Sebastian Wesselborg

A Caspase-Related Protease Regulates Apoptosis in Yeast.

Yeast can undergo cell death accompanied by cellular markers of apoptosis. However, orthologs of classical mammalian apoptosis regulators appeared to be missing from the yeast genome, challenging a common mechanism of yeast and mammalian apoptosis. Here we investigate Yor197w, a yeast protein with structural homology to mammalian caspases, and demonstrate caspase-like processing of the protein. Hydrogen peroxide treatment induces apoptosis together with a caspase-like enzymatic activity in yeast. This response is completely abrogated after disruption and strongly stimulated after overexpression of Yor197w. Yor197w also mediates the death process within chronologically aged cultures, pointing to a physiological role in elimination of overaged cells. We conclude that Yor197w indeed functions as a bona fide caspase in yeast and propose the name Yeast Caspase-1 (YCA1, gene YCA1).

Role of AMPK-mTOR-Ulk1/2 in the Regulation of Autophagy: Cross Talk, Shortcuts, and Feedbacks

ABSTRACT Living cells are adaptive self-sustaining systems. They strictly depend on the sufficient supply of oxygen, energy, and nutrients from the outside in order to sustain their internal organization. However, as autonomous entities they are able to monitor and appropriately adapt to any critical fluctuation in their environment. In the case of insufficient external nutrient supply or augmented energy demands, cells start to extensively digest their own interior. This process, known as macroautophagy, comprises the transport of cytosolic portions and entire organelles to the lysosomal compartment via specific double-membrane vesicles, called autophagosomes. Although extensively upregulated under nutrient restriction, a low level of basal autophagy is likewise crucial in order to sustain the cellular homeostasis. On the other hand, cells have to avoid excessive and enduring self-digestion. The delicate balance between external energy and nutrient supply and internal production and consumption is a demanding task. The complex protein network that senses and precisely reacts to environmental changes is thus mainly regulated by rapid and reversible posttranslational modifications such as phosphorylation. This review focuses on the serine/threonine protein kinases AMP-activated protein kinase, mammalian target of rapamycin (mTOR), and unc-51-like kinase 1/2 (Ulk1/2), three interconnected major junctions within the autophagy regulating signaling network.

The role of caspases in development, immunity, and apoptotic signal transduction: lessons from knockout mice.

The rapid discovery of a great number of caspases, together with multiple control points of their activation, proceeds well ahead of our knowledge of their physiological roles within the organism. There are still major gaps, but recent gene targeting of caspases provides us with several new and fundamental aspects of their physiological functions. The fact that different lines of KO mice exhibit preferential apoptosis defects rather than a global suppression of apoptosis indicates that caspases play a largely nonredundant apoptotic role in a tissue- and stimulus-dependent manner. Furthermore, despite compelling evidence for a key role of Casp8 and Casp9, the restricted phenotype of both Casp8- and Casp9-deficient mice suggests that other apical caspases must exist regulating apoptotic processes. Casp10, for instance, which is very similar in its structure to Casp8, has been shown to be recruited to death receptors (13xIn vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-like domains. Fernandes-Alnemri, T, Armstrong, R.C, Krebs, J, Srinivasula, S.M, Wang, L, Bullrich, F, Fritz, L.C, Trapani, J.A, Tomaselli, K.J, Litwack, G, and Alnemri, E.S. Proc. Natl. Acad. Sci. USA. 1996; 93: 7464–7469Crossref | PubMed | Scopus (653)See all References, 72xFas-associated death domain protein interleukin-1beta-converting enzyme 2 (FLICE2), an ICE/Ced-3 homologue, is proximally involved in CD95- and p55-mediated death signaling. Vincenz, C and Dixit, V.M. J. Biol. Chem. 1997; 272: 6578–6583Crossref | PubMed | Scopus (239)See all References). Although fibroblasts from Casp8 null mice are almost completely resistant to death receptor–mediated apoptosis, it cannot be excluded that Casp10, having little importance in fibroblasts, exerts crucial functions in other cell types. In addition, since certain cell types such as embryonic fibroblasts from Apaf1 KO mice are still considerably sensitive to a variety of apoptosis inducers, it is very likely that other yet undiscovered key regulators exist. We also have to be aware that the restricted phenotype of most KO mice may underestimate the role of the targeted caspases, because single caspases may substitute other family members. A major obstacle of most KO mice is their prenatal lethality, which precludes manifestations of caspase functions in the adult organism. Therefore, in future research, conditional disruption in a cell type–specific manner or in certain developmental stages will be required to elucidate more precisely the in vivo functional significance of individual caspases in development, immune functions, and pathological forms of apoptosis.*To whom correspondence should be addressed (e-mail: kso@uni-muenster.de).†Present address: Division of Cell Biology, University of Munster, Rontgenweg 21, D-48149 Munster, Germany.

