Quantitative Proteomics Analysis of Macrophage Rafts Reveals Compartmentalized Activation of the Proteasome and of Proteasome-mediated ERK Activation in Response to Lipopolysaccharide

Laboratories of Respiratory Biology, NIEHS, National Institutes of Health, United States Department of Health and Human Services, Research Triangle Park, North Carolina 27709, USA.
Molecular &amp Cellular Proteomics (Impact Factor: 6.56). 10/2008; 8(1):201-13. DOI: 10.1074/mcp.M800286-MCP200
Source: PubMed


Lipopolysaccharide (LPS), a glycolipid component of the outer membrane of Gram-negative bacteria, is a potent initiator of the innate immune response of the macrophage. LPS triggers downstream signaling by selectively recruiting and activating proteins in cholesterol-rich membrane microdomains called lipid rafts. We applied proteomics analysis to macrophage detergent-resistant membranes (DRMs) during an LPS exposure time course in an effort to identify and validate novel events occurring in macrophage rafts. Following metabolic incorporation in cell culture of heavy isotopes of amino acids arginine and lysine ([(13)C(6)]Arg and [(13)C(6)]Lys) or their light counterparts, a SILAC (stable isotope labeling with amino acids in cell culture)-based quantitative, liquid chromatography-tandem mass spectrometry proteomics approach was used to profile LPS-induced changes in the lipid raft proteome of RAW 264.7 macrophages. Unsupervised network analysis of the proteomics data set revealed a marked representation of the ubiquitin-proteasome system as well as changes in proteasome subunit composition following LPS challenge. Functional analysis of DRMs confirmed that LPS causes selective activation of the proteasome in macrophage rafts and proteasome inactivation outside of rafts. Given previous reports of an essential role for proteasomal degradation of IkappaB kinase-phosphorylated p105 in LPS activation of ERK mitogen-activated protein kinase, we tested for a role of rafts in compartmentalization of these events. Immunoblotting of DRMs revealed proteasome-dependent activation of MEK and ERK specifically occurring in lipid rafts as well as proteasomal activity upon raft-localized p105 that was enhanced by LPS. Cholesterol extraction from the intact macrophage with methyl-beta-cyclodextrin was sufficient to activate ERK, recapitulating the LPS-IkappaB kinase-p105-MEK-ERK cascade, whereas both it and the alternate raft-disrupting agent nystatin blocked subsequent LPS activation of the ERK cascade. Taken together, our findings indicate a critical, selective role for raft compartmentalization and regulation of proteasome activity in activation of the MEK-ERK pathway.

