Initially discovered as an initiator protease in apoptosis mediated by death receptors, caspase-8 is now known to have an apparently confounding opposing effect in securing cell survival. It is required to allow mouse embryo survival, and the survival of hematopoietic cells during their development and activation. Classic models in which caspase-8 is depleted or inhibited frequently result in inhibition of apoptosis, and conversion to death through a necrotic pathway. This bewildering switch is now known to be driven by activation of a pathway dependent on protein kinases of the RIP family, which engage a pathway known as necroptosis. If caspase-8 does not control this pathway, necrotic death results. The pro-apoptotic and pro-survival functions of caspase-8 are regulated by a specific interaction with the pseudo-caspase cFLIP, and it is thought that the heterocomplex between these two partners alters the substrate specificity of caspase-8 in favor of inactivating components of the RIP kinase pathway. The description of how caspase-8 and cFLIP coordinate the switch between apoptosis and survival is just beginning. The mechanism is not known, the differential targets are not known, and the reason of why an apoptotic initiator has been co-opted as a critical survival factor is only guessed at. Elucidating these unknowns will be important in understanding mechanisms and possible therapeutic targets in autoimmune, inflammatory, and metastatic diseases.

Initially discovered as an initiator protease in apoptosis mediated by death receptors, caspase-8 is now known to have an apparently confounding opposing effect in securing cell survival. It is required to allow mouse embryo survival, and the survival of hematopoietic cells during their development and activation. Classic models in which caspase-8 is depleted or inhibited frequently result in inhibition of apoptosis, and conversion to death through a necrotic pathway. This bewildering switch is now known to be driven by activation of a pathway dependent on protein kinases of the RIP family, which engage a pathway known as necroptosis. If caspase-8 does not control this pathway, necrotic death results. The pro-apoptotic and pro-survival functions of caspase-8 are regulated by a specific interaction with the pseudo-caspase cFLIP, and it is thought that the heterocomplex between these two partners alters the substrate specificity of caspase-8 in favor of inactivating components of the RIP kinase pathway. The description of how caspase-8 and cFLIP coordinate the switch between apoptosis and survival is just beginning. The mechanism is not known, the differential targets are not known, and the reason of why an apoptotic initiator has been co-opted as a critical survival factor is only guessed at. Elucidating these unknowns will be important in understanding mechanisms and possible therapeutic targets in autoimmune, inflammatory, and metastatic diseases. © 2014 Elsevier Ltd. All rights reserved.

Recently it has been reported that caspase-3 activation occurs in stimulated T-lymphocytes without associated apoptosis (Miossec, C., Dutilleul, V., Fassy, F., and Diu-Hercend, A. (1997) J. Biol. Chem. 272, 13459-13462). To explore this phenomenon, human peripheral blood lymphocytes (PBLs) were stimulated with mitogenic lectins or anti-CD3 antibody, and the proteolytic processing of different caspases and caspase substrates was analyzed by immunoblotting. Proteolytic processing of caspases-3 and -7 and the caspase substrates poly(ADP-ribose) polymerase, GDP dissociation inhibitor, and PKCdelta was observed when PBLs were activated in vitro, and lysates were prepared using RIPA buffer which contains 1% Nonidet P-40, 0.5% deoxycholate, and 0.1% SDS. In contrast, when a lysis buffer containing 2% SDS was used, the caspases remained in their zymogen pro-forms, and no proteolytic processing of caspase substrates was detected. Moreover, in experiments using intact cells and a cell-permeable fluorigenic caspase substrate, no caspase activity was observed in activated T-cells, whereas it was clearly detected when PBLs were treated with the apoptosis-inducing anticancer drug etoposide. Since the granzyme B is a direct activator of caspase-3 and its expression is induced following T-cell activation, we tested the effects of anti-GraB, an engineered serpin that specifically inhibits GraB. When the activated T-lymphocytes were lysed in RIPA buffer containing anti-GraB, no proteolytic processing or activation of caspase-3 was observed, strongly suggesting that release of GraB or similar proteases from their storage sites in cytotoxic granules during the lysis procedure is responsible for caspase activation. These findings demonstrate that T-cells do not process caspases upon activation and caution about the method of cell lysis used when studying granzyme-expressing cells.

