The CD95 (Fas/APO-1) death-inducing signaling complex (DISC) is essential for the initiation of CD95-mediated apoptotic and nonapoptotic responses. The CD95 DISC comprises CD95, FADD, procaspase-8, procaspase-10, and c-FLIP proteins. Procaspase-8 and procaspase-10 are activated at the DISC, leading to the formation of active caspases and apoptosis initiation. In this study we analyzed the stoichiometry of the CD95 DISC. Using quantitative western blots, mass spectrometry, and mathematical modeling, we reveal that the amount of DED proteins procaspase-8/procaspase-10 and c-FLIP at the DISC exceeds that of FADD by several-fold. Furthermore, our findings imply that procaspase-8, procaspase-10, and c-FLIP could form DED chains at the DISC, enabling the formation of dimers and efficient activation of caspase-8. Taken together, our findings provide an enhanced understanding of caspase-8 activation and initiation of apoptosis at the DISC.

Mapping of proteins involved in normal eye functions is a prerequisite to identify pathological changes during eye disease processes. We therefore analysed the proteome of human vitreous by applying in-depth proteomic screening technologies. For ethical reasons human vitreous samples were obtained by vitrectomy from “surrogate normal patients” with epiretinal gliosis that is considered to constitute only negligible pathological vitreoretinal changes. We applied different protein prefractionation strategies including liquid phase isoelectric focussing, 1D SDS gel electrophoresis and a combination of both and compared the number of identified proteins obtained by the respective method. Liquid phase isoelectric focussing followed by SDS gel electrophoresis increased the number of identified proteins by a factor of five compared to the analysis of crude unseparated human vitreous. Depending on the prefractionation method proteins were subjected to trypsin digestion either in-gel or in solution and the resulting peptides were analysed on a UPLC system coupled online to an LTQ Orbitrap XL mass spectrometer. The obtained mass spectra were searched against the SwissProt database using the Mascot search engine. Bioinformatics tools were used to annotate known biological functions to the detected proteins. Following this strategy we examined the vitreous proteomes of three individuals and identified 1111 unique proteins. Besides structural, transport and binding proteins, we detected 261 proteins with known enzymatic activity, 51 proteases, 35 protease inhibitors, 35 members of complement and coagulation cascades, 15 peptide hormones, 5 growth factors, 11 cytokines, 47 receptors, 30 proteins of visual perception, 91 proteins involved in apoptosis regulation and 265 proteins with signalling activity. This highly complex mixture strikingly differs from the human plasma proteome. Thus human vitreous fluid seems to be a unique body fluid. 262 unique proteins were detected which are present in all three patient samples indicating that these might represent the constitutive protein pattern of human vitreous. The presented catalogue of human vitreous proteins will enhance our understanding of physiological processes in the eye and provides the groundwork for future studies on pathological vitreous proteome changes.