RNA clamping by Vasa assembles a piRNA amplifier complex on transposon transcripts.

Germline-specific Piwi-interacting RNAs (piRNAs) protect animal genomes against transposons and are essential for fertility. piRNAs targeting active transposons are amplified by the ping-pong cycle, which couples Piwi endonucleolytic slicing of target RNAs to biogenesis of new piRNAs. Here, we describe the identification of a transient Amplifier complex that mediates biogenesis of secondary piRNAs in insect cells. Amplifier is nucleated by the DEAD box RNA helicase Vasa and contains the two Piwi proteins participating in the ping-pong loop, the Tudor protein Qin/Kumo and antisense piRNA guides. These components assemble on the surface of Vasa's helicase domain, which functions as an RNA clamp to anchor Amplifier onto transposon transcripts. We show that ATP-dependent RNP remodeling by Vasa facilitates transfer of 5' sliced piRNA precursors between ping-pong partners, and loss of this activity causes sterility in Drosophila. Our results reveal the molecular basis for the small RNA amplification that confers adaptive immunity against transposons.

The central role of initiator caspase-9 in apoptosis signal transduction and the regulation of its activation and activity on the apoptosome.

Key structural and catalytic features are conserved across the entire family of cysteine-dependent aspartate-specific proteases (caspases). Of the caspases involved in apoptosis signal transduction, the initiator caspases-2, -8 and -9 are activated at multi-protein activation platforms, and activation is thought to involve homo-dimerisation of the monomeric zymogens. Caspase-9, the essential initiator caspase required for apoptosis signalling through the mitochondrial pathway, is activated on the apoptosome complex, and failure to activate caspase-9 has profound pathophysiological consequences. Here, we review the pertinent literature on which the currently prevalent understanding of caspase-9 activation is based, extend this view by insight obtained from recent structural and kinetic studies on caspase-9 signalling, and describe an emerging model for the regulation of caspase-9 activation and activity that arise from the complexity of multi-protein interactions at the apoptosome. This integrated view allows us to postulate and to discuss functional consequences for caspase-9 activation and apoptosis execution that may take centre stage in future experimental cell research on apoptosis signalling.

Proteasome inhibition can impair caspase-8 activation upon submaximal stimulation of apoptotic tumor necrosis factor-related apoptosis inducing ligand (TRAIL) signaling.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) can induce extrinsic apoptosis, resulting in caspase-8 activation, but may also initiate transcription-dependent prosurvival signaling. Proteasome inhibitors were suggested to promote TRAIL signal transduction through the death-inducing signaling complex (DISC) by modulating the relative abundance of core DISC components, thereby enhancing caspase-8 activation and apoptosis. To test this hypothesis, we quantified the changes in DISC protein levels as an early consequence of proteasome inhibition in HeLa cervical cancer cells and, based on these data, mathematically modeled the proapoptotic TRAIL signaling toward caspase-8 activation. Modeling results surprisingly suggested that caspase-8 activation might be delayed in presence of proteasome inhibitors, in particular at submaximal TRAIL doses. Subsequent FRET-based single cell time-lapse imaging at conditions where transcription dependent prosurvival signaling was blocked confirmed this hypothesis: caspase-8 activity was delayed by hours in the presence of proteasome inhibitors epoxomicin or bortezomib. Corresponding delays were detected for effector caspase processing and cell death. Contrary to current models, we therefore provide evidence that synergies between TRAIL and proteasome inhibitors do not result from changes in the levels of core DISC signaling proteins.

The caspase-8 dimerization/dissociation balance is a highly potent regulator of caspase-8, -3, -6 signaling.

Apoptosis is driven by positive feedback activation between aspartate-specific cysteinyl proteases (caspases). These feedback loops ensure the swift and efficient elimination of cells upon initiation of apoptosis execution. At the same time, the signaling network must be insensitive to erroneous, mild caspase activation to avoid unwanted, excessive cell death. Sublethal caspase activation in fact was shown to be a requirement for the differentiation of multiple cell types but might also occur accidentally during short, transient cellular stress conditions. Here we carried out an in silico comparison of the molecular mechanisms that so far have been identified to impair the amplification of caspase activities via the caspase-8, -3, -6 loop. In a systems model resembling HeLa cervical cancer cells, the dimerization/dissociation balance of caspase-8 potently suppressed the amplification of caspase responses, surprisingly outperforming or matching known caspase-8 and -3 inhibitors such as bifunctional apoptosis repressor or x-linked inhibitor of apoptosis protein. These findings were further substantiated in global sensitivity analyses based on combinations of protein concentrations from the sub- to superphysiological range to screen the full spectrum of biological variability that can be expected within cell populations and between distinct cell types. Additional modeling showed that the combined effects of x-linked inhibitor of apoptosis protein and caspase-8 dimerization/dissociation processes can also provide resistance to larger inputs of active caspases. Our study therefore highlights a central and so far underappreciated role of caspase-8 dimerization/dissociation in avoiding unwanted cell death by lethal amplification of caspase responses via the caspase-8, -3, -6 loop.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.