Impact of the ubiquitin system on the orchestration of signal transduction during continuous TNF challenge
Rheumatology
Cell Biology
Final Report Abstract
Ubiquitin (Ub) is a small protein and one posttranslational principle involved in numerous cellular functions within all kingdoms of life. The orchestration of external stimuli such as the coordination of TNF signaling requires various posttranslational modifications. A disturbed signal perception is a hallmark of numerous human diseases, such as SIRS and Sepsis or chronic inflammatory maladies like rheumatoid arthritis or inflammatory bowel disease as well as malignant processes. We identified a phosphorylation site at serin-20 of ubiquitin, which is so far, functionally uncharacterized. We further observed a significant reduction of phosphorylation at serin-20 within ubiquitin after long-term TNF exposition. Along this funding period we verified the existence of Ub-serine-20 phosphorylation in human cells and established the fundamentals to explore the molecular function of this regulatory principle. We could show that ubiquitin is phosphorylated in dependency of external stimuli. The development of a phospho-site specific antibody to Ub-serine-20 enabled us to study changes in this modification in more direct manner as before. Additionally we developed mutant variants of Ub deficient for the phosphorylation site at serine-20 as well as one that should mimic constant phosphorylation. These variants were overexpressed in human cells and we could show that they introduced to the ubiquitination reaction similar to endogenous Ub. This allowed us to start co-purification experiments in order to identify enzymes and substrate proteins, that depend on serine-20 phosphorylation in order to fulfill their function or being ubiquitinated. Thus, we could already show that the TRAF2 protein, a crucial component of the TNF signaling complex, co-purified with the phospho-deficient variant but not with wildtype-Ub when the cells are not challenged to any external stimulus. Altogether we established a stable basis for future projects, that will need to continue the experiments developed here to finally allow a more comprehensive picture of ubiquitin phosphorylation and its function for signal transduction. Furthermore, the potential of Ubiquitin serin-20 as a marker and/or target for diagnosis and prognosis of inflammatory diseases as well as malignant processes is still an issue worthwhile to be explored.
Publications
- TNF Tolerance in Monocytes and Macrophages: Characteristics and Molecular Mechanisms. J Immunol Res, 2017. 2017: p. 9570129
Huber R., Bikker R., Welz B., Christmann M., Brand K.
(See online at https://doi.org/10.1155/2017/9570129) - (Phospho-)Proteome analysis of TNF long term-incubated primary human monocytes. DGKL-P071. Annual meeting of the DGKL 2018 in Mannheim
R. Bikker, B. Welz, M. Christmann, M. Weber, L. Hoffmeister, K. Neumann, K. Preuß, J. Junemann, A. Pich, R. Huber, K. Brand
- Impact of the ubiquitin system on the orchestration of signal transduction during continuous TNF challenge. DGKL-P073. Annual meeting of the DGKL 2018 in Mannheim
M. Christmann, R. Bikker, K. Brand
- Phosphorylation of Ubiquitin at serin-20 and its impact on the coordination of cellular functions in human cells. DGKL-PO-58. Annual meeting of the DGKL 2019 in Magdeburg
M. Christmann, R. Bikker, K. Brand
- Proteome and Phosphoproteome Analysis in TNF Long Term-Exposed Primary Human Monocytes. Int J Mol Sci, 2019. 20(5)
Welz B., Bikker R., Junemann J., Christmann M., Neumann K., Weber M., Hoffmeister L., Preuss K., Pich A., Huber R., Brand K.
(See online at https://doi.org/10.3390/ijms20051241)
