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The estrogen-responsive B box protein (EBBP) and Pyrin belong to a family of structurally related proteins. While mutations in the pyrin gene cause an autoinflammatory disease, the biological function of EBBP is unknown. In this study, we identified the proinflammatory cytokine interleukin-1beta (IL-1beta) as an EBBP-binding partner. Furthermore, caspase-1 and NACHT, LRR and Pyrin domain containing protein (NALP) 1, two components of the recently identified inflammasome, a platform for the activation of caspase-1, also interact with EBBP. These proteins bind to the RFP domain of EBBP, suggesting that this domain of so far unknown function is an important protein-binding domain. EBBP was secreted in a caspase-1-dependent manner from cultured cells, and its secretion was enhanced by IL-1beta. Vice versa, endogenous and overerexpressed EBBP increased IL-1beta secretion. These results provide evidence for a role of EBBP in innate immunity by enhancing the alternative secretion pathway of IL-1beta.

Sympathetic neurons undergo programmed cell death (PCD) upon deprivation of nerve growth factor (NGF). PCD of neurons is blocked by inhibitors of the interleukin-1beta converting enzyme (ICE)/Ced-3-like cysteine protease, indicating involvement of this class of proteases in the cell death programme. Here we demonstrate that the proteolytic activities of the proteasome are also essential in PCD of neurons. Nanomolar concentrations of several proteasome inhibitors, including the highly selective inhibitor lactacystin, not only prolonged survival of NGF-deprived neurons but also prevented processing of poly(ADP-ribose) polymerase which is known to be cleaved by an ICE/Ced-3 family member during PCD. These results demonstrate that the proteasome is a key regulator of neuronal PCD and that, within this process, it is involved upstream of proteases of the ICE/Ced-3 family. This order of events was confirmed in macrophages where lactacystin inhibited the proteolytic activation of precursor ICE and the subsequent generation of active interleukin-1beta.

International audience; Different types of cell death are often defined by morphological criteria, without a clear reference to precise biochemical mechanisms. The Nomenclature Committee on Cell Death (NCCD) proposes unified criteria for the definition of cell death and of its different morphologies, while formulating several caveats against the misuse of words and concepts that slow down progress in the area of cell death research. Authors, reviewers and editors of scientific periodicals are invited to abandon expressions like 'percentage apoptosis' and to replace them with more accurate descriptions of the biochemical and cellular parameters that are actually measured. Moreover, at the present stage, it should be accepted that caspase-independent mechanisms can cooperate with (or substitute for) caspases in the execution of lethal signaling pathways and that 'autophagic cell death' is a type of cell death occurring together with (but not necessarily by) autophagic vacuolization. This study details the 2009 recommendations of the NCCD on the use of cell death-related terminology including 'entosis', 'mitotic catastrophe', 'necrosis', 'necroptosis' and 'pyroptosis'.

Upon DNA damage, a complex called the PIDDosome is formed and either signals NF-kappaB activation and thus cell survival or alternatively triggers caspase-2 activation and apoptosis. PIDD (p53-induced protein with a death domain) is constitutively processed giving rise to a 48-kDa N-terminal fragment containing the leucine-rich repeats (LRRs, PIDD-N) and a 51-kDa C-terminal fragment containing the death domain (DD, PIDD-C). The latter undergoes further cleavage resulting in a 37-kDa fragment (PIDD-CC). Here we show that processing occurs at S446 (generating PIDD-C) and S588 (generating PIDD-CC) by an auto-processing mechanism similar to that found in the nuclear pore protein Nup98/96 and inteins. Auto-cleavage of PIDD determines the outcome of the downstream signaling events. Whereas initially formed PIDD-C mediates the activation of NF-kappaB via the recruitment of RIP1 and NEMO, subsequent formation of PIDD-CC causes caspase-2 activation and thus cell death. A non-cleavable PIDD mutant is unable to translocate from the cytoplasm to the nucleus and loses both activities. In this way, auto-proteolysis of PIDD might participate in the orchestration of the DNA damage-induced life and death signaling pathways.

Members of the viral Flice/caspase-8 inhibitory protein (v-FLIP) family prevent induction of apoptosis by death receptors through inhibition of the processing and activation of procaspase-8 and -10 at the level of the receptor-associated death-inducing signaling complex (DISC). Here, we have addressed the molecular function of the v-FLIP member MC159 of the human molluscum contagiosum virus. MC159 FLIP powerfully inhibited both caspase-dependent and caspase-independent cell death induced by Fas. The C-terminal region of MC159 bound TNF receptor-associated factor (TRAF)3, was necessary for optimal TRAF2 binding, and mediated the recruitment of both TRAFs into the Fas DISC. TRAF-binding-deficient mutants of MC159 showed impaired inhibition of FasL-induced caspase-8 processing and Fas internalization, and had reduced antiapoptotic activity. Our findings provide evidence that a MC159/TRAF2/TRAF3 complex regulates a new aspect of Fas signaling, and identify MC159 FLIP as a molecule that targets multiple features of Fas-induced cell death.

