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Caspase-1 location in cells has been studied with fluorochrome-labeled inhibitors of caspase-1 (FLICA reagents). We report that FLICA reagents have limited cell-membrane permeability. This impacts experimental design as cells with intact membranes, including caspase-1 knockout cells, are not appropriate controls for cells with inflammasome-induced gasdermin D membrane pores., (© 2024 The Authors. European Journal of Immunology published by Wiley‐VCH GmbH.)

The migrational propensity of neuroblastoma is affected by cell identity, but the mechanisms behind the divergence remain unknown. Using RNAi and time-lapse imaging, we show that ADRN-type NB cells exhibit RAC1- and kalirin-dependent nucleokinetic (NUC) migration that relies on several integral components of neuronal migration. Inhibition of NUC migration by RAC1 and kalirin-GEF1 inhibitors occurs without hampering cell proliferation and ADRN identity. Using three clinically relevant expression dichotomies, we reveal that most of up-regulated mRNAs in RAC1- and kalirin-GEF1-suppressed ADRN-type NB cells are associated with low-risk characteristics. The computational analysis shows that, in a context of overall gene set poverty, the upregulomes in RAC1- and kalirin-GEF1-suppressed ADRN-type cells are a batch of AU-rich element-containing mRNAs, which suggests a link between NUC migration and mRNA stability. Gene set enrichment analysis-based search for vulnerabilities reveals prospective weak points in RAC1- and kalirin-GEF1-suppressed ADRN-type NB cells, including activities of H3K27- and DNA methyltransferases. Altogether, these data support the introduction of NUC inhibitors into cancer treatment research., (© 2021 Afanasyeva et al.)

Canonical inflammasomes are multiprotein complexes that can activate both caspase-1 and caspase-8. Caspase-1 drives rapid lysis of cells by pyroptosis and maturation of interleukin (IL)-1β and IL-18. In caspase-1-deficient cells, inflammasome formation still leads to caspase-3 activation and slower apoptotic death, dependent on caspase-8 as an apical caspase. A role for caspase-8 directly upstream of caspase-1 has also been suggested, but here we show that caspase-8-deficient macrophages have no defect in AIM2 inflammasome-mediated caspase-1 activation, pyroptosis, and IL-1β cleavage. In investigating the inflammasome-induced apoptotic pathway, we previously demonstrated that activated caspase-8 is essential for caspase-3 cleavage and apoptosis in caspase-1-deficient cells. However, here we found that AIM2 inflammasome-initiated caspase-3 cleavage was maintained in Ripk3 -/- Casp8 -/- macrophages. Gene knockdown showed that caspase-1 was required for the caspase-3 cleavage. Thus inflammasomes activate a network of caspases that can promote both pyroptotic and apoptotic cell death. In cells where rapid pyroptosis is blocked, delayed inflammasome-dependent cell death could still occur due to both caspase-1- and caspase-8-dependent apoptosis. Initiation of redundant cell death pathways is likely to be a strategy for coping with pathogen interference in death processes., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Caspase-8 is required for extrinsic apoptosis, but is also central for preventing a pro-inflammatory receptor interacting protein kinase (RIPK) 3-mixed lineage kinase domain-like (MLKL)-dependent cell death pathway termed necroptosis. Despite these critical cellular functions, the impact of capase-8 deletion in the myeloid cell lineage, which forms the basis for innate immune responses, has remained unclear. In a recent article in Arthritis Research & Therapy, Cuda et al. report that myeloid cell-restricted caspase-8 loss leads to a very mild RIPK3-dependent inflammatory phenotype. The presented results suggest that inflammation does not arise exclusively because of RIPK3-mediated necroptotic death but that, in the absence of caspase-8, RIPK1 and RIPK3 enhance microbiome-driven Toll-like receptor-induced pro-inflammatory cytokine production.

