Emad S. Alnemri

Cytochrome c and dATP-Dependent Formation of Apaf-1/Caspase-9 Complex Initiates an Apoptotic Protease Cascade

We report here the purification of the third protein factor, Apaf-3, that participates in caspase-3 activation in vitro. Apaf-3 was identified as a member of the caspase family, caspase-9. Caspase-9 and Apaf-1 bind to each other via their respective NH2-terminal CED-3 homologous domains in the presence of cytochrome c and dATP, an event that leads to caspase-9 activation. Activated caspase-9 in turn cleaves and activates caspase-3. Depletion of caspase-9 from S-100 extracts diminished caspase-3 activation. Mutation of the active site of caspase-9 attenuated the activation of caspase-3 and cellular apoptotic response in vivo, indicating that caspase-9 is the most upstream member of the apoptotic protease cascade that is triggered by cytochrome c and dATP.

Human ICE/CED-3 Protease Nomenclature

It is now clear that members of the ICE/CED-3 protease family play key biological roles in inflammation and mammalian apoptosis. To date, ten homologs of human origin have been published (Figure 1Figure 1). The frenetic pace of identification of new homologs has led to inconsistent and multiple names for many of these enzymes. As a consequence, the general scientific community is finding it increasingly difficult to follow this provocative and rapidly moving field. In an effort to remedy this situation, several of us who have been involved in the identification and characterization of these enzymes have formed a committee, with the objective of proposing a nomenclature for the human members of this protease family that is sensible and easy to use. The purpose of this letter is to outline our recommendations.Figure 1Caspase Designations for Human Members of the ICE/CED3 Protease Family and Phylogenic Relationships among these ProteasesThe phylogenic relationships were determined using the PILEUP algorithm (version 8.1; gap weight = 3.0; gap length weight = 0.1) (Program Manual for the Wisconsin Package, Version 8, September 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin 53711). This figure was contributed by Don Nicholson, Merck Frosst.View Large Image | View Hi-Res Image | Download PowerPoint SlideWe propose to use the trivial name “caspase” as a root for serial names for all family members. The selection of caspase was based on two catalytic properties of these enzymes. The “c” is intended to reflect a cysteine protease mechanism, and “aspase” refers to their ability to cleave after aspartic acid, the most distinctive catalytic feature of this protease family. To designate individual family members, caspase will be followed by an arabic numeral, which will be assigned based on its date of publication. Current assignments are shown in Figure 1Figure 1. The root name for the corresponding gene will be CASP.Each of these enzymes is synthesized as a proenzyme that is proteolytically activated to form a heterodimeric catalytic domain. Proenzymes will be referred to as pro-“enzyme name” (e.g. pro-caspase-1). Subunits of the heterodimer will be described by the enzyme name, followed by the correct molecular size of the subunit (e.g. caspase-1-p10, caspase-1-p20). In a general sense, subunits may be referred to as large and small. The N-terminal peptide that is removed during proteolytic activation of these proteases will be referred to as such.Splice variants will be given a small English character suffix, separated from the name of the protease by a slash, which will be assigned based on order of publication (e.g. caspase-1/a).Please consult one of the members of the committee prior to final publication of any new homolog, so that the appropriate number can be assigned. A new homolog is defined as a fully sequenced protein, cDNA, or gene that has a statistically significant relationship to the large and small subunits of one of the established family members. We strongly encourage all investigators of these proteases to adopt these nomenclature recommendations. It is only through compliance that we will achieve our goal of improving communication between scientists both inside and outside this exciting field.

Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012

In 2009, the Nomenclature Committee on Cell Death (NCCD) proposed a set of recommendations for the definition of distinct cell death morphologies and for the appropriate use of cell death-related terminology, including ‘apoptosis’, ‘necrosis’ and ‘mitotic catastrophe’. In view of the substantial progress in the biochemical and genetic exploration of cell death, time has come to switch from morphological to molecular definitions of cell death modalities. Here we propose a functional classification of cell death subroutines that applies to both in vitro and in vivo settings and includes extrinsic apoptosis, caspase-dependent or -independent intrinsic apoptosis, regulated necrosis, autophagic cell death and mitotic catastrophe. Moreover, we discuss the utility of expressions indicating additional cell death modalities. On the basis of the new, revised NCCD classification, cell death subroutines are defined by a series of precise, measurable biochemical features.

