Oxidation of caspase-8 by hypothiocyanous acid enables TNF-mediated necroptosis.

Necroptosis is a form of regulated cell death triggered by various host and pathogen-derived molecules during infection and inflammation. The essential step leading to necroptosis is phosphorylation of the mixed lineage kinase domain-like protein by receptor-interacting protein kinase 3. Caspase-8 cleaves receptor-interacting protein kinases to block necroptosis, so synthetic caspase inhibitors are required to study this process in experimental models. However, it is unclear how caspase-8 activity is regulated in a physiological setting. The active site cysteine of caspases is sensitive to oxidative inactivation, so we hypothesized that oxidants generated at sites of inflammation can inhibit caspase-8 and promote necroptosis. Here, we discovered that hypothiocyanous acid (HOSCN), an oxidant generated in vivo by heme peroxidases including myeloperoxidase and lactoperoxidase, is a potent caspase-8 inhibitor. We found HOSCN was able to promote necroptosis in mouse fibroblasts treated with tumor necrosis factor. We also demonstrate purified caspase-8 was inactivated by low concentrations of HOSCN, with the predominant product being a disulfide-linked dimer between Cys360 and Cys409 of the large and small catalytic subunits. We show oxidation still occurred in the presence of reducing agents, and reduction of the dimer was slow, consistent with HOSCN being a powerful physiological caspase inhibitor. While the initial oxidation product is a dimer, further modification also occurred in cells treated with HOSCN, leading to higher molecular weight caspase-8 species. Taken together, these findings indicate major disruption of caspase-8 function and suggest a novel mechanism for the promotion of necroptosis at sites of inflammation.

Induction of the reactive chlorine-responsive transcription factor RclR in Escherichia coli following ingestion by neutrophils.

Neutrophils generate hypochlorous acid (HOCl) and related reactive chlorine species as part of their defence against invading microorganisms. In isolation, bacteria respond to reactive chlorine species by upregulating responses that provide defence against oxidative challenge. Key questions are whether these responses are induced when bacteria are phagocytosed by neutrophils, and whether this provides them with a survival advantage. We investigated RclR, a transcriptional activator of the rclABC operon in Escherichia coli that has been shown to be specifically activated by reactive chlorine species. We first measured induction by individual reactive chlorine species, and showed that HOCl itself activates the response, as do chloramines (products of HOCl reacting with amines) provided they are cell permeable. Strong RclR activation was seen in E. coli following phagocytosis by neutrophils, beginning within 5 min and persisting for 40 min. RclR activation was suppressed by inhibitors of NOX2 and myeloperoxidase, providing strong evidence that it was due to HOCl production in the phagosome. RclR activation demonstrates that HOCl, or a derived chloramine, enters phagocytosed bacteria in sufficient amount to induce this response. Although RclR was induced in wild-type bacteria following phagocytosis, we detected no greater sensitivity to neutrophil killing of mutants lacking genes in the rclABC operon.

Peroxiredoxin interaction with the cytoskeletal-regulatory protein CRMP2: Investigation of a putative redox relay.

Hydrogen peroxide (H2O2) acts as a signaling molecule in cells by oxidising cysteine residues in regulatory proteins such as phosphatases, kinases and transcription factors. It is unclear exactly how many of these proteins are specifically targeted by H2O2 because they appear too unreactive to be directly oxidised. One proposal is that peroxiredoxins (Prxs) initially react with H2O2 and then oxidise adjacent proteins via a thiol relay mechanism. The aim of this study was to identify constitutive interaction partners of Prx2 in Jurkat T-lymphoma cells, in which thiol protein oxidation occurs at low micromolar concentrations of H2O2. Immunoprecipitation and proximity ligation assays identified a physical interaction between collapsin response mediator protein 2 (CRMP2) and cytoplasmic Prx2. CRMP2 regulates microtubule structure during lymphocyte migration and neuronal development. Exposure of Jurkat cells to low micromolar levels of H2O2 caused rapid and reversible oxidation of CRMP2, in parallel with Prx2 oxidation, despite purified recombinant CRMP2 protein reacting slowly with H2O2 (k~1 M-1s-1). Lowering Prx expression should inhibit oxidation of proteins oxidised by a relay mechanism, however knockout of Prx2 had no effect on CRMP2 oxidation. CRMP2 also interacted with Prx1, suggesting redundancy in single knockout cells. Prx 1 and 2 double knockout Jurkat cells were not viable. An interaction between Prx2 and CRMP2 was also detected in other human and rodent cells, including primary neurons. However, low concentrations of H2O2 did not cause CRMP2 oxidation in these cells. This indicates a cell-type specific mechanism for promoting CRMP2 oxidation in Jurkat cells, with insufficient evidence to attribute oxidation to a Prx-dependent redox relay.

Superoxide dismutase protects ribonucleotide reductase from inactivation in yeast.

Ribonucleotide reductase (RNR) catalyses the rate limiting step of DNA synthesis utilising a mechanism that requires a tyrosyl radical. We have previously shown that superoxide can quench protein tyrosyl radicals in vitro, either by oxidative addition, or reduction of the radical to tyrosine. Here, we observe that Saccharomyces cerevisiae strains lacking either copper-zincSOD (SOD1) or manganese SOD (SOD2) had decreased RNR activity compared to SOD-competent yeast. When superoxide production was increased by treatment with paraquat, RNR activity was further decreased, with yeast lacking SOD1 being the most sensitive. The growth of yeast lacking SOD1 was also the most sensitive to paraquat treatment. Using expressed recombinant RNR, superoxide addition was not detectable using mass-spectrometry. This suggests that oxidative addition is not the major route of inhibition in our system, but does not rule out reduction by superoxide as a possible mechanism. Our results demonstrate that protection of RNR from inactivation by superoxide is an important function of SOD, particularly cytoplasmic SOD1.

Structure/function analysis of Ca2+ binding to the C2A domain of synaptotagmin 1.

Synaptotagmin 1, a Ca2+ sensor for fast synaptic vesicle exocytosis, contains two C2 domains that form Ca2+-dependent complexes with phospholipids. To examine the functional importance of Ca2+ binding to the C2A domain of synaptotagmin 1, we studied two C2A domain mutations, D232N and D238N, using recombinant proteins and knock-in mice. Both mutations severely decreased intrinsic Ca2+ binding and Ca2+-dependent phospholipid binding by the isolated C2A domain. Both mutations, however, did not alter the apparent Ca2+ affinity of the double C2 domain fragment, although both decreased the tightness of the Ca2+/phospholipid/double C2 domain complex. When introduced into the endogenous synaptotagmin 1 gene in mice, the D232N and D238N mutations had no apparent effect on morbidity and mortality and caused no detectable alteration in the Ca2+-dependent properties of synaptotagmin 1. Electrophysiological recordings of cultured hippocampal neurons from knock-in mice revealed that neither mutation induced major changes in synaptic transmission. The D232N mutation, however, caused increased synaptic depression during repetitive stimulation, whereas the D238N mutation did not exhibit this phenotype. Our data indicate that Ca2+ binding to the C2A domain of synaptotagmin 1 may be important but not essential, consistent with the finding that the two C2 domains cooperate and may be partially redundant in Ca2+-dependent phospholipid binding. Moreover, although the apparent Ca2+ affinity of the synaptotagmin 1/phospholipid complex is critical, the tightness of the Ca2+/phospholipid complex is not. Our data also demonstrate that subtle changes in the biochemical properties of synaptotagmin 1 can result in significant alterations in synaptic responses.