Cytosolic DNA can indicate infection and induces type I interferon (IFN) and AIM2 inflammasome responses. Characterization of these responses has required introduction of DNA into the cytosol of macrophages by either chemical transfection or electroporation, each of which has advantages in different applications. We describe here optimized procedures for both electroporation and chemical transfection, including the centrifugation of chemical transfection reagent onto cells, which greatly increases the speed and strength of responses. Appropriate choice of DNA and use of these methods allow study of either the cytosolic DNA responses in isolation or the simultaneous stimulation of cytosolic receptors and the CpG DNA receptor toll-like receptor 9 (TLR9) in the endosomes.

Inflammasomes mediate inflammatory and cell death responses to pathogens and cellular stress signals via activation of procaspases-1 and -8. During inflammasome assembly, activated receptors of the NLR or PYHIN family recruit the adaptor protein ASC and initiate polymerization of its pyrin domain (PYD) into filaments. We show that ASC filaments in turn nucleate procaspase-8 death effector domain (DED) filaments in vitro and in vivo. Interaction between ASC PYD and procaspase-8 tandem DEDs optimally required both DEDs and represents an unusual heterotypic interaction between domains of the death fold superfamily. Analysis of ASC PYD mutants showed that interaction surfaces that mediate procaspase-8 interaction overlap with those required for ASC self-association and interaction with the PYDs of inflammasome initiators. Our data indicate that multiple types of death fold domain filaments form at inflammasomes and that PYD/DED and homotypic PYD interaction modes are similar. Interestingly, we observed condensation of procaspase-8 filaments containing the catalytic domain, suggesting that procaspase-8 interactions within and/or between filaments may be involved in caspase-8 activation. Procaspase-8 filaments may also be relevant to apoptosis induced by death receptors., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Inflammasomes are protein complexes that promote caspase activation, resulting in processing of IL-1β and cell death, in response to infection and cellular stresses. Inflammasomes have been anticipated to contribute to autoimmunity. The New Zealand Black (NZB) mouse develops anti-erythrocyte Abs and is a model of autoimmune hemolytic anemia. These mice also develop anti-nuclear Abs typical of lupus. In this article, we show that NZB macrophages have deficient inflammasome responses to a DNA virus and fungal infection. Absent in melanoma 2 (AIM2) inflammasome responses are compromised in NZB by high expression of the AIM 2 antagonist protein p202, and consequently NZB cells had low IL-1β output in response to both transfected DNA and mouse CMV infection. Surprisingly, we also found that a second inflammasome system, mediated by the NLR family, pyrin domain containing 3 (NLRP3) initiating protein, was completely lacking in NZB cells. This was due to a point mutation in an intron of the Nlrp3 gene in NZB mice, which generates a novel splice acceptor site. This leads to incorporation of a pseudoexon with a premature stop codon. The lack of full-length NLRP3 protein results in NZB being effectively null for Nlrp3, with no production of bioactive IL-1β in response to NLRP3 stimuli, including infection with Candida albicans. Thus, this autoimmune strain harbors two inflammasome deficiencies, mediated through quite distinct mechanisms. We hypothesize that the inflammasome deficiencies in NZB alter the interaction of the host with both microflora and pathogens, promoting prolonged production of cytokines that contribute to development of autoantibodies., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Inflammasomes are large protein complexes induced by a wide range of microbial, stress, and environmental stimuli that function to induce cell death and inflammatory cytokine processing. Formation of an inflammasome involves dramatic relocalization of the inflammasome adapter protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) into a single speck. We have developed a flow cytometric assay for inflammasome formation, time of flight inflammasome evaluation, which detects the change in ASC distribution within the cell. The transit of ASC into the speck is detected by a decreased width or increased height of the pulse of emitted fluorescence. This assay can be used to quantify native inflammasome formation in subsets of mixed cell populations ex vivo. It can also provide a rapid and sensitive technique for investigating molecular interactions in inflammasome formation, by comparison of wild-type and mutant proteins in inflammasome reconstitution experiments., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Mouse p202 containing two hemopoietic expression, interferon inducibility, nuclear localization (HIN) domains antagonizes AIM2 inflammasome signaling and potentially modifies lupus susceptibility. We found that only HIN1 of p202 binds double-stranded DNA (dsDNA), while HIN2 forms a homotetramer. Crystal structures of HIN1 revealed that dsDNA is bound on face opposite the site used in AIM2 and IFI16. The structure of HIN2 revealed a dimer of dimers, the face analogous to the HIN1 dsDNA binding site being a dimerization interface. Electron microscopy imaging showed that HIN1 is flexibly linked to HIN2 in p202, and tetramerization provided enhanced avidity for dsDNA. Surprisingly, HIN2 of p202 interacts with the AIM HIN domain. We propose that this results in a spatial separation of the AIM2 pyrin domains, and indeed p202 prevented the dsDNA-dependent clustering of apoptosis-associated speck-like protein containing caspase recruitment domain (ASC) and AIM2 inflammasome activation. We hypothesize that while p202 was evolutionarily selected to limit AIM2-mediated inflammation in some mouse strains, the same mechanism contributes to increased interferon production and lupus susceptibility., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Cell death is an effective strategy to limit intracellular infections. Canonical inflammasomes, including NLRP3, NLRC4, and AIM2, recruit and activate caspase-1 in response to a range of microbial stimuli and endogenous danger signals. Caspase-1 then promotes the secretion of IL-1β and IL-18 and a rapid form of lytic programmed cell death termed pyroptosis. A second inflammatory caspase, mouse caspase-11, mediates pyroptotic death through an unknown non-canonical inflammasome system in response to cytosolic bacteria. In addition, recent work shows that inflammasomes can also recruit procaspase-8, initiating apoptosis. The induction of multiple pathways of cell death has probably evolved to counteract microbial evasion of cell death pathways., (Published by Elsevier Ltd.)

