Ubiquitination and phosphorylation modifications mediate the macrophage lipid metabolism elicited by poly(I:C)

Lipid droplets (LDs) are important organelles in the viral lifecycle, and viruses can regulate lipid metabolism and LD synthesis through various mechanisms. Ubiquitination, phosphorylation, and glycosylation, as classic protein modifications participating in the regulation of host lipid metabolism. However, it remains unclear whether dsRNA viruses can manipulate lipid metabolism through protein modifications. To investigate the roles of ubiquitination, phosphorylation, and N/O-glycosylation modifications in the lipid metabolism process induced by dsRNA viruses, we conducted preliminary explorations using proteomics and modificationomics techniques. Initially, primary kidney macrophages were isolated from Ctenopharyngodon idella using a percoll density gradient centrifugation and stimulated with poly(I:C) to simulate dsRNA virus infection. Subsequently, differential proteins in the three modificationomics types were invesitigated, and GO function and KEGG pathway enrichment were performed. The results revealed that 2 080 ubiquitinated proteins including 224 phosphorylation-modified differential proteins were identified in ubiquitination mass spectrometry. 8 lipid metabolism-related pathways encompassing 15 proteins were enriched in ubiquitination-modified differential proteins. Among all pathways enriched in ubiquitin modification proteomics, lipid metabolism pathways were significantly enriched. Phosphorylation modification mass spectrometry detected 1 415 phosphorylated proteins, including 139 differential phosphorylated proteins. The phosphorylated differential proteins were enriched in only 1 lipid metabolism pathway and 1 significantly upregulated protein cytosolic phospholipase A2 (cPLA2). N/O-glycosylation mass spectrometry analysis identified 371 N-glycosylated proteins and 243 O-glycosylated proteins. Differential expression analysis revealed 368 N-glycosylated differential proteins and 120 O-glycosylated differential proteins but no major lipid metabolism pathway was enriched among these differential proteins. Further, the expression levels of the 16 differential modification proteins showed that ACOX1 and HADHA were the most significantly down-regulated proteins in terms of ubiquitination modification following poly(I:C) stimulation, while DAGLα, ABCA1, and PLD1 were the most significantly up-regulated proteins. The results suggested that ACOX1, HADHA, DAGLα, ABCA1, PLD1, and cPLA2 may be the key modification proteins in the lipid metabolism induced by poly(I:C). In summary, the present study demonstrated that ubiquitination and phosphorylation modifications play a crucial role in the lipid metabolism and lipid droplet synthesis in viral dsRNA stimulation, underlying the important role of lipid metabolism-related protein modifications in viral dsRNA infections.