IAPs block apoptotic events induced by caspase‐8 and cytochrome c by direct inhibition of distinct caspases

Inhibitor of apoptosis (IAP) gene products play an evolutionarily conserved role in regulating programmed cell death in diverse species ranging from insects to humans. Human XIAP, cIAP1 and cIAP2 are direct inhibitors of at least two members of the caspase family of cell death proteases: caspase‐3 and caspase‐7. Here we compared the mechanism by which IAPs interfere with activation of caspase‐3 and other effector caspases in cytosolic extracts where caspase activation was initiated by caspase‐8, a proximal protease activated by ligation of TNF‐family receptors, or by cytochrome c, which is released from mitochondria into the cytosol during apoptosis. These studies demonstrate that XIAP, cIAP1 and cIAP2 can prevent the proteolytic processing of pro‐caspases ‐3, ‐6 and ‐7 by blocking the cytochrome c‐induced activation of pro‐caspase‐9. In contrast, these IAP family proteins did not prevent caspase‐8‐induced proteolytic activation of pro‐caspase‐3; however, they subsequently inhibited active caspase‐3 directly, thus blocking downstream apoptotic events such as further activation of caspases. These findings demonstrate that IAPs can suppress different apoptotic pathways by inhibiting distinct caspases and identify pro‐caspase‐9 as a new target for IAP‐mediated inhibition of apoptosis.

Autoactivation of Procaspase-9 by Apaf-1-Mediated Oligomerization

Activation of procaspase-9 by Apaf-1 in the cytochrome c/dATP-dependent pathway requires proteolytic cleavage to generate the mature caspase molecule. To elucidate the mechanism of activation of procaspase-9 by Apaf-1, we designed an in vitro Apaf-1-procaspase-9 activation system using recombinant components. Here, we show that deletion of the Apaf-1 WD-40 repeats makes Apaf-1 constitutively active and capable of processing procaspase-9 independent of cytochrome c an dATP. Apaf-1-mediated processing of procaspase-9 occurs at Asp-315 by an intrinsic autocatalytic activity of procaspase-9 itself. We provide evidence that Apaf-1 can form oligomers and may facilitate procaspase-9 autoactivation by oligomerizing its precursor molecules. Once activated, caspase-9 can initiate a caspase cascade involving the downstream executioners caspase-3, -6, and -7.

AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA.

Host- and pathogen-associated cytoplasmic double-stranded DNA triggers the activation of a NALP3 (also known as cryopyrin and NLRP3)-independent inflammasome, which activates caspase-1 leading to maturation of pro-interleukin-1β and inflammation. The nature of the cytoplasmic-DNA-sensing inflammasome is currently unknown. Here we show that AIM2 (absent in melanoma 2), an interferon-inducible HIN-200 family member that contains an amino-terminal pyrin domain and a carboxy-terminal oligonucleotide/oligosaccharide-binding domain, senses cytoplasmic DNA by means of its oligonucleotide/oligosaccharide-binding domain and interacts with ASC (apoptosis-associated speck-like protein containing a CARD) through its pyrin domain to activate caspase-1. The interaction of AIM2 with ASC also leads to the formation of the ASC pyroptosome, which induces pyroptotic cell death in cells containing caspase-1. Knockdown of AIM2 by short interfering RNA reduced inflammasome/pyroptosome activation by cytoplasmic DNA in human and mouse macrophages, whereas stable expression of AIM2 in the non-responsive human embryonic kidney 293T cell line conferred responsiveness to cytoplasmic DNA. Our results show that cytoplasmic DNA triggers formation of the AIM2 inflammasome by inducing AIM2 oligomerization. This study identifies AIM2 as an important inflammasome component that senses potentially dangerous cytoplasmic DNA, leading to activation of the ASC pyroptosome and caspase-1.

