Composition stoichiometry of CD95/Fas complexes as a switch between life and death

• Applicant: Professorin Dr. Inna Lavrik
• Subject Area: Cell Biology
• Term: since 2014
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 260615047

Programmed cell death is essential for tissue homeostasis, immune regulation, and the elimination of damaged or malignant cells. Two types of programmed cell death, apoptosis and necroptosis, are of particular importance in both health and disease. The stimulation of CD95 (Fas/APO-1) leads to the induction of distinct macromolecular complexes such as the DISC (Death-Inducing Signaling Complex), complex IIa (ripoptosome), and complex IIb (necrosome). These complexes orchestrate life-versus-death decisions in cells and share a core set of components, including FADD, procaspase-8/10, RIPK1, and the c-FLIP isoforms. A key regulatory element within these platforms is the procaspase-8/c-FLIPL heterodimer, which balances apoptotic and necroptotic signaling outcomes. This proposal aims to dissect the quantitative composition, structural organization, and functional regulation of CD95-induced signaling platforms, with a central focus on the procaspase-8/c-FLIPL heterodimer and its modulation by the small molecule inhibitor FLIPinB, developed in our laboratory. Building upon a robust foundation of quantitative mass spectrometry, biochemical systems biology, and structure-based modeling established during the previous funding period, we will first define the stoichiometric organization and structural features of CD95-induced complex IIa using AQUA-based proteomics, AlphaFold-Multimer predictions, and targeted mutagenesis to clarify how domain interactions and component stoichiometry determine complex function. In parallel, we will investigate the role of the procaspase-8/c-FLIPL heterodimer in complex II formation and cell death regulation by introducing mutations at FLIPinB-interacting sites and assessing the effects of FLIPinB treatment in cellular and 3D tumor models to evaluate therapeutic relevance, particularly in cancer. Furthermore, we will explore whether PRMT5-mediated methylation extends to c-FLIP upon CD95 activation and how this influences the balance between apoptosis and necroptosis, building on our prior identification of procaspase-8 methylation and using combined genetic, biochemical, and pharmacological approaches. By integrating proteomics, structural prediction, functional assays, and advanced 3D models, this project aims to provide new mechanistic insights into the regulation of death receptor signaling and to uncover novel therapeutic strategies for diseases involving dysregulated cell death, including cancer and immune disorders.

• DFG Programme: Research Grants