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

Intracellular lipopolysaccharide from Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, Shigella flexneri, and Burkholderia thailandensis activates mouse caspase-11, causing pyroptotic cell death, interleukin-1β processing, and lethal septic shock. How caspase-11 executes these downstream signalling events is largely unknown. Here we show that gasdermin D is essential for caspase-11-dependent pyroptosis and interleukin-1β maturation. A forward genetic screen with ethyl-N-nitrosourea-mutagenized mice links Gsdmd to the intracellular lipopolysaccharide response. Macrophages from Gsdmd(-/-) mice generated by gene targeting also exhibit defective pyroptosis and interleukin-1β secretion induced by cytoplasmic lipopolysaccharide or Gram-negative bacteria. In addition, Gsdmd(-/-) mice are protected from a lethal dose of lipopolysaccharide. Mechanistically, caspase-11 cleaves gasdermin D, and the resulting amino-terminal fragment promotes both pyroptosis and NLRP3-dependent activation of caspase-1 in a cell-intrinsic manner. Our data identify gasdermin D as a critical target of caspase-11 and a key mediator of the host response against Gram-negative bacteria.

Apolipoprotein E is immunochemically localized to the senile plaques, vascular amyloid, and neuroflbrillary tangles of Alzheimer diwase. In

Caspases are intracellular proteases that function as initiators and effectors of apoptosis. The kinase Akt and p21-Ras, an Akt activator, induced phosphorylation of pro-caspase-9 (pro-Casp9) in cells. Cytochrome c-induced proteolytic processing of pro-Casp9 was defective in cytosolic extracts from cells expressing either active Ras or Akt. Akt phosphorylated recombinant Casp9 in vitro on serine-196 and inhibited its protease activity. Mutant pro-Casp9(Ser196Ala) was resistant to Akt-mediated phosphorylation and inhibition in vitro and in cells, resulting in Akt-resistant induction of apoptosis. Thus, caspases can be directly regulated by protein phosphorylation.

Although the mechanism of mammalian apoptosis has not been elucidated, a protease of the CED-3/ICE family is anticipated to be a component of the death machinery. Several lines of evidence predict that this protease cleaves the death substrate poly(ADP-ribose) polymerase (PARP) to a specific 85 kDa form observed during apoptosis, is inhibitable by the CrmA protein, and is distinct from ICE. We cloned a ced-3/ICE-related gene, designated Yama, that encodes a protein identical to CPP32 beta. Purified Yama was a zymogen that, when activated, cleaved PARP to generate the 85 kDa apoptotic fragment. Cleavage of PARP by Yama was inhibited by CrmA but not by an inactive point mutant of CrmA. Furthermore, CrmA blocked cleavage of PARP in cells undergoing apoptosis. We propose that Yama may represent an effector component of the mammalian cell death pathway and suggest that CrmA blocks apoptosis by inhibiting Yama.

The 'inhibitor of apoptosis' (IAP) gene family, which was discovered less than a decade ago, encodes a group of structurally related proteins that, in addition to their ability to suppress apoptotic cell death, are involved in an increasing number of seemingly unrelated cellular functions. Here, we review the functional and structural properties of this fascinating group of proteins, and of several recently identified IAP-binding factors that regulate IAP function.

endopeptidases. Caspase 1 is remarkably specific for Genentech Inc.the precursors of IL-1␤ and IL-18 (interferon-␥-inducing South San Francisco, California 94080 factor), making a single initial cut in each procytokine that activates them and allows exit from the cytosol. Ectopic expression of caspase 1 in some cells can result The discovery of a novel proteolytic network in the cytoin apoptosis, but a role in developmentally programmed sol of metazoan cells comes as a surprise to a field that cell death is unlikely given the normal phenotype of previously considered intracellular proteolysis to mainly knockout mice (see for example, Ghayur et al., 1997). be a mechanism to process antigen or remove effete Processing of the caspase 1 precursor, required for actiproteins. The network, supported by caspases, a family vation, is rarely seen in experimental models of apopof cysteine proteases that specifically cleave proteins tosis, and thus, though it is clearly required for activation after Asp residues, involves limited proteolysis of a of the cytokines mentioned above, the role of this casgrowing number of cellular substrates. It is not surprising pase in apoptosis, if any, is still uncertain. The close that a signaling pathway utilizes proteolysis, for this is relationship in sequence identity and predicted subthe essence of the blood coagulation cascade. What is strate specificity of caspases 4 and 5 implicate them as surprising is that the caspase pathway takes place in effectors of cytokine activation, possibly as upstream the interior of a cell. Caspase precursors are usually activators of caspase 1 itself. activated at internal conserved Asp residues, with the Whereas caspase 1 (and possibly 4 and 5) is primarily result that most activated caspases can process their involved in procytokine activation, other caspases, notaown and other caspase zymogens given sufficient time bly 2, 3, 6, 7, 8, and 10, are considered to promote and a high enough concentration in vitro (see for exampathways to apoptosis. This conclusion is based largely ple, Srinivasula et al., 1996). This suggests a cascade on the following observations: (1) the zymogens are seen mechanism for transmission of signals, but the extent to be processed during apoptosis, or in vitro models of to which this happens in vivo is currently an enigma, apoptosis, and (2) at least in vitro, they cut proteins and a fertile area of research.whose cleavage is associated with apoptotic cell death. The caspases seem to be a development of multicellu-So far, about a dozen proteins have been shown to lar organisms, and in humans at least seven of the ten be specifically cleaved during apoptosis. It is relatively currently known family members participate in one of simple to demonstrate which clips are caused by castwo distinct signaling pathways: (1) activation of proinpases (it turns out to be almost all of them) through flammatory cytokines, and (2) promotion of apoptotic analysis of the cleavage site, but more difficult to detercell death. Recent publications h...

The inhibitor-of-apoptosis (IAP) family of genes has an evolutionarily conserved role in regulating programmed cell death in animals ranging from insects to humans. 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 apoptosis, our findings suggest how IAPs might inhibit cell death, providing evidence for a mechanism of action for these mammalian cell-death suppressors.