No abstract

Current first-line artemisinin antimalarials are threatened by the emergence of resistant Plasmodium falciparum. Decreased sensitivity is evident in the initial (early ring) stage of intraerythrocytic development, meaning that it is crucial to understand the action of artemisinins at this stage. Here, we examined the roles of iron (Fe) ions and haem in artemisinin activation in early rings using Fe ion chelators and a specific haemoglobinase inhibitor (E64d). Quantitative modelling of the antagonism accounted for its complex dependence on the chemical features of the artemisinins and on the drug exposure time, and showed that almost all artemisinin activity in early rings (>80%) is due to haem-mediated activation. The surprising implication that haemoglobin uptake and digestion is active in early rings is supported by identification of active haemoglobinases (falcipains) at this stage. Genetic down-modulation of the expression of the two main cysteine protease haemoglobinases, falcipains 2 and 3, renders early ring stage parasites resistant to artemisinins. This confirms the important role of haemoglobin-degrading falcipains in artemisinin activation, and shows that changes in the rate of artemisinin activation could mediate high-level artemisinin resistance.

Highlights d Lysosomal RNase T2 activity functions upstream of TLR8 d RNase T2 cleaves ssRNA between purines and uridine (U) d This releases U and purine-2 0 ,3 0 -cyclophosphate-terminated RNAs activating TLR8 d Staphylococcus aureus is sensed in a manner dependent on RNase T2 and TLR8

Intracellular nucleotide binding and oligomerization domain (NOD) receptors recognize antigens including bacterial peptidoglycans and initiate immune responses by triggering the production of pro-inflammatory cytokines through activating NF-kB and MAP kinases. Receptor interacting protein kinase 2 (RIPK2) is critical for NOD-mediated NF-kB activation and cytokine production. Here we develop and characterize a selective RIPK2 kinase inhibitor, WEHI-345, which delays RIPK2 ubiquitylation and NF-kB activation downstream of NOD engagement. Despite only delaying NF-kB activation on NOD stimulation, WEHI-345 prevents cytokine production in vitro and in vivo and ameliorates experimental autoimmune encephalomyelitis in mice. Our study highlights the importance of the kinase activity of RIPK2 for proper immune responses and demonstrates the therapeutic potential of inhibiting RIPK2 in NOD-driven inflammatory diseases.

The E3 ubiquitin ligase Parkin is a key effector of the removal of damaged mitochondria by mitophagy. Parkin determines cell fate in response to mitochondrial damage, with its loss promoting early onset Parkinson's disease and potentially also cancer progression. Controlling a cell's apoptotic response is essential to co‐ordinate the removal of damaged mitochondria. We report that following mitochondrial damage‐induced mitophagy, Parkin directly ubiquitinates the apoptotic effector protein BAK at a conserved lysine in its hydrophobic groove, a region that is crucial for BAK activation by BH3‐only proteins and its homo‐dimerisation during apoptosis. Ubiquitination inhibited BAK activity by impairing its activation and the formation of lethal BAK oligomers. Parkin also suppresses BAX‐mediated apoptosis, but in the absence of BAX ubiquitination suggesting an indirect mechanism. In addition, we find that BAK‐dependent mitochondrial outer membrane permeabilisation during apoptosis promotes PINK1‐dependent Parkin activation. Hence, we propose that Parkin directly inhibits BAK to suppress errant apoptosis, thereby allowing the effective clearance of damaged mitochondria, but also promotes clearance of apoptotic mitochondria to limit their potential pro‐inflammatory effect.

Protein ubiquitination is involved in virtually all cellular processes. Enrichment strategies employing antibodies targeting ubiquitin-derived diGly remnants combined with mass spectrometry (MS) have enabled investigations of ubiquitin signaling at a large scale. However, so far the power of data independent acquisition (DIA) with regards to sensitivity in single run analysis and data completeness have not yet been explored. Here, we develop a sensitive workflow combining diGly antibody-based enrichment and optimized Orbitrap-based DIA with comprehensive spectral libraries together containing more than 90,000 diGly peptides. This approach identifies 35,000 diGly peptides in single measurements of proteasome inhibitor-treated cells – double the number and quantitative accuracy of data dependent acquisition. Applied to TNF signaling, the workflow comprehensively captures known sites while adding many novel ones. An in-depth, systems-wide investigation of ubiquitination across the circadian cycle uncovers hundreds of cycling ubiquitination sites and dozens of cycling ubiquitin clusters within individual membrane protein receptors and transporters, highlighting new connections between metabolism and circadian regulation.

Highlights d The release of conventionally secreted cytokines is reduced in necroptotic cells d Receptor shedding occurs during TNF-induced necroptosis and apoptosis d Lysosomal components are released via lysosomal exocytosis during necroptosis

Innate immune signaling and programmed cell death are intimately linked, and many signaling pathways can regulate and induce both, transcription of inflammatory mediators or autonomous cell death. The best-characterized examples for these dual outcomes are members of the TNF superfamily, the inflammasome receptors, and the toll-like receptors. Signaling via the intracellular peptidoglycan receptors NOD1 and NOD2, however, does not appear to follow this trend, despite involving signaling proteins, or proteins with domains that are linked to programmed cell death, such as RIP kinases, inhibitors of apoptosis (IAP) proteins or the CARD domains on NOD1/2. To better understand the connections between NOD signaling and cell death induction, we here review the latest findings on the molecular regulation of signaling downstream of the NOD receptors and explore the links between this immune signaling pathway and the regulation of cell death.