Cutting edge: NF-kappaB activating pattern recognition and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression

The IL-1 family cytokines are regulated on transcriptional and posttranscriptional levels. Pattern recognition and cytokine receptors control pro-IL-1β transcription whereas inflammasomes regulate the proteolytic processing of pro-IL-1β. The NLRP3 inflammasome, however, assembles in response to extracellular ATP, pore-forming toxins, or crystals only in the presence of proinflammatory stimuli. How the activation of gene transcription by signaling receptors enables NLRP3 activation remains elusive and controversial. In this study, we show that cell priming through multiple signaling receptors induces NLRP3 expression, which we identified to be a critical checkpoint for NLRP3 activation. Signals provided by NF-κB activators are necessary but not sufficient for NLRP3 activation, and a second stimulus such as ATP or crystal-induced damage is required for NLRP3 activation.

A conserved XIAP-interaction motif in caspase-9 and Smac/DIABLO regulates caspase activity and apoptosis.

X-linked inhibitor-of-apoptosis protein (XIAP) interacts with caspase-9 and inhibits its activity, whereas Smac (also known as DIABLO) relieves this inhibition through interaction with XIAP. Here we show that XIAP associates with the active caspase-9–Apaf-1 holoenzyme complex through binding to the amino terminus of the linker peptide on the small subunit of caspase-9, which becomes exposed after proteolytic processing of procaspase-9 at Asp 315. Supporting this observation, point mutations that abrogate the proteolytic processing but not the catalytic activity of caspase-9, or deletion of the linker peptide, prevented caspase-9 association with XIAP and its concomitant inhibition. We note that the N-terminal four residues of caspase-9 linker peptide share significant homology with the N-terminal tetra-peptide in mature Smac and in the Drosophila proteins Hid/Grim/Reaper, defining a conserved class of IAP-binding motifs. Consistent with this finding, binding of the caspase-9 linker peptide and Smac to the BIR3 domain of XIAP is mutually exclusive, suggesting that Smac potentiates caspase-9 activity by disrupting the interaction of the linker peptide of caspase-9 with BIR3. Our studies reveal a mechanism in which binding to the BIR3 domain by two conserved peptides, one from Smac and the other one from caspase-9, has opposing effects on caspase activity and apoptosis.

Negative regulation of the Apaf-1 apoptosome by Hsp70.

Release of cytochrome c from mitochondria by apoptotic signals induces ATP/dATP-dependent formation of the oligomeric Apaf-1–caspase-9 apoptosome. Here we show that the documented anti-apoptotic effect of the principal heat-shock protein, Hsp70, is mediated through its direct association with the caspase-recruitment domain (CARD) of Apaf-1 and through inhibition of apoptosome formation. The interaction between Hsp70 and Apaf-1 prevents oligomerization of Apaf-1 and association of Apaf-1 with procaspase-9. On the basis of these results, we propose that resistance to apoptosis exhibited by stressed cells and some tumours, which constitutively express high levels of Hsp70, may be due in part to modulation of Apaf-1 function by Hsp70.

Identification and molecular cloning of two novel receptors for the cytotoxic ligand TRAIL.

A human receptor for the cytotoxic ligand TRAIL (TRAIL receptor-1, designated DR4) was identified recently as a member of the tumor necrosis factor receptor family. In this report we describe the identification of two additional human TRAIL receptors, TRAIL receptor-2 and TRAIL receptor-3, that belong to the tumor necrosis factor receptor family. Interestingly, TRAIL receptor-2 but not TRAIL receptor-3 contains a cytoplasmic “death domain” necessary for induction of apoptosis and is hence designated death receptor-5 (DR5). Like DR4, DR5 engages the apoptotic pathway independent of the adaptor molecule FADD/MORT1. Because of its lack of a death domain, TRAIL receptor-3 is not capable of inducing apoptosis. However, by competing for TRAIL, it is capable of inhibiting TRAIL-induced apoptosis. Thus, TRAIL receptor-3 may function as an antagonistic decoy receptor to attenuate the cytotoxic effect of TRAIL in most tissues that are TRAIL+, DR4+, and DR5+.