolysaccharide A (LPS)-stimulated human MH-S alveolar macrophages in vitro , and its interaction with pro-inflammatory IL-12 and anti-inflammatory IL-10. Similarly, our previous work using the broad spectrum HDAC inhibitor trichostatin A has demonstrated therapeutic efficacy reducing the pathogenesis of acute gram negative pneumonia in-vivo . With the current project, we aim to identify the HDAC3 interactome in LPS-stimulated human pulmonary epithelial cells type II (A549), the first immune defense in pathogens invading the lungs.
Methods:: Human A549 cells were transfected with a BioID2-tagged HDAC3 plasmid, and were divided in three groups: a. Untreated, b. stimulated with LPS, and c. stimulated with LPS and treated with Entinostat (MS275, a HDAC1/3 inhibitor). Cells were lysed, and total and biotinylated protein were submitted for quantitative Liquid Chromatography/Mass Spectrometry analysis to identify the peptides that directly bind with HDAC3. Data were searched against the Swiss Protein Human database, and False Discovery Rate (FDR) was set at 1%.
Results:: The dataset yielded 395,776 peptide matches. After peptide sequencing by database searching and validation, and FDR exclusion, we identified 2,666 proteins. Of these, 33 have been annotated to function in the NF-kB signaling pathway and/or NF-kB-regulated transcriptional activity.
Conclusion:Protein acetylation is a reversible post-translational modification that is known to stabilize protein structure and modify function and localization. With the current project we have identified several potential proteins in the HDAC3 interactome that may be targeted early in the NF-κB activation process to disrupt HDAC3-substrate interaction and potentially downstream signaling. Nuclear activation of NF-κB is known to be involved in the early stages of Acute Respiratory Distress Syndrome and is a target of intense study for the development of novel therapeutics.
- Stanfield, B; Soderblom, E; Palmer, S; Sullenger, B; Purves, T; Welty-Wolf, K; Agarwal, S; Kasotakis, G
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