Proteome and Phosphoproteome Analysis in TNF Long Term-Exposed Primary Human Monocytes

Welz, Bastian; Bikker, Rolf; Junemann, Johannes ORCID; Christmann, Martin ORCID; Neumann, Konstantin ORCID; Weber, Mareike ORCID; Hoffmeister, Leonie; Preuß, Katharina; Pich, Andreas; Huber, René ORCID; Brand, Korbinian

To better understand the inflammation-associated mechanisms modulating and terminating tumor necrosis factor (TNF-)induced signal transduction and the development of TNF tolerance, we analyzed both the proteome and the phosphoproteome in TNF long term-incubated (i.e., 48 h) primary human monocytes using liquid chromatography-mass spectrometry. Our analyses revealed the presence of a defined set of proteins characterized by reproducible changes in expression and phosphorylation patterns in long term TNF-treated samples. In total, 148 proteins and 569 phosphopeptides were significantly regulated (103 proteins increased, 45 proteins decreased; 377 peptides with increased and 192 peptides with decreased phosphorylation). A variety of these proteins are associated with the non-canonical nuclear factor kappaB (NF-kappaB) pathway (nuclear factor kappaB (NFKB) 2, v-rel reticuloendotheliosis viral oncogene homolog (REL) B, indolamin-2,3-dioxygenase (IDO), kynureninase (KYNU)) or involved in the negative regulation of the canonical NF-kappaB system. Within the phosphopeptides, binding motifs for specific kinases were identified. Glycogen synthase kinase (GSK) 3 proved to be a promising candidate, since it targets NF-kappaB inhibiting factors, such as CCAAT/enhancer binding protein (C/EBP) beta. Our experiments demonstrate that both proteome and phosphoproteome analysis can be effectively applied to study protein/phosphorylation patterns of primary monocytes. These results provide new regulatory candidates and evidence for a complex network of specific but synergistically acting/cooperating mechanisms enabling the affected cells to resist sustained TNF exposure and resulting in the resolution of inflammation

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Welz, Bastian / Bikker, Rolf / Junemann, Johannes / et al: Proteome and Phosphoproteome Analysis in TNF Long Term-Exposed Primary Human Monocytes. 2019.

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