Multi-omic Data Integration Links Deleted in Breast Cancer 1 (DBC1) Degradation to Chromatin Remodeling in Inflammatory Response

Ernesto S Nakayasu,Roslyn N Brown,Charles Ansong,Michael A Sydor,Sayed Imtiaz,Cosmin Mihai,Ryan Sontag,Kim K Hixson,Matthew E Monroe,Tiago J.P Sobreira,Galya Orr,Vladislav A Petyuk,Feng Yang,Richard D Smith,Joshua N Adkins

doi:10.1074/mcp.m112.026138

Abstract

This study investigated the dynamics of ubiquitinated proteins after the inflammatory stimulation of RAW 264.7 macrophage-like cells with bacterial lipopolysaccharide. Ubiquitination is a common protein post-translational modification that regulates many key cellular functions. We demonstrated that levels of global ubiquitination and K48 and K63 polyubiquitin chains change after lipopolysaccharide stimulation. Quantitative proteomic analysis identified 1199 ubiquitinated proteins, 78 of which exhibited significant changes in ubiquitination levels following stimulation. Integrating the ubiquitinome data with global proteomic and transcriptomic results allowed us to identify a subset of 88 proteins that were targeted for degradation after lipopolysaccharide stimulation. Using cellular assays and Western blot analyses, we biochemically validated DBC1 (a histone deacetylase inhibitor) as a degradation substrate that is targeted via an orchestrated mechanism utilizing caspases and the proteasome. The degradation of DBC1 releases histone deacetylase activity, linking lipopolysaccharide activation to chromatin remodeling in caspase- and proteasome-mediated signaling.

Highlights

Ubiquitination is a versatile protein post-translational modification related to many key cellular functions
We investigated global protein ubiquitination dynamics in host immune cells induced by S
We show that one of these targeted proteins, a histone deacetylase (HDAC) inhibitor known as deleted in breast cancer 1 (DBC1), provides a signaling mechanism that controls chromatin remodeling in an inflammatory response

Summary

Introduction

Ubiquitination is a versatile protein post-translational modification related to many key cellular functions. Typhimurium) interferes with these pathways by expressing and secreting four known E3 ubiquitin ligases (SopA, SspH1, SspH2, and SlrP) and two known DUBs (SseL and AvrA) into host cells [5] Of these proteins, SseL was shown to deubiquitinate I␬B, which prevents it from being degraded by the proteasome and leads to inhibition of NF-␬B activation [6]. We used a RAW 264.7 macrophage-like cell line that retains many common functions of primary macrophages [7] as our model host immune cells The results from this investigation demonstrate that global levels of protein ubiquitination change after LPS stimulation and that this change modulates the activity of DUBs. We took advantage of previously published transcriptomics and proteomics data that, when integrated with our ubiquitinome data, allowed us to identify proteins degraded in response to LPS stimulus. We show that one of these targeted proteins, a histone deacetylase (HDAC) inhibitor known as deleted in breast cancer 1 (DBC1), provides a signaling mechanism that controls chromatin remodeling in an inflammatory response

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