Pan-Caspase Inhibitor zVAD Induces Necroptotic and Autophagic Cell Death in TLR3/4-Stimulated Macrophages.

In addition to inducing apoptosis, caspase inhibition contributes to necroptosis and/or autophagy depending on the cell type and cellular context. In macrophages, necroptosis can be induced by co-treatment with Toll-like receptor (TLR) ligands (lipopolysaccharide [LPS] for TLR4 and polyinosinic-polycytidylic acid [poly I:C] for TLR3) and a cell-permeable pan-caspase inhibitor zVAD. Here, we elucidated the signaling pathways and molecular mechanisms of cell death. We showed that LPS/zVAD- and poly I:C/zVAD-induced cell death in bone marrow-derived macrophages (BMDMs) was inhibited by receptor-interacting protein kinase 1 (RIP1) inhibitor necrostatin-1 and autophagy inhibitor 3-methyladenine. Electron microscopic images displayed autophagosome/autolysosomes, and immunoblotting data revealed increased LC3II expression. Although zVAD did not affect LPS- or poly I:C-induced activation of IKK, JNK, and p38, it enhanced IRF3 and STAT1 activation as well as type I interferon (IFN) expression. In addition, zVAD inhibited ERK and Akt phosphorylation induced by LPS and poly I:C. Of note, zVAD-induced enhancement of the IRF3/IFN/STAT1 axis was abolished by necrostatin-1, while zVAD-induced inhibition of ERK and Akt was not. Our data further support the involvement of autocrine IFNs action in reactive oxygen species (ROS)-dependent necroptosis, LPS/zVAD-elicited ROS production was inhibited by necrostatin-1, neutralizing antibody of IFN receptor (IFNR) and JAK inhibitor AZD1480. Accordingly, both cell death and ROS production induced by TLR ligands plus zVAD were abrogated in STAT1 knockout macrophages. We conclude that enhanced TRIF-RIP1-dependent autocrine action of IFNß, rather than inhibition of ERK or Akt, is involved in TLRs/zVAD-induced autophagic and necroptotic cell death via the JAK/STAT1/ROS pathway.

Characterization of site-specific mutants of alkylhydroperoxide reductase with dual functionality from Helicobacter pylori.

Alkylhydroperoxide reductase (AhpC) is an abundant and important antioxidant protein present in Helicobacter pylori (HP), a spiral Gram-negative microaerophilic bacterium. By sequence alignment and structure comparison, HP-AhpC was found to be more homologous to human peroxiredoxins (hPrx) than to other eubacterial AhpC proteins. Similar to hPrxI, native HP-AhpC existed as a dimer of single subunit, comprising alpha-helix and beta-sheet domains with low surface hydrophobicity. AhpC can form high-molecular-weight (HMW) aggregates ranging from 700 to higher than 2,000 kDa under oxidative stress, possessing chaperone activity in the presence of thioredoxin (Trx). Further analysis of peroxide-reductase activities showed that HP-AhpC was more resistant to H(2)O(2) than hPrxI. However, the mechanism of enzyme inactivation to H(2)O(2) appeared to be similar for both HP-AhpC and hPrxI as revealed by native gel electrophoresis followed by proteomic identification using two-dimensional gel electrophoresis (2-DE) and LC-MS/MS. In contrast to T90D-hPrxI mutant with chaperone activity, site-specific mutant T87D-HP-AhpC did not form HMW chaperone complexes. The comparison of these two evolutionarily distant and yet functionally related enzymes may shed some light on the mechanism(s) underlying the evolution and development of the dual functionality in HP-AhpC and hPrxI with similar protein structure.