Pathogenic intracellular bacteria often hijack macrophages for their propagation. The infected macrophages release IL-1β and IL-18 and simultaneously commit suicide, which is called pyroptosis; both responses require caspase-1. Here, we found that pyroptotic cells induced by microbial infection were efficiently engulfed by human monocytic THP-1-cell-derived macrophages or mouse peritoneal macrophages. This engulfment was inhibited by the D89E mutant of milk fat globule (MFG) epidermal growth factor (EGF) factor 8 (MFG-E8; a phosphatidylserine-binding protein) that has been shown previously to inhibit phosphatidylserine-dependent engulfment of apoptotic cells by macrophages, suggesting that the engulfment of pyroptotic cells by macrophages was also phosphatidylserine dependent. Using a pair of cell lines that respectively exhibited pyroptosis or apoptosis after muramyl dipeptide treatment, we showed that both pyroptotic and apoptotic cells bound to a T-cell immunoglobulin and mucin domain-containing 4 (Tim4; another phosphatidylserine-binding protein)-coated plate, whereas heat-killed necrotic cells did not, indicating that phosphatidylserine was externalized in pyroptosis and apoptosis but not in accidental necrosis. Macrophages engulfed apoptotic cells most efficiently, followed by pyroptotic and then heat-killed necrotic cells. Pyroptotic cells also released a macrophage attractant(s), 'find-me' signal, whose activity was diminished by apyrase that degrades nucleoside triphosphate to nucleoside monophosphate. Heat-killed necrotic cells and pyroptotic cells released ATP much more efficiently than apoptotic cells. These results suggest that pyroptotic cells, like apoptotic cells, actively induce phagocytosis by macrophages using 'eat-me' and find-me signals. Based on these results, a possible role of coordinated induction of pyroptosis and inflammatory cytokine production is discussed.

Proximity biotinylation based on Escherichia coli BirA enzymes such as BioID (BirA*) and TurboID is a key technology for identifying proteins that interact with a target protein in a cell or organism. However, there have been some improvements in the enzymes that are used for that purpose. Here, we demonstrate a novel BirA enzyme, AirID (ancestral BirA for proximity-dependent biotin identification), which was designed de novo using an ancestral enzyme reconstruction algorithm and metagenome data. AirID-fusion proteins such as AirID-p53 or AirID-IκBα indicated biotinylation of MDM2 or RelA, respectively, in vitro and in cells, respectively. AirID-CRBN showed the pomalidomide-dependent biotinylation of IKZF1 and SALL4 in vitro. AirID-CRBN biotinylated the endogenous CUL4 and RBX1 in the CRL4CRBN complex based on the streptavidin pull-down assay. LC-MS/MS analysis of cells that were stably expressing AirID-IκBα showed top-level biotinylation of RelA proteins. These results indicate that AirID is a novel enzyme for analyzing protein–protein interactions.

Neuroblastoma is the most common solid pediatric tumor and remarkable for its clinical heterogeneity. Despite recent advances in chemotherapy, the prognosis of advanced neuroblastoma is still very poor. However, some favorable types of neuroblastoma, especially in infants under 1 year of age, are known to regress spontaneously or mature even if widespread metastases to bone marrow, skin and/or liver (special stage: stage IVS) are present. Apoptosis is known to occur in normal development of nervous systems, and neuroblastoma is generated from neural crest cells when the apoptotic systems do not carry out. Delayed implementation of the normal apoptotic pathway has been proposed as an explanation for the spontaneous regression of favorable neuroblastoma.1) It is reported that resistance to apoptosis plays a contributory role in the mechanism of the aggressive behavior shown by advanced neuroblastoma.2) Acute lymphocytic leukemia, like advanced neuroblastoma, is also a pediatric disease that is difficult to treat, especially in older children or those with a high amount of leukemic cells in the peripheral blood.Angelica keiskei has been used traditionally in Japan as a diuretic, laxative, analeptic and galactagogue, and an A. keiskei extract was previously reported to affect metabolic activity 3,4) and vasoconstriction 5) in rats. Moreover, A. keiskei and a major chalcone constituent of this plant, xanthoangelol, reportedly have inhibitory effects against tumor promoter activity 6,7) and metastasis. 8) Xanthoangelol possesses a chalcone structure, and some compounds related to calchones are known to have antitumor activity and to induce apoptosis. Quercetin chalcone was reported to reduce the size of implanted colon-25 tumors in vivo. 9) However, there has been no report on the effects of chalcones, including xanthoangelol, on neuroblastoma.In this study, we examined the antitumor effect and apoptosis-inducing activity of xanthoangelol against a human neuroblastoma cell line (IMR-32), and also a leukemia cell line (Jurkat) which have been widely used in previous studies of apoptosis. MATERIALS AND METHODS Materials Xanthoangelol(3Ј-C-geranyl-2Ј,4,4Ј-trihydroxychalcone) was isolated from the stem exudate of A. keiskei 6) and dissolved in dimethyl sulfoxide (DMSO) (final concentration 0.2%). IMR-32 and Jurkat were maintained in RPMI-1640 medium (Invitrogen) supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin and 10% fetal bovine serum (FBS) (Invitrogen). The cells were maintained at 37°C/5% CO 2 in a humid environment.Trypan Blue Exclusion Assay The cells ( 1ϫ10 6 ) were plated into a 60-mm dish and maintained for 24 h. Xanthoangelol (final concentrations 10 Ϫ6 , 10 Ϫ5, 10 Ϫ4 M) and vehicle were applied for 48 h. For the IMR-32 cell protocol, the cells were stripped using 0.05% trypsin-EDTA solution after washing them in phosphate-buffered saline (Ϫ). They were then washed in RPMI-1640 medium (with 10% FBS) and counted with a phase-contrast microscope immediately after addition of an equal volume of 1% tr...

Background: ASC mediates apoptosis and necrosis of tumor cells and necrosis of microbe-infected macrophages. Results: ASC mediates necrosis only when cells express caspase-1; however, inhibition of caspase-1 proteolytic activity did not suppress the necrosis. Conclusion: Caspase-1 but not its proteolytic activity is essential for ASC-mediated necrosis. Significance: This study explains why ASC induces apoptosis or necrosis depending on the cell type.