The inhibitor-of-apoptosis (IAP) family of genes has an evolutionarily conserved role in regulating programmed cell death in animals ranging from insects to humans1,2,3,4,5,6. Ectopic expression of human IAP proteins can suppress cell death induced by a variety of stimuli, but the mechanism of this inhibition was previously unknown. Here we show that human X-chromosome-linked IAP directly inhibits at least two members of the caspase family of cell-death proteases, caspase-3 and caspase-7. As the caspases are highly conserved throughout the animal kingdom and are the principal effectors of apoptosis7, our findings suggest how IAPs might inhibit cell death, providing evidence for a mechanism of action for these mammalian cell-death suppressors.

The inhibitor of apoptosis (IAP) family of proteins are highly conserved through evolution. However, the mechanisms by which these proteins interfere with apoptotic cell death have been enigmatic. Recently, we showed that one of the human IAP family proteins, XIAP, can bind to and potently inhibit specific cell death proteases (caspases) that function in the distal portions of the proteolytic cascades involved in apoptosis. In this study, we investigated three of the other known members of the human IAP family, c-IAP-1, c-IAP-2 and NAIP. Similarly to XIAP, in vitro binding experiments indicated that c-IAP-1 and c-IAP-2 bound specifically to the terminal effector cell death proteases, caspases-3 and -7, but not to the proximal protease caspase-8, caspases-1 or -6. In contrast, NAIP failed to bind tightly to any of these proteases. Recombinant c-IAP-1 and c-IAP-2 also inhibited the activity of caspases-3 and -7 in vitro, with estimated Kis of <=0.1 microM, whereas NAIP did not. The BIR domain-containing region of c-IAP-1 and c-IAP-2 was sufficient for inhibition of these caspases, though proteins that retained the RING domain were somewhat more potent. Utilizing a cell-free system in which caspases were activated in cytosolic extracts by addition of cytochrome c, c-IAP-1 and c-IAP-2 inhibited both the generation of caspase activities and proteolytic processing of pro-caspase-3. Similar results were obtained in intact cells when c-IAP-1 and c-IAP-2 were overexpressed by gene transfection, and apoptosis was induced by the anticancer drug, etoposide. Cleavage of c-IAP-1 or c-IAP-2 was not observed when interacting with the caspases, implying a different mechanism from the baculovirus p35 protein, the broad spectrum suicide inactivator of caspases. Taken together, these findings suggest that c-IAP-1 and c-IAP-2 function similarly to XIAP by inhibiting the distal cell death proteases, caspases-3 and -7, whereas NAIP presumably inhibits apoptosis via other targets.

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.

Proteases are fundamental to successful parasitism, including that of the schistosome flatworm parasite, which causes the disease schistosomiasis in 200 million people worldwide. The proteasome is receiving attention as a potential drug target for treatment of a variety of infectious parasitic diseases, but it has been understudied in the schistosome. Adult Schistosoma mansoni were incubated with 1 μM concentrations of the proteasome inhibitors bortezomib, carfilzomib, and MG132. After 24 h, bortezomib and carfilzomib decreased worm motility by more than 85% and endogenous proteasome activity by >75%, and after 72 h, they increased caspase activity by >4.5-fold. The association between the engagement of the proteasome target and the phenotypic and biochemical effects recorded encouraged the chromatographic enrichment of the S. mansoni proteasome (Sm20S). Activity assays with fluorogenic proteasome substrates revealed that Sm20S contains caspase-type (β1), trypsin-type (β2), and chymotrypsin-type (β5) activities. Sm20S was screened with 11 peptide epoxyketone inhibitors derived from the marine natural product carmaphycin B. Analogue 17 was 27.4-fold less cytotoxic to HepG2 cells than carmaphycin B and showed equal potency for the β5 subunits of Sm20S, human constitutive proteasome, and human immunoproteasome. However, this analogue was 13.2-fold more potent at targeting Sm20S β2 than it was at targeting the equivalent subunits of the human enzymes. Furthermore, 1 μM 17 decreased both worm motility and endogenous Sm20S activity by more than 90% after 24 h. We provide direct evidence of the proteasome's importance to schistosome viability and identify a lead for which future studies will aim to improve the potency, selectivity, and safety.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.