Fas activation triggers apoptosis in many cell types. Studies with anti-Fas antibodies have produced conflicting results on Fas signaling, particularly the role of the Bcl-2 family in this process. Comparison between physiological ligand and anti-Fas antibodies revealed that only extensive Fas aggregation, by membrane bound FasL or aggregated soluble FasL consistently triggered apoptosis, whereas antibodies could act as death agonists or antagonists. Studies on Fas signaling in cell lines and primary cells from transgenic mice revealed that FADD/MORT1 and caspase-8 were required for apoptosis. In contrast, Bcl-2 or Bcl-xLdid not block FasL-induced apoptosis in lymphocytes or hepatocytes, demonstrating that signaling for cell death induced by Fas and the pathways to apoptosis regulated by the Bcl-2 family are distinct.

The killing mediated by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells represents an important mechanism in the immune defense against tumors, virus-infected cells, parasites and other foreign invaders. The deleterious effects observed in the absence of a properly working cellular immune response have been characterized extensively in immunodeficient patients (for a review see Matsumoto et al. 1992) and naturally occurring animal models such as SCID (severe combined immunodeficiency) or nude mice. In general, immunodeficient individuals are predisposed to develop severe opportunistic infections which ultimately can lead to their death. Recently, gene targeting technology has been used repeatedly to generate mice with defined mutations in genes implicated in T and NK cell function (for a review see Yeung et al. 1993), in an attempt to dissect the individual pathways involved in the lymphocyte-mediated immune response.

Apoptosis is critically dependent on the presence of the ced-3 gene in Caenorhabditis elegans, which encodes a protein homologous to the mammalian interleukin (IL)-1 beta-converting enzyme (ICE). Overexpression of ICE or ced-3 promotes apoptosis. Cytotoxic T lymphocyte-mediated rapid apoptosis is induced by the proteases granzyme A and B. ICE and granzyme B share the rare substrate site of aspartic acid, after which amino acid cleavage of precursor IL-1 beta (pIL-1 beta) occurs. Here we show that granzyme A, but not granzyme B, converts pIL-1 beta to its 17-kD mature form. Major cleavage occurs at Arg120, four amino acids downstream of the authentic processing site, Asp116. IL-1 beta generated by granzyme A is biologically active. When pIL-1 beta processing is monitored in lipopolysaccharide-activated macrophage target cells attacked by cytotoxic T lymphocytes, intracellular conversion precedes lysis. Prior granzyme inactivation blocks this processing. We conclude that the apoptosis-inducing granzyme A and ICE share at least one downstream target substrate, i.e., pIL-1 beta. This suggests that lymphocytes, by means of their own converting enzyme, could initiate a local inflammatory response independent of the presence of ICE.

Publisher Summary This chapter highlights purification and assay methods for granzyme B. The highest levels of granzyme B are found in the granules of cytotoxic T lymphocytes (CTLs). Lower levels of granzyme B are found in helper T cells and in thymocytes. The proposed physiological roles of granzyme B include the inhibition of colon cancer cell growth and the induction of chromatin DNA degradation during T cell-mediated apoptosis. Human and mouse granzyme B are isolated from cytoplasmic granules of activated CTL lines grown in the presence of recombinant interleukin 2. Granzyme B preferentially cleaves substrates with negatively charged side chains, and prefers aspartic over glutamic acid, as aspartic acid has a shorter side chain. Granzyme B also exhibits a weak esterase activity toward substrates containing a serine or an asparagine, as these two residues can form hydrogen bonds with the Arg-208. Granzyme B is inhibited by a variety of inhibitors, such as lima bean trypsin, soybean trypsin, and chymostatin.

Bcl-2 does not inhibit cell death induced by the physiological Fas ligand: implications for the existence of type I and type II cells

Non-immune activation of the first component of complement (C1) by the heart mitochondrial inner membrane has been investigated. Cardiolipin, the only strong activator of C1 among phospholipids, is present in large amounts in the heart mitochondrial inner membrane. We therefore studied its contribution to C1 activation by mitochondria. The proteins of the mitochondrial inner membrane were found to activate C1 only weakly, in contrast with the phospholipid fraction which induces strong C1 activation. Furthermore, the digestion of mitochondrial inner membranes with proteolytic enzymes did not affect C1 activation. Additional support in favour of cardiolipin being the responsible activator came from competition experiments with mitochondrial creatine kinase (mt-CPK) and adriamycin, known to bind to cardiolipin. Both mt-CPK and adriamycin displaced C1q from the mitochondrial inner membrane. In addition, C1q displaced mt-CPK bound to mitoplasts.

Examining a CTL-line (B6.1) with specific cytolytic activity, we have confirmed the ability of CTL-lines to produce poly C9-like, tubular complexes of two sizes (polyperforin 1 and 2). Isolated cytoplasmic granules from this cell line can induce hemolysis and contain the lysosomal marker arylsulfatase. Electron microscopial studies show that granules contain precursor molecules which are assembled into polyperforin upon addition of Ca2+. Antibodies raised against granules predominantly react with a 27-kd protein. We compared the B6.1 cell with a hybrid, PC60, derived from a cross between B6.1 and a non-cytolytic rat-thymoma. This hybrid line exhibits inducible CTL-activity. In PC60 with induced cytolytic activity, polyperforins 1 and 2, cytolytic granules and granule-associated proteins are detected. Non-cytolytic PC60 cells lack cytolytic granules and polyperforin 1, but contain polyperforin 2. These results suggest that cytolytic granules appear in parallel with the expression of cytotoxicity during T-lymphocyte differentiation.