High-risk neuroblastomas often harbor structural chromosomal alterations, including amplified MYCN, and usually have a near-di/tetraploid DNA index, but the mechanisms creating tetraploidy remain unclear. Gene-expression analyses revealed that certain MYCN/MYC and p53/pRB-E2F target genes, especially regulating mitotic processes, are strongly expressed in near-di/tetraploid neuroblastomas. Using a functional RNAi screening approach and live-cell imaging, we identified a group of genes, including MAD2L1, which after knockdown induced mitotic-linked cell death in MYCN-amplified and TP53-mutated neuroblastoma cells. We found that MYCN/MYC-mediated overactivation of the metaphase-anaphase checkpoint synergizes with loss of p53-p21 function to prevent arrest or apoptosis of tetraploid neuroblastoma cells., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Macrophage pre-treatment with bacterial lipopolysaccharide (LPS) boosts subsequent activation of the NLRP3 inflammasome, which controls caspase-1-dependent pro-inflammatory cytokine maturation. Previous work has attributed this phenomenon (known as LPS 'priming') to LPS-dependent induction of NLRP3 expression. Whilst this plays a role, here we demonstrate that rapid LPS priming of NLRP3 inflammasome activation can occur independently of NLRP3 induction, since the priming effect of LPS is still apparent at short pre-treatment times in which NLRP3 protein expression remains unchanged. Furthermore, rapid LPS priming is still evident in Nlrp3(-/-) primary macrophages with NLRP3 expression reconstituted using a constitutive promoter. Similarly, we found that LPS potentiates AIM2 inflammasome activation to submaximal doses of cytosolic DNA without concomitant upregulation of AIM2 protein expression. Our data suggest that, in addition to augmenting NLRP3 inflammasome activity via NLRP3 induction, LPS boosts caspase-1 activation by the NLRP3 and AIM2 inflammasomes by an acute mechanism that is independent of inflammasome sensor induction., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Motivation: Gene expression profiling by microarrays or transcript sequencing enables observing the pathogenic function of tumors on a mesoscopic level., Results: We investigated neuroblastoma tumors that clinically exhibit a very heterogeneous course ranging from rapid growth with fatal outcome to spontaneous regression and detected regulatory oncogenetic shifts in their metabolic networks. In contrast to common enrichment tests, we took network topology into account by applying adjusted wavelet transforms on an elaborated and new 2D grid representation of curated pathway maps from the Kyoto Enzyclopedia of Genes and Genomes. The aggressive form of the tumors showed regulatory shifts for purine and pyrimidine biosynthesis as well as folate-mediated metabolism of the one-carbon pool in respect to increased nucleotide production. We spotted an oncogentic regulatory switch in glutamate metabolism for which we provided experimental validation, being the first steps towards new possible drug therapy. The pattern recognition method we used complements normal enrichment tests to detect such functionally related regulation patterns., Availability and Implementation: PathWave is implemented in a package for R (www.r-project.org) version 2.6.0 or higher. It is freely available from http://www.ichip.de/software/pathwave.html.

