TY  - GEN
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/10443/
ID  - heidok10443
KW  - TNF-alpha 
KW  -  genom-weiter siRNA ScreenTNF-alpha 
KW  -  genome-wide siRNA screening
N2  - Signaling pathways are crucial for multicellular organisms: they are necessary for cell communication and development, they enable cells to specialize and act together. TNF-alpha signaling is one such pathway crucial for orchestrating the body's response to cellular stress or invasion by pathogens. TNF- signaling mediates inflammation, a key event for an efficient innate immune response. As inflammation has to be tightly controlled in order to avoid damage to the host, deregulation of TNF-alpha signaling has been implicated in the pathogenesis of many inflammatory diseases and cancer. TNF-alpha exerts inflammatory effects by binding to its receptor, TNFR1. Upon binding, several proteins including DD (death domain) proteins are recruited to TNFR1 to form the TNFR complex, resulting in the activation of the IkappaB kinase complex (IKK). Subsequently, inhibitor of kappaB (IkappaB) proteins are phosphorylated and degraded, releasing transcription factors of the NF-kappaB family. NF-kappaB then controls the expression of hundreds of different genes required for inflammation and innate immunity. The aim of my PhD project was to identify new factors required for TNF-alpha signaling. In order to monitor NF-kappaB transcriptional activity upon stimulation with TNF-alpha, I established a cell-based dual luciferase assay. This assay was suited to measure TNF-alpha signaling activity in miniaturized format necessary for large-scale experiments. For finding novel regulators of TNF-alpha signaling, I used the cell-based dual luciferase assay in two genome-wide RNAi screens. These screens identified several candidates potentially implicated in NF-kappaB activation by TNF-alpha. I next established secondary assays for confirming the requirement of these candidates in TNF-signaling. On the basis of the results of these secondary assays, I selected three candidates, SPP1, GAB3 and CASP4, for further characterization.  Epistasis experiments revealed that SPP1, GAB3 and CASP4 are required for the activation of the IkappaB kinase complex. Further experiments demonstrated that the candidates are not essential for proper TNFR1 cell surface expression. SPP1, a multifunctional protein, has been described to interact with the DD protein MyD88 during Toll-like receptor signaling. It could thus interact with DD proteins present in the TNFR complex. GAB3 belongs to a family of scaffold proteins of which one member, GAB2, has been shown to interact with RANK, a TNFR family member. Analogously, GAB3 could act as a scaffold at TNFR1 supporting the recruitment of signaling molecules. CASP4 is an inflammatory caspase. My results indicate that CASP4 catalytical activity is dispensable for its role in TNF-alpha signaling. CASP4 could thus serve as another scaffold protein in the TNFR complex as described for other caspases. Future experiments will identify the interaction partners of SPP1, GAB3 and CASP4, clarifying the molecular details of their mode of action in TNF-alpha-induced activation of NF-kappaB.
A1  - Nickles, Dorothee
AV  - public
TI  - Identification of Novel Regulators of TNF-alpha Signaling using Genome-wide RNAi Screens
Y1  - 2009///
ER  -