Communicated by A.M.MichelsonRecently, the existence of 40-kd and 46-kd 2-5A synthetases in interferon-treated cells has been confirmed by cloning and characterization of cDNA corresponding to these small size enzymes. By the use of specific monoclonal antibodies, we describe here two forms of high mol. wt 2-5A synthetases of 69 and 100 kd in human cells. The monoclonal antibodies immunoprecipitate either a 69-or a 100-kd 2-5A synthetase. These purified 2-5A synthetases in immune complex preparations are active, i.e. addition of poly(l).poly(C) and ATP results in the synthesis of 2-5A. Both 2-5A synthetases are composed of several subspecies with similar isoelectric points in the range of 7-8 but have different subcellular localizations: 100-kd synthetase is recovered from the microsomal pellet whereas 69-kd synthetase is found to be associated with cell membranes as well as with the microsomal pellet. Different types of interferon-treated human cells express both or either forms of these enzymes. The 69and 100-kd 2-5A synthetases were also identified by electrophoretic transfer inmmunoblot analysis using rabbit polyclonal antibodies against a synthetic peptide common on both 46and 40-kd 2-5A synthetases. These results indicate that small and large size isozymes share a common peptide sequence.

Historically, two main forms of cell death have been distinguished: apoptosis and necrosis. Apoptosis was initially considered as the only physiological and programmed form of cell death. This type of death is recurrently associated with caspases, a family of cysteine proteases activated in apoptotic conditions. However, it is now widely recognized that programmed cell death (PCD) can also occur in the complete absence of caspase activation. The existence of non-caspase PCD pathways was corroborated by the discovery of caspase-independent executioners, such as the mitochondrial protein Apoptosis-Inducing Factor (AIF). Necrosis has often been viewed as an accidental and uncontrolled cell death process. Nevertheless, increasing evidence shows that, like apoptosis, necrosis could be a highly orchestrated type of PCD. Indeed, apoptosis and necrosis present more similarities than it has been originally thought. Here, we summarize the different classifications of PCD and the current knowledge of a necrotic PCD pathway mediated by AIF: alkylating DNA-damage mediated death. We also outline the molecular mechanisms controlling this form of PCD and discuss their potential relevance in physiological and pathological settings. These emerging data on the molecular mechanisms regulating programmed necrosis may certainly have potent therapeutic consequences in treating both apoptotic-resistant tumors and degenerating adult neurons.

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The protein kinase from human cells dependent on double-stranded (ds) RNA is a 68-kDa protein (p68 kinase), the level of which is enhanced significantly in cells treated with interferon. When activated by low concentrations of dsRNA, the p68 kinase becomes phosphorylated and thereby catalyzes the phosphorylation of the protein-synthesis initiation factor, eIF2. Here, we have purified the p68 kinase to homogeneity using a specific monoclonal antibody to investigate its capacity to bind dsRNA, poly(1). poly(C). Our study suggest that p68 kinase has high-and low-affinity binding sites: the high-affinity binding site is responsible for the activation and the low-affinity binding site for the inhibition of kinase activity. This is in accord with the fact that autophosphorylation of p68 kinase occurs at low concentrations of dsRNA whereas high concentrations of dsRNA inhibit its autophosphorylation.We have also investigated the binding of adenoviral VAI RNA to the purified p68 kinase and have found that the affinity of this binding is lower than that of poly(1). poly(C). We show that VAL RNA can activate or inhibit autophosphorylation of p68 kinase in a dose-dependent manner, i. e. activation at < 1 pg/ml or inhibition at > 1 pg/ml of VAI RNA. In spite of its lower affinity of binding, VAI RNA cannot be displaced by poly(1) . poly(C) or reovirus dsRNA. These data confirm our previous results to illustrate that VAI RNA can bind p68 kinase and cause its inactivation irreversably.Treatment of cells with interferon results in the induction of a specific protein kinase, dependent on double-stranded (ds) RNA for its activity [l-41. The protein kinase from human cells is a M,-68000 protein (p68 kinase) which has been recently characterized using specific monoclonal antibodies [5]. The p68 kinase is characterized by two distinct protein kinase activities [6]. The first one is functional for its autophosphorylation whereas the second one is responsible for the phosphorylation of exogenous substrates such as calf thymus histones and the cr subunit of the eukaryotic proteinsynthesis initiation factor, eIF2. When activated by dsRNA in the presence of MnZf and ATP, p68 kinase is autophosphorylated. This phosphorylated p68 kinase is then capable of catalyzing phosphorylation of eIF2. Phosphorylation of exogenous substrates (histone, eIF2) is not dependent on dsRNA [7]. It might occur as long as p68 kinase is phosphorylated, i. e. there is a strong correlation between the degree of phosphate content of p68 kinase and its kinase activity on exogenous substrates [6, 71. The protein kinase activities mediating autophosphorylation of p68 kinase and phosphorylation of exogenous substrates are independent of cyclic AMP or cyclic GMP and are markedly stimulated by M n Z + . Abbreviations. dsRNA, double-stranded RNA; p68 kinase, human dsRNA-dependent protein kinase; eIF2, eukaryotic initiation factor 2; PhMeS02F, phenylmethylsulfonyl fluoride; mAb, monoclonal antibody; p65 kinase, murine dsRNA-dependent protein kinase; ssRNA, single-standed...

Apoptosis-inducing factor (AIF) is a bifunctional NADH oxidase involved in mitochondrial respiration and caspase-independent apoptosis. Three alternatively spliced mRNA isoforms of AIF have been identified previously: AIF, AIF-exB, and AIFsh. Here, we report the cloning and the biochemical characterization of a new isoform named AIF short 2 (AIFsh2). AIFsh2 transcript includes a previously unknown exon placed between exons 9 and 10 of AIF. The resulting AIFsh2 protein, which localizes in mitochondria, corresponds to the oxidoreductase domain of AIF. In this way, AIFsh2 exhibits similar NADH oxidase activity to AIF and generates reactive oxygen species. Like AIF, AIFsh2 is released from mitochondria to cytosol after an apoptotic insult in a calpain or cathepsin-dependent manner. However, in contrast to AIF, AIFsh2 does not induce nuclear apoptosis. Thus, it seems that the reactive oxygen species produced by the oxidoreductase domain of AIF/AIFsh2 are not important for AIF-dependent nuclear apoptosis. In addition, we demonstrate that the AIFsh2 mRNA is absent in normal brain tissue, whereas it is expressed in neuroblastoma-derived cells, suggesting a different regulation in normal and transformed cells from the brain lineage. Together, our results reveal that AIF yields an original and independent genetic regulation of the two AIF functions. This is an important issue to understand the physiological role of this protein.