Human mature red blood cells express caspase-3 and caspase-8, but are devoid of mitochondrial regulators of apoptosis.

Although proteases of the caspase family are essential mediators of apoptosis in nucleated cells, in anucleate cells their presence and potential functions are almost completely unknown. Human erythrocytes are a major cell population that does not contain a cell nucleus or other organelles. However, during senescence they undergo certain morphological alterations resembling apoptosis. In the present study, we found that mature erythrocytes contain considerable amounts of caspase-3 and -8, whereas essential components of the mitochondrial apoptotic cascade such as caspase-9, Apaf-1 and cytochrome c were missing. Strikingly, although caspases of erythrocytes were functionally active in vitro, they failed to become activated in intact erythrocytes either during prolonged storage or in response to various proapoptotic stimuli. Following an increase of cytosolic calcium, instead the cysteine protease calpain but not caspases became activated and mediated fodrin cleavage and other morphological alterations such as cell shrinkage. Our results therefore suggest that erythrocytes do not have a functional death system. In addition, because of the presence of procaspases and the absence of a cell nucleus and mitochondria erythrocytes may be an attractive system to dissect the role of certain apoptosis-regulatory pathways. Cell Death and Differentiation (2001) 8, 1197–1206

P2Z purinoreceptor ligation induces activation of caspases with distinct roles in apoptotic and necrotic alterations of cell death

Myeloic cells express a peculiar surface receptor for extracellular ATP, called the P2Z/P2X7 purinoreceptor, which is involved in cell death signalling. Here, we investigated the role of caspases, a family of proteases implicated in apoptosis and the cytokine secretion. We observed that extracellular ATP induced the activation of multiple caspases including caspase‐1, ‐3 and ‐8, and subsequent cleavage of the caspase substrates PARP and lamin B. Using caspase inhibitors, it was found that caspases were specifically involved in ATP‐induced apoptotic damage such as chromatin condensation and DNA fragmentation. In contrast, inhibition of caspases only marginally affected necrotic alterations and cell death proceeded normally whether or not nuclear damage was blocked. Our results therefore suggest that the activation of caspases by the P2Z receptor is required for apoptotic but not necrotic alterations of ATP‐induced cell death.

Cancer stem cell markers in common cancers – therapeutic implications

Rapid advances in the cancer stem cell (CSC) field have provided cause for optimism for the development of more reliable cancer therapies in the future. Strategies aimed at efficient targeting of CSCs are becoming important for monitoring the progress of cancer therapy and for evaluating new therapeutic approaches. Here, we characterize and compare the specific markers that have been found to be present on stem cells, cancer cells and CSCs in selected tissues (colon, breast, liver, pancreas and prostate). We then discuss future directions of this intriguing new research field in the context of new diagnostic and therapeutic opportunities.

Caspase-8/FLICE functions as an executioner caspase in anticancer drug-induced apoptosis

