Regulation of CD95-induced NF-[Kappa]B activation

Abstract

It is well understood that the death receptor CD95 induces apoptotic cell death but also pro-survival signaling. Defective regulation of CD95 signaling is connected to a number of diseases. While the induction of CD95-induced apoptosis is dependent on caspases, the molecular mechanisms leading to activation of the CD95-mediated pro-survival NF-κB pathway are not yet fully understood. The protein c-FLIP is known to block activation of caspases at the CD95 death inducing signaling complex (DISC) and plays an important role in driving CD95-induced NF-κB activation. The present study was designed to get new insights into the mechanism of CD95-mediated NF-κB activation. In the course of this study, a new imaging flow cytometry-based method to analyze apoptosis and the NF-κB pathway on single cell level was established. This new method uncovered that both pathways are activated in parallel, which provided further insights into the mechanism of NF-κB activation. It was shown that the DISC protein c-FLIP interacts with a central regulator of the NF-κB pathway, NEMO. Peptides derived from an in silico model of the c-FLIP-NEMO interaction were able to reduce CD95-induced NF-κB activation in cells expressing high levels of c-FLIPL. In addition, the effect of the CBM complex on CD95-induced NF-κB activation was analyzed. The core components of CBM complex were found to be associated to c-FLIP and NEMO in a mass spectrometry screen. Finally, the autophagy receptor NDP52 was described as a negative regulator of CD95- and TNF-R-induced NF-κB activation. Furthermore, this study showed that NDP52 interacts with NEMO and the DUB A20 and thereby limits NF-κB activation by reducing NEMO ubiquitination. Taken together, this study uncovered new regulatory mechanisms of CD95-induced NF-κB activity. The identification and further improvement of NEMO-derived peptides that block tumor-promoting CD95-induced NF-κB activation without blocking of CD95-mediated apoptosis has potential for new anti-cancer treatment.