The term apoptosis often has been used interchangeably with the term programmed cell death. Here we describe a form of programmed cell death that is distinct from apoptosis by the criteria of morphology, biochemistry, and response to apoptosis inhibitors. Morphologically, this alternative form of programmed cell death appears during development and in some cases of neurodegeneration. Despite its lack of response to caspase inhibitors and Bcl-x L, we show that this form of cell death is driven by an alternative caspase-9 activity that is Apaf-1-independent. Characterization of this alternative form of programmed cell death should lead to new insight into cell death programs and their roles in development and degeneration.

Apoptosis is orchestrated by the concerted action of caspases, activated in a minimal two-step proteolytic cascade. Existing data suggests that apical caspases are activated by adaptor-mediated clustering of inactive zymogens. However, the mechanism by which apical caspases achieve catalytic competence in their recruitment/activation complexes remains unresolved. We explain that proximity-induced activation of apical caspases is attributable to dimerization. Internal proteolysis does not activate these apical caspases but is a secondary event resulting in partial stabilization of activated dimers. Activation of caspases-8 and -9 occurs by dimerization that is fully recapitulated in vitro by kosmotropes, salts with the ability to stabilize the structure of proteins. Further, single amino acid substitutions at the dimer interface abrogate the activity of caspases-8 and -9 introduced into recipient mammalian cells. We propose a unified caspase activation hypothesis whereby apical caspases are activated by dimerization of monomeric zymogens.

Programmed cell death (pcd) may take the form of apoptotic or nonapoptotic pcd. Whereas cysteine aspartyl-specific proteases (caspases) mediate apoptosis, the mediators of nonapoptotic cell death programs are much less well characterized. Here, we report that paraptosis, an alternative, nonapoptotic cell death program that may be induced by the insulin-like growth factor I receptor (among other inducers), is mediated by mitogen-activated protein kinases (MAPKs) and inhibited by AIP-1/Alix. The inhibition by AIP-1/Alix is specific for paraptosis since apoptosis was not inhibited. Caspases were not activated in this paradigm, nor were caspase inhibitors effective in blocking cell death. However, insulinlike growth factor I receptor (IGFIR)-induced paraptosis was inhibited by MEK-2-specific inhibitors and by antisense oligonucleotides directed against c-jun N-terminal kinase-1 (JNK-1). These results suggest that IGFIR-induced paraptosis is mediated by MAPKs, and inhibited by AIP-1/Alix.

The apical protease of the human intrinsic apoptotic pathway, caspase-9, is activated in a polymeric activation platform known as the apoptosome. The mechanism has been debated, and two contrasting hypotheses have been suggested. One of these postulates an allosteric activation of monomeric caspase-9; the other postulates a dimer-driven assembly at the surface of the apoptosome--the "induced proximity" model. We show that both Hofmeister salts and a reconstituted mini-apoptosome activate caspase-9 by a second-order process, compatible with a conserved dimer-driven process. Significantly, replacement of the recruitment domain of the apical caspase of the extrinsic apoptotic pathway, caspase-8, by that of caspase-9 allows activation of this hybrid caspase by the apoptosome. Consequently, apical caspases can be activated simply by directing their zymogens to the apoptosome, ruling out the requirement for allosteric activation and supporting an induced proximity dimerization model for apical caspase activation in vivo.

Cells depend on specific stimuli, such as trophic factors, for survival and in the absence of such stimuli, undergo apoptosis. How do cells initiate apoptosis in response to the withdrawal of trophic factors or other dependent stimuli? Recent studies of apoptosis induction by neurotrophin withdrawal argue for a novel form of pro-apoptotic signal transduction ±`negative signal transduction' ± in which the absence of ligand-receptor interaction induces cell death. We have found that the prototype for this form of signaling ± the common neurotrophin receptor, p75 NTR ± creates a state of cellular dependence (or addiction) on neurotrophins, and that this effect requires an`addiction/dependence domain' (ADD) in the intracytoplasmic region of p75 NTR . We have recently found other receptors that include dependence domains, arguing that dependence receptors, and their associated dependence domains, may be involved in a rather general mechanism to create cellular states of dependence on trophic factors, cytokines, adhesion, electrical activity and other dependent stimuli.

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This study describes the first proteomic analysis of paraptosis—a non-apoptotic form of programmed cell death. As with apoptosis, the first description of paraptosis was based on morphological criteria. Since there are no known markers for paraptosis, the purpose of this study was to dissect changes in the proteome profile occurring during paraptosis. Using one- and two-dimensional SDS–PAGE, Western analysis, and mass spectrometry, we show that during paraptosis, alterations occur mainly in cytoskeletal proteins, signal transduction proteins, mitochondrial proteins, and some metabolic proteins. We also report the identification of: (1) a paraptosis inhibitor, phosphatidylethanolamine binding protein (PEBP-1), and (2) a candidate mediator of paraptosis, prohibitin. Identification of specific paraptotic changes will ultimately lead to tools to detect this type of programmed cell death in in vivo systems and allow for its further characterization.

The type 1 insulin-like growth factor receptor (IGF-IR