The trichothecene mycotoxin deoxynivalenol (DON) is a common food contaminant that is of public health significance (Pestka, 2010) because it is a translational inhibitor that targets the innate immune system. 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). While it is recognized that DON-induced RSR involves protein phosphorylation and that DON targets the ribosome, a comprehensive assessment of how these events contribute to signaling, modulation of ribosome function and regulation of key biological processes is lacking.To encapture global signaling events mediating DON-induced RSR and immunotoxicity, we employed quantitative proteomics to evaluate the dynamics of protein phosphorylation during early (≤30min) DON-induced RSR in RAW 264.7 murine macrophage treated with a toxicologically relevant concentration of DON (250ng/mL) and in the spleens of mice orally exposed to 5 mg/kg body weight DON. Large-scale phosphoproteomic analysis employing stable isotope labeling of amino acids in cell culture (SILAC) for RAW 264.7 or stable isotope dimethyl labeling for mouse spleen, in conjunction with titanium dioxide chromatography revealed that DON-induced RSR involves extensive phosphorylation alterations. In RAW 264.7, transcriptional regulation was the main target during early DON-induced RSR involving transcription factors/cofactors and epigenetic modulators. Other biological processes impacted included cell cycle, RNA processing, translation, ribosome biogenesis, monocyte differentiation and cytoskeleton organization. Some of these processes could be mediated by signaling networks involving MAPK-, NFκB-, AKT- and AMPK-linked pathways.To understand the role of the ribosome in the spatiotemporal regulation of translational inhibition and the RSR, we evaluated dynamic changes in ribosome-associated proteome and phosphoproteome in RAW 264.7 cells similarly labeled and treated with DON. There was an overall decrease in translation-related proteins interacting with the ribosome, concurrently with a compensatory increase in proteins that mediate protein folding, biosynthetic pathways, and cellular organization. Alterations in the ribosome-associated phosphoproteome reflected proteins known to modulate translational and transcriptional regulation, as well as others that converged with known signaling pathways. These overlapped with phosphoproteins previously characterized in intact RAW 264.7 cells, suggesting the role of the ribosome as a platform for interaction and phosphorylation of proteins involved in the coordination of the early translation inhibition as well as recruitment and maintenance of stress-related proteins.In vivo DON exposure in the mouse affected biological processes such as cytoskeleton organization, regulation of apoptosis, and lymphocyte activation and development in the spleen, which likely contribute to immune dysregulation previously reported for the toxin. 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, which could be mediated by the MAPK and PI3K/AKT pathways.Taken together, the proteomic analyses presented in this dissertation revealed extensive phosphorylation events mediate signaling and regulation key biological processes including transcriptional regulation leading to DON-induced RSR and immunotoxicity. Serving as a platform for stress-related proteins and phosphoproteins, the ribosome facilitates spatiotemporal regulation of translation inhibition and RSR at the subcellular level.
Read
