Subcellular Quantitative Proteomics Reveals Multiple Pathway Cross-Talk That Coordinates Specific Signaling and Transcriptional Regulation for the Early Host Response to LPS

Abstract

Toll-like receptor 4 (TLR4) specifically recognizes lipopolysaccharide (LPS) to initiate signal transduction events that modulate host inflammatory responses. Although increasing numbers of genes have been characterized individually for their involvement in TLR4 signaling, the LPS-induced TLR4-mediated signaling pathway and connected networks are incompletely delineated. Given that most components of signaling pathways are activated at an early phase of the LPS-induced response, we have employed a subcellular, SILAC-based quantitative proteomics approach to identify proteins in LPS-stimulated macrophages showing either cytosolic- or nuclear-specific changes in abundance. Subcellular fractionation not only reduces the spectral complexity for identifying maximum numbers of proteins but also enriches for low-abundance proteins within the compartment in which they function. Following 10 min LPS stimulation, the abundances of 508 proteins were found elevated in the cytosol, while the elevated levels of 678 proteins together with the decreased abundances of another 80 proteins were quantified in nuclei. Coincident with observations that many key proteins involved in signal relays in the MAPK and NF-kappaB cascades were found simultaneously regulated in the cytosol, various transcriptional factors (TFs) such as IRFs were found activated in the nuclei. We also extended links between these intracellular pathways and various biological processes by identifying multiple pathway modules. For the first time, our combined data sets from quantitative proteomics and bioinformatics analyses provide a direct, system-wide insight into how cross-talk between upstream signaling pathways modulates the activities of particular TFs for regulating sets of pro-inflammatory genes.