Modulation of apoptosis signalling by proteasome inhibition : a single cell analysis

The proteasome inhibitor bortezomib has been successfully used in cancer therapy. Proteasome inhibition modulates various signalling pathways and causes cell death in cancer cells while sparing normal cells. In this thesis we characterise the apoptotic signalling kinetics and sequential events in response to proteasome inhibition in single cells. We identified a novel non-canonical pathway to apoptosis induction in cells with an inhibited intrinsic apoptotic pathway. Proteasome inhibition promoted the apical activation of caspase-8 in these cells. For the first time we demonstrated that autophagy induction in response to proteasome inhibition is critical for caspase-8 activation. Caspase-8 activation resulted in limited apoptosis, which can be further increased by antagonism of the endogenous caspase inhibitor XIAP. Therefore our findings provide an alternative treatment strategy to restore apoptosis susceptibility in highly resistant cancer cells. Proteasome inhibitors have also been used to re-sensitise resistant cancer cells to TRAIL treatment. Since no kinetic data from single cells are available yet, it is not known whether, when and where the intracellular signalling kinetics of TRAIL-induced apoptosis are affected by proteasome inhibition. We therefore quantified the signalling kinetics of TRAIL-induced apoptosis in response to proteasome inhibition in single cells. Depending on the TRAIL concentration we found two modulation sites, which interfered with TRAIL signalling kinetics upon proteasome inhibition: caspase-8 activation at low doses and the threshold for mitochondria1 permeabilisation at high doses. Our results suggest that upregulation of cFLIP and Mcl-1 by impaired protein degradation were responsible for delayed caspase-8 activation and prolonged caspase-8 activation before MOMP, respectively. Our findings therefore indicate that the synergy between TRAIL and proteasome inhibition is probably based on the stabilisation of active caspases rather than enhanced DISC formation, as is often proposed in the literature

Modulation of apoptosis sensitivity through the interplay with autophagic and proteasomal degradation pathways.

Autophagic and proteasomal degradation constitute the major cellular proteolysis pathways. Their physiological and pathophysiological adaptation and perturbation modulates the relative abundance of apoptosis-transducing proteins and thereby can positively or negatively adjust cell death susceptibility. In addition to balancing protein expression amounts, components of the autophagic and proteasomal degradation machineries directly interact with and co-regulate apoptosis signal transduction. The influence of autophagic and proteasomal activity on apoptosis susceptibility is now rapidly gaining more attention as a significant modulator of cell death signalling in the context of human health and disease. Here we present a concise and critical overview of the latest knowledge on the molecular interplay between apoptosis signalling, autophagy and proteasomal protein degradation. We highlight that these three pathways constitute an intricate signalling triangle that can govern and modulate cell fate decisions between death and survival. Owing to rapid research progress in recent years, it is now possible to provide detailed insight into the mechanisms of pathway crosstalk, common signalling nodes and the role of multi-functional proteins in co-regulating both protein degradation and cell death

Diffusion is capable of translating anisotropic apoptosis initiation into a homogeneous execution of cell death

<p>Abstract</p> <p>Background</p> <p>Apoptosis is an essential cell death process throughout the entire life span of all metazoans and its deregulation in humans has been implicated in many proliferative and degenerative diseases. Mitochondrial outer membrane permeabilisation (MOMP) and activation of effector caspases are key processes during apoptosis signalling. MOMP can be subject to spatial coordination in human cancer cells, resulting in intracellular waves of cytochrome-c release. To investigate the consequences of these spatial anisotropies in mitochondrial permeabilisation on subsequent effector caspase activation, we devised a mathematical reaction-diffusion model building on a set of partial differential equations.</p> <p>Results</p> <p>Reaction-diffusion modelling suggested that even if strong spatial anisotropies existed during mitochondrial cytochrome c release, these would be eliminated by free diffusion of the cytosolic proteins that instantiate the apoptosis execution network. Experimentally, rapid sampling of mitochondrial permeabilisation and effector caspase activity in individual HeLa cervical cancer cells confirmed predictions of the reaction-diffusion model and demonstrated that the signalling network of apoptosis execution could efficiently translate spatial anisotropies in mitochondrial permeabilisation into a homogeneous effector caspase response throughout the cytosol. Further systems modelling suggested that a more than 10,000-fold impaired diffusivity would be required to maintain spatial anisotropies as observed during mitochondrial permeabilisation until the time effector caspases become activated.</p> <p>Conclusions</p> <p>Multi-protein diffusion efficiently contributes to eliminating spatial asynchronies which are present during the initiation of apoptosis execution and thereby ensures homogeneous apoptosis execution throughout the entire cell body. For previously reported biological scenarios in which effector caspase activity was shown to be targeted selectively to specific subcellular regions additional mechanisms must exist that limit or spatially coordinate caspase activation and/or protect diffusing soluble caspase substrates from unwanted proteolysis.</p

RNA Clamping by Vasa Assembles a piRNA Amplifier Complex on Transposon Transcripts

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