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Available from: Kenneth Tomer, Oct 02, 2015
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    • "It has been reported that LPS, known to efficiently induce ALIS in macrophages [6], [60], interacts directly with proteasome which may contribute to LPS-induced inflammatory responses of macrophages [61], and that UPS components localized in lipid rafts may be instrumental to this action [62]. It should be noted that we never observed any direct physical contact between UPS-storing PaCSs and the plasma membrane, where lipid rafts are to be found, and that in macrophages we failed to detect PaCSs, even after LPS treatment. "
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    ABSTRACT: A variety of ubiquitinated protein-containing cytoplasmic structures has been reported, from aggresomes to aggresome-like induced structures/sequestosomes or particle-rich cytoplasmic structures (PaCSs) that we recently observed in some human diseases. Nevertheless, the morphological and cytochemical patterns of the different structures remain largely unknown thus jeopardizing their univocal identification. Here, we show that PaCSs resulted from proteasome and polyubiquitinated protein accumulation into well-demarcated, membrane-free, cytoskeleton-poor areas enriched in glycogen and glycosaminoglycans. A major requirement for PaCS detection by either electron or confocal microscopy was the addition of osmium to aldehyde fixatives. However, by analyzing living cells, we found that proteasome chymotrypsin-like activity concentrated in well-defined cytoplasmic structures identified as PaCSs by ultrastructural morphology and immunocytochemistry of the same cells. PaCSs differed ultrastructurally and cytochemically from sequestosomes which may coexist with PaCSs. In human dendritic or natural killer cells, PaCSs were induced in vitro by cytokines/trophic factors during differentiation/activation from blood progenitors. Our results provide evidence that PaCS is indeed a novel distinctive cytoplasmic structure which may play a critical role in the ubiquitin-proteasome system response to immune, infectious or proneoplastic stimuli.
    PLoS ONE 12/2013; 8(12):e82560. DOI:10.1371/journal.pone.0082560 · 3.23 Impact Factor
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    • "While these studies are important for identification of biomarkers of effect, they are not informative from perspective of early events and signaling within the macrophage, a primary target of DON which mediates innate immune activation (Pestka, 2008; Zhou et al., 2003). Stable isotope labeling of amino acids in cell culture (SILAC) has been successfully used to characterize the signaling and subcellular compartmentalization for global delineation of macrophage behavior during phagocytosis and upon toll-like receptor stimulation (Dhungana et al., 2009; Rogers and Foster, 2007), suggesting the applicability of this strategy to the study of DON-induced RSR. "
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    ABSTRACT: The trichothecene mycotoxin deoxynivalenol (DON) targets the innate immune system and is of public health significance because of its frequent presence in human and animal food. DON-induced proinflammatory gene expression and apoptosis in the lymphoid tissue have been associated with a ribotoxic stress response (RSR) that involves rapid phosphorylation of mitogen-activated protein kinases (MAPKs). To better understand the relationship between protein phosphorylation and DON's immunotoxic effects, stable isotope dimethyl labeling-based proteomics in conjunction with titanium dioxide chromatography was employed to quantitatively profile the immediate (≤30 min) phosphoproteome changes in the spleens of mice orally exposed to 5 mg/kg body weight DON. A total of 90 phosphoproteins indicative of novel phosphorylation events were significantly modulated by DON. In addition to critical branches and scaffolds of MAPK signaling being affected, DON exposure also altered phosphorylation of proteins that mediate PI3K/AKT pathways. Gene ontology analysis revealed that DON exposure affected biological processes such as cytoskeleton organization, regulation of apoptosis, and lymphocyte activation and development, which likely contribute to immune dysregulation associated with DON-induced RSR. Consistent with these findings, DON impacted phosphorylation of proteins within diverse immune cell populations, including monocytes, macrophages, T cells, B cells, dendritic cells and mast cells. Fuzzy c-means clustering analysis further indicated that DON evoked several distinctive temporal profiles of regulated phosphopeptides. Overall, the findings from this investigation can serve as a template for future focused exploration and modeling of cellular responses associated with the immunotoxicity evoked by DON and other ribotoxins.
    Toxicological Sciences 06/2013; DOI:10.1093/toxsci/kft145 · 3.85 Impact Factor
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    • "Commonly, only a limited number of proteins have been used to assign cellular phenotype, and the specificity of such proteins for any given subpopulation has not been rigorously demonstrated [9]. Mass spectrometry has been widely used to investigate the proteomes of macrophages and dendritic cells [10]–[12], but much less is known about the plasma membrane proteomes of these cells. Moreover, no studies have directly compared the proteomes of macrophages and DCs. "
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    ABSTRACT: Monocytes differentiate into heterogeneous populations of tissue macrophages and dendritic cells (DCs) that regulate inflammation and immunity. Identifying specific populations of myeloid cells in vivo is problematic, however, because only a limited number of proteins have been used to assign cellular phenotype. Using mass spectrometry and bone marrow-derived cells, we provided a global view of the proteomes of M-CSF-derived macrophages, classically and alternatively activated macrophages, and GM-CSF-derived DCs. Remarkably, the expression levels of half the plasma membrane proteins differed significantly in the various populations of cells derived in vitro. Moreover, the membrane proteomes of macrophages and DCs were more distinct than those of classically and alternatively activated macrophages. Hierarchical cluster and dual statistical analyses demonstrated that each cell type exhibited a robust proteomic signature that was unique. To interrogate the phenotype of myeloid cells in vivo, we subjected elicited peritoneal macrophages harvested from wild-type and GM-CSF-deficient mice to mass spectrometric and functional analysis. Unexpectedly, we found that peritoneal macrophages exhibited many features of the DCs generated in vitro. These findings demonstrate that global analysis of the membrane proteome can help define immune cell phenotypes in vivo.
    PLoS ONE 03/2012; 7(3):e33297. DOI:10.1371/journal.pone.0033297 · 3.23 Impact Factor
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