High incidence of chemotherapy resistance is the primary cause of treatment failure in a subset of neuroblastomas with amplified MYCN. We have reported previously that ectopic MYCN expression promotes proliferation of neuroblastoma Tet21N cells and simultaneously sensitizes them to the drug-induced apoptosis. In search for genes that are involved in MYCN-dependent regulation of drug resistance, we used a function-based gene cloning approach and identified CTSD encoding for a lysosomal aspartyl protease cathepsin D. Downregulation of cathepsin D expression by RNA interference or inhibition of its enzymatic activity increased sensitivity of MYCN-expressing Tet21N cells to doxorubicin. Overexpression of cathepsin D in Tet21N cells attenuated doxorubicin-induced apoptosis. It was accompanied by activation of protein kinase B (Akt) and persistent antiapoptotic activity of Bcl-2. In primary neuroblastomas, high CTSD messenger RNA (mRNA) levels were associated with amplified MYCN, a strong predictive marker of adverse outcome. Chromatin immunoprecipitation and luciferase promoter assays revealed that MYCN protein binds to the CTSD promoter and activates its transcription, suggesting a direct link between deregulated MYCN and CTSD mRNA expression. We further show that neuroblastoma cells can secrete mitogenic procathepsin D and that MYCN expression and especially doxorubicin treatment promote procathepsin D secretion. Extracellular exogenous cathepsin D induces Akt-1 phosphorylation and doxorubicin resistance in sensitive cells. These results demonstrate an important role of cathepsin D in antiapoptotic signaling in neuroblastoma cells and suggest a novel mechanism for the development of chemotherapy resistance in neuroblastoma.

Mounting evidence implicates BRCA2 not only in maintenance of genome integrity but also in cell-cycle checkpoints. However, the contribution of BRCA2 in the checkpoints is still far from being understood. Here, we demonstrate that breast cancer cells MX-1 are unable to maintain genome integrity, which results in gross polyploidization. We generated MX-1 clones, stably expressing BRCA2, and found that BRCA2 acts to suppress polyploidy. Compared with MX-1, the ectopically BRCA2-expressing cells had different intracellular levels of Aurora A, Aurora B, p21, E2F-1, and pRb, suggesting a BRCA2-mediated suppression of polyploidy via stabilization of the checkpoint proteins levels.

Instability of cryptic plasmids in Sinorhizobium meliloti laboratory strains SKhM1-188, DM7-R, and P108 as well as in their clones isolated from nodules of alfalfa grown during a long-term microvegetation experiment (120 days) was studied. The isolated clones of strains SKhM1-188 and DM7-R manifested stable inheritance of plasmids, whereas 12.7-14.0% of clones with changed plasmid profile were detected in a population of clones from strain P108. These segregants were designated as P108c. Segregants P108c exhibited significantly decreased symbiotic effectiveness, nitrogenase activity, and the competitiveness with respect to alfalfa, compared to the original strain P108. It was established that a 80-kb deletion occurred in a larger of two cryptic plasmids (240 and 230 kb) of segregants P108c. It was concluded that genetic rearrangements are possible in rhizobial clones that did not undergo structural transformation and retained viability in the nodule during the natural vegetation period of alfalfa.

