title: Dynamic changes in ribosome-associated proteome and phosphoproteome during deoxynivalenol-induced translation inhibition and ribotoxic stress.
abstract: Deoxynivalenol (DON), a trichothecene mycotoxin produced by Fusarium that commonly contaminates cereal-based food, interacts with the ribosome to cause translation inhibition and activate stress kinases in mononuclear phagocytes via the ribotoxic stress response (RSR). The goal of this study was to test the hypothesis that the ribosome functions as a platform for spatiotemporal regulation of translation inhibition and RSR. Specifically, we employed stable isotope labeling of amino acids in cell culture (SILAC)-based proteomics to quantify the early (≤ 30 min) DON-induced changes in ribosome-associated proteins in RAW 264.7 murine macrophage. Changes in the proteome and phosphoproteome were determined using off-gel isoelectric focusing and titanium dioxide chromatography, respectively, in conjunction with LC-MS/MS. Following exposure of RAW 264.7 to a toxicologically relevant concentration of DON (250 ng/ml), we observed an overall decrease in translation-related proteins interacting with the ribosome, concurrently with a compensatory increase in proteins that mediate protein folding, biosynthesis, and cellular organization. Alterations in the ribosome-associated phosphoproteome reflected proteins that modulate translational and transcriptional regulation, and others that converged with signaling pathways known to overlap with phosphorylation changes characterized previously in intact RAW 264.7 cells. These results suggest that the ribosome plays a central role as a hub for association and phosphorylation of proteins involved in the coordination of early translation inhibition as well as recruitment and maintenance of stress-related proteins-both of which enable cells to adapt and respond to ribotoxin exposure. This study provides a template for elucidating the molecular mechanisms of DON and other ribosome-targeting agents.
authors.affiliation: * Department of Biochemistry and Molecular Biology;
authors.forename: Xiao
authors.initials: X
authors.lastname: Pan
authors.forename: Douglas A
authors.initials: DA
authors.lastname: Whitten
authors.forename: Curtis G
authors.initials: CG
authors.lastname: Wilkerson
authors.forename: James J
authors.initials: JJ
authors.lastname: Pestka
mesh_terms: D000818:Animals; D002460:Cell Line; D063990:Gene Ontology; D007700:Kinetics; D051379:Mice; D010750:Phosphoproteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D014176:Protein Biosynthesis / Q000187:drug effects*; D020543:Proteome / Q000235:genetics / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects* / Q000378:metabolism; D013312:Stress, Physiological / Q000187:drug effects*; D014255:Trichothecenes / Q000633:toxicity*
chemical_list: D010750:Phosphoproteins; D020543:Proteome; D012269:Ribosomal Proteins; D014255:Trichothecenes; C007262:deoxynivalenol
keywords: deoxynivalenol; proteomics.; ribosome; ribotoxic stress response; translation