Caspase-10 Negatively Regulates Caspase-8-Mediated Cell Death, Switching the Response to CD95L in Favor of NF-κB Activation and Cell Survival.

Formation of the death-inducing signaling complex (DISC) initiates extrinsic apoptosis. Caspase-8 and its regulator cFLIP control death signaling by binding to death-receptor-bound FADD. By elucidating the function of the caspase-8 homolog, caspase-10, we discover that caspase-10 negatively regulates caspase-8-mediated cell death. Significantly, we reveal that caspase-10 reduces DISC association and activation of caspase-8. Furthermore, we extend our co-operative/hierarchical binding model of caspase-8/cFLIP and show that caspase-10 does not compete with caspase-8 for binding to FADD. Utilizing caspase-8-knockout cells, we demonstrate that caspase-8 is required upstream of both cFLIP and caspase-10 and that DISC formation critically depends on the scaffold function of caspase-8. We establish that caspase-10 rewires DISC signaling to NF-κB activation/cell survival and demonstrate that the catalytic activity of caspase-10, and caspase-8, is redundant in gene induction. Thus, our data are consistent with a model in which both caspase-10 and cFLIP coordinately regulate CD95L-mediated signaling for death or survival.

Organization of Mitochondrial Gene Expression in Two Distinct Ribosome-Containing Assemblies.

Mitochondria contain their own genetic system that provides subunits of the complexes driving oxidative phosphorylation. A quarter of the mitochondrial proteome participates in gene expression, but how all these factors are orchestrated and spatially organized is currently unknown. Here, we established a method to purify and analyze native and intact complexes of mitochondrial ribosomes. Quantitative mass spectrometry revealed extensive interactions of ribosomes with factors involved in all the steps of posttranscriptional gene expression. These interactions result in large expressosome-like assemblies that we termed mitochondrial organization of gene expression (MIOREX) complexes. Superresolution microscopy revealed that most MIOREX complexes are evenly distributed throughout the mitochondrial network, whereas a subset is present as nucleoid-MIOREX complexes that unite the whole spectrum of organellar gene expression. Our work therefore provides a conceptual framework for the spatial organization of mitochondrial protein synthesis that likely developed to facilitate gene expression in the organelle.

Characterization of the ripoptosome and its components: implications for anti-inflammatory and cancer therapy.

Most intracellular signaling cascades rely on the formation of multiprotein signaling complexes assembled in large protein signaling platforms. Especially in cell death signaling, there is a large variety of these complexes, including the apoptosome, the necrosome, or the death-inducing signaling complex (DISC), to name only a few. During the last years, a number of cellular conditions were identified that lead to the formation of another signaling platform, the so-called ripoptosome. Diverse stimuli such as genotoxic stress, death receptor or Toll-like-receptor (TLR) ligation, or degradation of cellular inhibitor of apoptosis proteins (cIAPs) are able to induce ripoptosome formation. The ripoptosome is tightly regulated by cIAPs that control intracellular RIP1 assembly and the association with other cell death-regulating proteins, most likely by ubiquitin linkage. The suppression of cIAP activity results in accumulation of RIP1 platforms that ultimately triggers necroptosis by activation of RIP3-MLKL-dependent necrosis signaling pathways. The ripoptosome is a 2-MDa protein complex, which consists of the core components caspase-8, FADD, different cFLIP isoforms, and RIP1. It represents one of the rheostats in cell death signaling, as it can activate apoptotic and necroptotic cell death responses. The specific formation and activation of the ripoptosome in cancer but not in primary cells suggests that this complex is a potential novel target for cancer or anti-inflammatory therapy, as suggested by the potential proinflammatory effects of necroptosis. Therefore, the better understanding and characterization of this signaling platform is of enormous importance for the development of novel cancer therapeutics. In this chapter, we describe several methods for purification and investigation of the ripoptosome in human cells. We also describe methods for monitoring apoptotic as well as necroptotic cell death.