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Identification of novel molecular components in Ripoptosome-mediated signaling pathways

Schilling, Ramon

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Programmed cellular execution is one of the hallmarks of cellular homeostasis as well as the development of multicellular organisms. To date, two major signaling pathways for programmed cell death have been elucidated, namely apoptosis and necroptosis. The induction of these two regulated cell death programs can be initiated by a variety of extrinsic as well as intrinsic stimuli, like genotoxic stress, death receptor activation, pathogen infection or the depletion cIAPs. Apoptosis thereby relies on the activation of caspase-8, which induces a downstream caspase cascade, finally leading to apoptotic execution. In contrast, caspase-independent necroptosis relies on the activation of the kinases RIP1 and RIP3, resulting in the phosphorylation of the pseudokinase MLKL. This finally leads to necroptotic plasma membrane permeabilization. Additionally, recent studies highlighted that certain cellular conditions can induce the formation of an intracellular 2 MDa multi-protein complex, called Ripoptosome, which can lead to apoptosis as well as necroptosis based on the stoichiometric composition. This protein complex consists of FADD, caspase-8, cFLIP and the name giving RIP1. However, it is still unclear, if all components or interacting proteins have been identified. In this study purified Ripoptosome complexes were analyzed for the identification of novel components by mass spectrometry. Interestingly, the non-canonical IKKs; TBK1 and IKK-ε as well as TRAF2 could be identified as integral elements of the complex. The association of these molecules with the Ripoptosome was observed upon TNF treatment or TLR3 stimulation or the overexpression of RIP1 as the initial stimulus. Moreover, the non-canonical IKKs were highly phosphorylated in the complex, arguing for substantial activation. The cell death regulating function of the Ripoptosome was not affected by the loss of TBK1 and IKK-ε, indicating that these molecules could play a pivotal role in non-cell death signaling pathways. In contrast, decrease of TRAF2 resulted in a sensitization against apoptotic cells death. This was true for the expression of RIP1 as well as the combined treatment of poly (I:C) or TNF with IAP antagonists. Additionally, RIP1-induced Ripoptosome formation could be linked to the activation of NF-κB and the induction of inflammatory cytokines. The reduction in TBK1 and IKK-ε protein levels was leading to a decrease in the RIP1-regulated gene induction. Additionally, the formation of the Ripoptosome was inducing the phosphorylation of autophagy receptors optineurin and sequestosome-1. This activation was highly depending on the kinases TBK1 and IKK-ε, indicating a possible Ripoptosome-dependent activation of the autophagy machinery. The novel identified components highlighted the Ripoptosome as a key complex not only for cell death regulation, but also for cell death-independent signaling pathways such as inflammatory responses and autophagy.

Item Type: Dissertation
Supervisor: Umansky, Prof. Dr. Viktor
Date of thesis defense: 4 August 2016
Date Deposited: 05 Sep 2016 09:04
Date: 2016
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Medizinische Fakultät Mannheim > Hautklinik
Subjects: 500 Natural sciences and mathematics
570 Life sciences
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