Cellular serine proteases are most active at neutral, physiological pH and have been implicated in a variety of different processes such as cellular Chemotaxis, protein turnover in tissues, endocytosis and exocytosis, or tumorigenesis (Neurath 1984). In addition, there is also considerable evidence that cellular proteases play an important role in cell-mediated cytotoxicity: T-cell killing can be inhibited by diisopropylfluorophosphate (DFP) or PMSF (Chang and Eisen 1980; Quan et al. 1982), suggesting DFP- or PMSF-inactivation of functionally important trypsin-like proteases. Antibody-dependent cell-mediated cytotoxicity is abolished by protease substrates such as acetyl tyrosine ester in a competitive manner, and by chloromethyl ketone derivatives of amino acids. Macromolecular antiproteases like α 1-antitrypsin and α 1-antichymotrypsin also suppress lysis when present in natural killer (NK) cell assays (Redelman and Hudig 1980; Hudig et al. 1981, 1984), further implicating the involvement of proteases in the cytotoxic event. While pretreatment of the effector cell with protease inhibitors has no effect on the lytic activity of NK cells, cytotoxicity is highly sensitive to the presence of inhibitors for a short period of time after the addition of the target cell (Lavie et al. 1985), suggesting that target cell binding triggers the exposure of enzymes to the external environment.

Cytoplasmic granules from cytolytic T-lymphocytes (CTL) contain two proteins which react with the serine esterase-specific affinity label diisopropylfluorophosphate (DFP). One of these is a trypsin-like esterase which consists of two disulfide-linked 35-kd subunits. The other consists of a single 29-kd chain. Both molecules are induced concomitantly with cytolytic activity and perforin activity in a CTL-derived T-cell hybrid.

We show that among the subsets of peripheral T lymphocytes (Lyt2+ L3T4- and L3T4+ Lyt2- cells) activated in short-term cultures by stimulation with H-2 incompatible leukocytes 97% of the cytolytic activity and all detectable perforin activity resides in the Lyt2+ cells. But both populations contain approximately equal amounts of a serine protease, granzyme A, the expression of which was previously thought to be restricted to cytolytic T lymphocytes.

Jun kinase signaling can be elicited by death receptor activation, but the mechanism and significance of this event are still unclear. It has been reported that cross-linking Abs to Fas trigger c-Jun N-terminal kinase (JNK) signaling via caspase-mediated activation of MEKK1 (JNK kinase kinase), elevation of ceramide levels or by recruitment of death domain associated protein (DAXX) to Fas. The effect of physiological ligand for Fas on JNK signaling was never investigated, although evidence is accumulating that Fas ligand is able to induce cellular responses distinct from those evoked by Ab-mediated cross-linking of Fas. Therefore, we investigated the effect of Fas ligand on JNK signaling. Like its ability to induce cell death, Fas ligand reliably activated JNK only upon extensive aggregation of the receptor. Although this was partially dependent on caspase activation, DAXX was not required. DAXX and other death receptor-associated proteins, which have been reported to bind directly or indirectly to Fas, such as receptor interacting protein (RIP) and RIP-associated ICH-1/CED-3-homologous protein with a death domain (RAIDD), were shown to be dispensable for Fas ligand-induced apoptosis.

Cell death by apoptosis occurs in a wide range of physiological events including repertoire selection of lymphocytes and during immune responses in vivo. A hallmark of apoptosis is the internucleosomal DNA degradation for which a Ca2+,Mg(2+)-dependent endonuclease has been postulated. This nuclease activity was extracted from both rat thymocyte and lymph node cell nuclei. When incubated with nuclei harbouring only limited amounts of endogenous nuclease activity, the ladder pattern of DNA fragments characteristic of apoptosis was induced. This extractable nucleolytic activity was immunoprecipitated with antibodies specific for rat deoxyribonuclease I (DNase I) and was inhibited by actin in complex with gelsolin segment 1, strongly pointing to the presence of a DNase I-type enzyme in the nuclear extracts. COS cells transiently transfected with the cDNA of rat parotid DNase I expressed the enzyme, and their nuclei were able to degrade their DNA into oligosome-sized fragments. PCR analysis of mRNA isolated from thymus, lymph node cells and kidney yielded a product identical in size to that from rat parotid DNase I. Immunohistochemical staining with antibodies to rat DNase I confirmed the presence of DNase I antigen in thymocytes and lymph node cells. The tissue distribution of DNase I is thus extended to tissues with no digestive function and to cells which are known to be susceptible to apoptosis. We propose that during apoptosis, an endonuclease indistinguishable from DNase I gains access to the nucleus due to the breakdown of the ER and the nuclear membrane.