Inhibitor of Apoptosis (IAP) proteins as drug targets for the treatment of cancer

Three companies, Genentech, Aegera Therapeutics/Human Genome Sciences, and Novartis, have commenced phase 1 clinical trials of inhibitor of apoptosis (IAP) antagonist ‘Smac mimetic’ compounds for the treatment of cancer. These trials represent the culmination of a line of research that commenced with analysis of how insect viruses stop host cells from killing themselves and led to the discovery of a family of proteins that regulate development in insects and signalling by tumour necrosis factor superfamily members in mammals, which prompted development of drugs that mimic natural IAP-binding proteins to promote cell death

Error bars in experimental biology

Error bars commonly appear in figures in publications, but experimental biologists are often unsure how they should be used and interpreted. In this article we illustrate some basic features of error bars and explain how they can help communicate data and assist correct interpretation. Error bars may show confidence intervals, standard errors, standard deviations, or other quantities. Different types of error bars give quite different information, and so figure legends must make clear what error bars represent. We suggest eight simple rules to assist with effective use and interpretation of error bars

Inhibitor of apoptosis proteins and their relatives: IAPs and other BIRPs

Apoptosis is a physiological cell death process important for development, homeostasis and the immune defence of multicellular animals. The key effectors of apoptosis are caspases, cysteine proteases that cleave after aspartate residues. The inhibitor of apoptosis (IAP) family of proteins prevent cell death by binding to and inhibiting active caspases and are negatively regulated by IAP-binding proteins, such as the mammalian protein DIABLO/Smac. IAPs are characterized by the presence of one to three domains known as baculoviral IAP repeat (BIR) domains and many also have a RING-finger domain at their carboxyl terminus. More recently, a second group of BIR-domain-containing proteins (BIRPs) have been identified that includes the mammalian proteins Bruce and Survivin as well as BIR-containing proteins in yeasts and Caenorhabditis elegans. These Survivin-like BIRPs regulate cytokinesis and mitotic spindle formation. In this review, we describe the IAPs and other BIRPs, their evolutionary relationships and their subcellular and tissue localizations

A novel Apaf-1–independent putative caspase-2 activation complex

CVaspase activation is a key event in apoptosis execution. In stress-induced apoptosis, the mitochondrial pathway of caspase activation is believed to be of central importance. In this pathway, cytochrome c released from mitochondria facilitates the formation of an Apaf-1 apoptosome that recruits and activates caspase-9. Recent data indicate that in some cells caspase-9 may not be the initiator caspase in stress-mediated apoptosis because caspase-2 is required upstream of mitochondria for the release of cytochrome c and other apoptogenic factors. To determine how caspase-2 is activated, we have studied the formation of a complex that mediates caspase-2 activation. Using gel filtration analysis of cell lysates, we show that caspase-2 is spontaneously recruited to a large protein complex independent of cytochrome c and Apaf-1 and that recruitment of caspase-2 to this complex is sufficient to mediate its activation. Using substrate-binding assays, we also provide the first evidence that caspase-2 activation may occur without processing of the precursor molecule. Our data are consistent with a model where caspase-2 activation occurs by oligomerization, independent of the Apaf-1 apoptosome

Bcl-2–regulated apoptosis and cytochrome c release can occur independently of both caspase-2 and caspase-9

Apoptosis in response to developmental cues and stress stimuli is mediated by caspases that are regulated by the Bcl-2 protein family. Although caspases 2 and 9 have each been proposed as the apical caspase in that pathway, neither is indispensable for the apoptosis of leukocytes or fibroblasts. To investigate whether these caspases share a redundant role in apoptosis initiation, we generated caspase-2−/−9−/− mice. Their overt phenotype, embryonic brain malformation and perinatal lethality mirrored that of caspase-9−/− mice but were not exacerbated. Analysis of adult mice reconstituted with caspase-2−/−9−/− hematopoietic cells revealed that the absence of both caspases did not influence hematopoietic development. Furthermore, lymphocytes and fibroblasts lacking both remained sensitive to diverse apoptotic stimuli. Dying caspase-2−/−9−/− lymphocytes displayed multiple hallmarks of caspase-dependent apoptosis, including the release of cytochrome c from mitochondria, and their demise was antagonized by several caspase inhibitors. These findings suggest that caspases other than caspases 2 and 9 can promote cytochrome c release and initiate Bcl-2–regulated apoptosis