The VirB11 ATPase is a subunit of the Agrobacterium tumefaciens transfer DNA (T-DNA) transfer system, a type IV secretion pathway required for delivery of T-DNA and effector proteins to plant cells during infection. In this study, we examined the effects of virB11 mutations on VirB protein accumulation, T-pilus production, and substrate translocation. Strains synthesizing VirB11 derivatives with mutations in the nucleoside triphosphate binding site (Walker A motif) accumulated wild-type levels of VirB proteins but failed to produce the T-pilus or export substrates at detectable levels, establishing the importance of nucleoside triphosphate binding or hydrolysis for T-pilus biogenesis. Similar findings were obtained for VirB4, a second ATPase of this transfer system. Analyses of strains expressing virB11 dominant alleles in general showed that T-pilus production is correlated with substrate translocation. Notably, strains expressing dominant alleles previously designated class II (dominant and nonfunctional) neither transferred T-DNA nor elaborated detectable levels of the T-pilus. By contrast, strains expressing most dominant alleles designated class III (dominant and functional) efficiently translocated T-DNA and synthesized abundant levels of T pilus. We did, however, identify four types of virB11 mutations or strain genotypes that selectively disrupted substrate translocation or T-pilus production: (i) virB11/virB11* merodiploid strains expressing all class II and III dominant alleles were strongly suppressed for T-DNA translocation but efficiently mobilized an IncQ plasmid to agrobacterial recipients and also elaborated abundant levels of T pilus; (ii) strains synthesizing two class III mutant proteins, VirB11, V258G and VirB11.I265T, efficiently transferred both DNA substrates but produced low and undetectable levels of T pilus, respectively; (iii) a strain synthesizing the class II mutant protein VirB11.I103T/M301L efficiently exported VirE2 but produced undetectable levels of T pilus; (iv) strains synthesizing three VirB11 derivatives with a four-residue (HMVD) insertion (L75.i4, C168.i4, and L302.i4) neither transferred T-DNA nor produced detectable levels of T pilus but efficiently transferred VirE2 to plants and the IncQ plasmid to agrobacterial recipient cells. Together, our findings support a model in which the VirB11 ATPase contributes at two levels to type IV secretion, T-pilus morphogenesis, and substrate selection. Furthermore, the contributions of VirB11 to machine assembly and substrate transfer can be uncoupled by mutagenesis.

Agrobacterium tumefaciens transfers oncogenic T-DNA and effector proteins to plant cells via a type IV secretion pathway. This transfer system, assembled from the products of the virB operon, is thought to consist of a transenvelope mating channel and the T pilus. When screened for the presence of VirB and VirE proteins, material sheared from the cell surface of octopine strain A348 was seen to possess detectable levels of VirB2 pilin, VirB5, and the VirB7 outer membrane lipoprotein. Material sheared from the cell surface of most virB gene deletion mutants also possessed VirB7, but not VirB2 or VirB5. During purification of the T pilus from wild-type cells, VirB2, VirB5, and VirB7 cofractionated through successive steps of gel filtration chromatography and sucrose density gradient centrifugation. A complex containing VirB2 and VirB7 was precipitated from a gel filtration fraction enriched for T pilus with both anti-VirB2 and anti-VirB7 antiserum. Both the exocellular and cellular forms of VirB7 migrated as disulfide-cross-linked dimers and monomers when samples were electrophoresed under nonreducing conditions. A mutant synthesizing VirB7 with a Ser substitution of the lipid-modified Cys15 residue failed to elaborate the T pilus, whereas a mutant synthesizing VirB7 with a Ser substitution for the disulfide-reactive Cys24 residue produced very low levels of T pilus. Together, these findings establish that the VirB7 lipoprotein localizes exocellularly, it associates with the T pilus, and both VirB7 lipid modification and disulfide cross-linking are important for T-pilus assembly. T-pilus-associated VirB2 migrated in nonreducing gels as a monomer and a disulfide-cross-linked homodimer, whereas cellular VirB2 migrated as a monomer. A strain synthesizing a VirB2 mutant with a Ser substitution for the reactive Cys64 residue elaborated T pilus but exhibited an attenuated virulence phenotype. Dithiothreitol-treated T pilus composed of native VirB2 pilin and untreated T pilus composed of the VirB2C64S mutant pilin distributed in sucrose gradients more predominantly in regions of lower sucrose density than untreated, native T pili. These findings indicate that intermolecular cross-linking of pilin monomers is not required for T-pilus production, but cross-linking does contribute to T-pilus stabilization.