Caspase-8 plays an essential role in apoptosis triggered by death receptors. Through the cleavage of Bid, a proapoptotic Bcl-2 member, it further activates the mitochondrial cytochrome c/Apaf-1 pathway. Because caspase-8 can be processed also by anticancer drugs independently of death receptors, we investigated its exact role and order in the caspase cascade. We show that in Jurkat cells either deficient for caspase-8 or overexpressing its inhibitor c-FLIP apoptosis mediated by CD95, but not by anticancer drugs was inhibited. In the absence of active caspase-8, anticancer drugs still induced the processing of caspase-9, -3 and Bid, indicating that Bid cleavage does not require caspase-8. Overexpression of Bcl-xL prevented the processing of caspase-8 as well as caspase-9, -6 and Bid in response to drugs, but was less effective in CD95-induced apoptosis. Similar responses were observed by overexpression of a dominant-negative caspase-9 mutant. To further determine the order of caspase-8 activation, we employed MCF7 cells lacking caspase-3. In contrast to caspase-9 that was cleaved in these cells, anticancer drugs induced caspase-8 activation only in caspase-3 transfected MCF7 cells. Thus, our data indicate that, unlike its proximal role in receptor signaling, in the mitochondrial pathway caspase-8 rather functions as an amplifying executioner caspase.

Regulation of NF-κB Activation by MAP Kinase Cascades

Transcription factor NF-kappa B plays a crucial role in the regulation of numerous genes involved in the inflammatory response and control of cell death. Activation of NF-kappa B is mediated through the phosphorylation of its inhibitory subunit I kappa B, followed by the subsequent degradation of I kappa B at the proteasome. A second level of control involves phosphorylation events of NF-kappa B in the cell nucleus. The kinases that regulate these processes are rather undefined. NF-kappa B activation is induced by a great variety of predominantly pathogenic and noxious stimuli. A similar spectrum of conditions triggers the activation of two mitogen-activated protein (MAP) kinase cascades, designated as the JNK and p38 kinase pathways. Several points of evidence suggest that MAP kinases can participate in the regulation of NF-kappa B transcriptional activity. Here, we will review very recent data demonstrating that both the JNK and the p38 pathways are involved in the activation of NF-kappa B in the cytoplasm as well as in modulation of its transactivating potential in the nucleus.

S100A8/A9 at low concentration promotes tumor cell growth via RAGE ligation and MAP kinase-dependent pathway

The complex formed by two members of the S100 calcium‐binding protein family, S100A8/A9, exerts apoptosis‐inducing activity against various cells, especially tumor cells. Here, we present evidence that S100A8/A9 also has cell growth‐promoting activity at low concentrations. Receptor of advanced glycation end product (RAGE) gene silencing and cotreatment with a RAGE‐specific blocking antibody revealed that this activity was mediated via RAGE ligation. To investigate the signaling pathways, MAPK phosphorylation and NF‐κB activation were characterized in S100A8/A9‐treated cells. S100A8/A9 caused a significant increase in p38 MAPK and p44/42 kinase phosphorylation, and the status of stress‐activated protein kinase/JNK phosphorylation remained unchanged. Treatment of cells with S100A8/A9 also enhanced NF‐κB activation. RAGE small interfering RNA pretreatment abrogated the S100A8/A9‐induced NF‐κB activation. Our data indicate that S100A8/A9‐promoted cell growth occurs through RAGE signaling and activation of NF‐κB.

Migration to apoptotic "find-me" signals is mediated via the phagocyte receptor G2A

Phagocytosis of apoptotic cells is fundamentally important throughout life, because non-cleared cells become secondarily necrotic and release intracellular contents, thus instigating inflammatory and autoimmune responses. Secreted “find-me” and exposed “eat-me” signals displayed by the dying cell in concert with the phagocyte receptors comprise the phagocytic synapse of apoptotic cell clearance. In this scenario, lysophospholipids (lysoPLs) are assumed to act as find-me signals for the attraction of phagocytes. However, both the identity of the lyso-PLs released from apoptotic cells and the nature of the phagocyte receptor are largely unknown. By a detailed analysis of the structural requirements we show here that lysophosphatidylcholine (lysoPC), but none of the lysoPC metabolites or other lysoPLs, represents the essential apoptotic attraction signal able to trigger a phagocyte chemotactic response. Furthermore, using RNA interference and expression studies, we demonstrate that the G-protein-coupled receptor G2A, unlike its relative GPR4, is involved in the chemotaxis of monocytic cells. Thus, our study identifies lysoPC and G2A as the crucial receptor/ligand system for the attraction of phagocytes to apoptotic cells and the prevention of autoimmunity.