Vaccinia virus utilizes microtubules for movement to the cell surface

Vaccinia virus (VV) egress has been studied using confocal, video, and electron microscopy. Previously, intracellular-enveloped virus (IEV) particles were proposed to induce the polymerization of actin tails, which propel IEV particles to the cell surface. However, data presented support an alternative model in which microtubules transport virions to the cell surface and actin tails form beneath cell-associated enveloped virus (CEV) particles at the cell surface. Thus, VV is unique in using both microtubules and actin filaments for egress. The following data support this proposal. (a) Microscopy detected actin tails at the surface but not the center of cells. (b) VV mutants lacking the A33R, A34R, or A36R proteins are unable to induce actin tail formation but produce CEV and extracellular-enveloped virus. (c) CEV formation is inhibited by nocodazole but not cytochalasin D or 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine (PP1). (d) IEV particles tagged with the enhanced green fluorescent protein fused to the VV B5R protein moved inside cells at 60 μm/min. This movement was stop-start, was along defined pathways, and was inhibited reversibly by nocodazole. This velocity was 20-fold greater than VV movement on actin tails and consonant with the rate of movement of organelles along microtubules

The anti-apoptotic activity of XIAP is retained upon mutation of both the caspase 3– and caspase 9–interacting sites

The X-linked mammalian inhibitor of apoptosis protein (XIAP) has been shown to bind several partners. These partners include caspase 3, caspase 9, DIABLO/Smac, HtrA2/Omi, TAB1, the bone morphogenetic protein receptor, and a presumptive E2 ubiquitin-conjugating enzyme. In addition, we show here that XIAP can bind to itself. To determine which of these interactions are required for it to inhibit apoptosis, we generated point mutant XIAP proteins and correlated their ability to bind other proteins with their ability to inhibit apoptosis. ∂RING point mutants of XIAP were as competent as their full-length counterparts in inhibiting apoptosis, although impaired in their ability to oligomerize with full-length XIAP. Triple point mutants, unable to bind caspase 9, caspase 3, and DIABLO/HtrA2/Omi, were completely ineffectual in inhibiting apoptosis. However, point mutants that had lost the ability to inhibit caspase 9 and caspase 3 but retained the ability to inhibit DIABLO were still able to inhibit apoptosis, demonstrating that IAP antagonism is required for apoptosis to proceed following UV irradiation

Apaf-1 and caspase-9 accelerate apoptosis, but do not determine whether factor-deprived or drug-treated cells die

Apoptosis after growth factor withdrawal or drug treatment is associated with mitochondrial cytochrome c release and activation of Apaf-1 and caspase-9. To determine whether loss of Apaf-1, caspase-2, and caspase-9 prevented death of factor-starved cells, allowing them to proliferate when growth factor was returned, we generated IL-3–dependent myeloid lines from gene-deleted mice. Long after growth factor removal, cells lacking Apaf-1, caspase-9 or both caspase-9 and caspase-2 appeared healthy, retained intact plasma membranes, and did not expose phosphatidylserine. However, release of cytochrome c still occurred, and they failed to form clones when IL-3 was restored. Cells lacking caspase-2 alone had no survival advantage. Therefore, Apaf-1, caspase-2, and caspase-9 are not required for programmed cell death of factor-dependent cells, but merely affect its rate. In contrast, transfection with Bcl-2 provided long-term, clonogenic protection, and could act independently of the apoptosome. Unlike expression of Bcl-2, loss of Apaf-1, caspase-2, or caspase-9 would therefore be unlikely to enhance the survival of cancer cells

TWEAK-FN14 signaling induces lysosomal degradation of a cIAP1–TRAF2 complex to sensitize tumor cells to TNFα

Synthetic inhibitor of apoptosis (IAP) antagonists induce degradation of IAP proteins such as cellular IAP1 (cIAP1), activate nuclear factor κB (NF-κB) signaling, and sensitize cells to tumor necrosis factor α (TNFα). The physiological relevance of these discoveries to cIAP1 function remains undetermined. We show that upon ligand binding, the TNF superfamily receptor FN14 recruits a cIAP1–Tnf receptor-associated factor 2 (TRAF2) complex. Unlike IAP antagonists that cause rapid proteasomal degradation of cIAP1, signaling by FN14 promotes the lysosomal degradation of cIAP1–TRAF2 in a cIAP1-dependent manner. TNF-like weak inducer of apoptosis (TWEAK)/FN14 signaling nevertheless promotes the same noncanonical NF-κB signaling elicited by IAP antagonists and, in sensitive cells, the same autocrine TNFα-induced death occurs. TWEAK-induced loss of the cIAP1–TRAF2 complex sensitizes immortalized and minimally passaged tumor cells to TNFα-induced death, whereas primary cells remain resistant. Conversely, cIAP1–TRAF2 complex overexpression limits FN14 signaling and protects tumor cells from TWEAK-induced TNFα sensitization. Lysosomal degradation of cIAP1–TRAF2 by TWEAK/FN14 therefore critically alters the balance of life/death signals emanating from TNF-R1 in immortalized cells