Klaus Schulze-Osthoff

Many cuts to ruin: a comprehensive update of caspase substrates

AbstractApoptotic cell death is executed by the caspase-mediated cleavage of various vital proteins. Elucidating the consequences of this endoproteolytic cleavage is crucial for our understanding of cell death and other biological processes. Many caspase substrates are just cleaved as bystanders, because they happen to contain a caspase cleavage site in their sequence. Several targets, however, have a discrete function in propagation of the cell death process. Many structural and regulatory proteins are inactivated by caspases, while other substrates can be activated. In most cases, the consequences of this gain-of-function are poorly understood. Caspase substrates can regulate the key morphological changes in apoptosis. Several caspase substrates also act as transducers and amplifiers that determine the apoptotic threshold and cell fate. This review summarizes the known caspase substrates comprising a bewildering list of more than 280 different proteins. We highlight some recent aspects inferred by the cleavage of certain proteins in apoptosis. We also discuss emerging themes of caspase cleavage in other forms of cell death and, in particular, in apparently unrelated processes, such as cell cycle regulation and cellular differentiation.

SMALL STRESS PROTEINS AS NOVEL REGULATORS OF APOPTOSIS : HEAT SHOCK PROTEIN 27 BLOCKS FAS/APO-1- AND STAUROSPORINE-INDUCED CELL DEATH

Small stress protein expression enhances the survival of mammalian cells exposed to numerous injuries that induce necrotic cell death. The cell surface receptor Fas/APO-1 and its ligand have been recently identified as important mediators of apoptosis. Here, we show that constitutive expression of human heat shock protein (hsp)27 in murine L929 cells blocks Fas/APO-1-mediated cell death. Expression of human hsp27 prevented anti-APO-1-induced DNA fragmentation and morphological changes. These results strongly suggest that human hsp27 acts as a cellular inhibitor of Fas/APO-1-induced apoptosis. We also report that the expression of small stress proteins from different species, such as human hsp27, Drosophila Dhsp27, or human αB-crystallin, confers resistance to apoptotic cell death induced by staurosporine, a protein kinase C inhibitor. Hence, small stress proteins are novel regulators that are able to block apoptosis induced by different pathways.

Depletion of the mitochondrial electron transport abrogates the cytotoxic and gene-inductive effects of TNF.

Tumor necrosis factor (TNF) has cytotoxic and gene‐inductive activities on several cell types. Previous studies on L929 fibrosarcoma cells have revealed that the mitochondrial electron transport system plays a key role in inducing TNF cytotoxicity, presumably by the formation of reactive oxygen intermediates (ROI). Here we report that mitochondria‐derived intermediates are not only cytotoxic but, in addition, function as signal transducers of TNF‐induced gene expression. The activation of NF kappa B, which fulfills an important role in TNF‐induced gene transcription, could be blocked by interference with the mitochondrial electron transport system. Furthermore, antimycin A, a mitochondrial inhibitor that increases the generation of ROI, potentiated TNF‐triggered NF kappa B activation. The dual role of mitochondria‐derived intermediates in cytotoxicity and immediate‐early gene induction of TNF was further substantiated by isolating L929 subclones which lacked a functional respiratory chain. This depletion of the mitochondrial oxidative metabolism resulted in resistance towards TNF cytotoxicity, as well as in inhibition of NF kappa B activation and interleukin‐6 gene induction by TNF. These findings suggest that mitochondria are the source of second messenger molecules and serve as common mediators of the TNF‐cytotoxic and gene‐regulatory signaling pathways.

Functions of glutathione and glutathione disulfide in immunology and immunopathology.

Even a moderate increase in the cellular cysteine supply elevates the intracellular glutathione (GSH) and glutathione disulfide (GSSG) levels and potentiates immunological functions of lymphocytes in vitro. At low GSSG levels, T cells cannot optimally activate the immunologically important transcription factor NFxB, whereas high GSSG levels inhibit the DNA binding activity of NFxB. The effects of GSSG are antagonized by reduced thioredoxin (TRX). As the protein tyrosine kinase activities p56lck and p59fyn are activated in intact cells by hydrogen peroxide, they are likely targets for GSSG action. These redox‐regulated enzymes trigger signal cascades for NFxB activation and transduce signals from the T cell antigen receptor, from CD4 and CD8 molecules, and from the IL‐2 receptor β‐chain. The effector phase of cytotoxic T cell responses and IL‐2‐dependent functions are inhibited even by a partial depletion of the intracellular GSH pool. As signal transduction is facilitated by prooxidant conditions, we propose that the well‐known immunological consequences of GSH depletion ultimately may be results of the accompanying GSSG deficiency. As HIV‐infected patients and SIV‐infected rhesus macaques have, on the average, significantly decreased plasma cyst(e)ine and intracellular GSH levels, we also hypothesize that AIDS may be the consequence of a GSSG deficiency as well.—Dröge, W., Schulze‐Osthoff, K., Mihm, S., Galter, D., Schenk, H., Eck, H.‐P., Roth, S., Gmünder, H. Functions of glutathione and glutathione disulfide in immunology and immunopathology. FASEB J. 8, 1131‐1138 (1994)

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.

HIV-1 Tat potentiates TNF-induced NF-kappa B activation and cytotoxicity by altering the cellular redox state.

This study demonstrates that human immunodeficiency virus type 1 (HIV‐1) Tat protein amplifies the activity of tumor necrosis factor (TNF), a cytokine that stimulates HIV‐1 replication through activation of NF‐kappa B. In HeLa cells stably transfected with the HIV‐1 tat gene (HeLa‐tat cells), expression of the Tat protein enhanced both TNF‐induced activation of NF‐kappa B and TNF‐mediated cytotoxicity. A similar potentiation of TNF effects was observed in Jurkat T cells and HeLa cells treated with soluble Tat protein. TNF‐mediated activation of NF‐kappa B and cytotoxicity involves the intracellular formation of reactive oxygen intermediates. Therefore, Tat‐mediated effects on the cellular redox state were analyzed. In both T cells and HeLa cells HIV‐1 Tat suppressed the expression of Mn‐dependent superoxide dismutase (Mn‐SOD), a mitochondrial enzyme that is part of the cellular defense system against oxidative stress. Thus, Mn‐SOD RNA protein levels and activity were markedly reduced in the presence of Tat. Decreased Mn‐SOD expression was associated with decreased levels of glutathione and a lower ratio of reduced:oxidized glutathione. A truncated Tat protein (Tat1‐72), known to transactivate the HIV‐1 long terminal repeat (LTR), no longer affected Mn‐SOD expression, the cellular redox state or TNF‐mediated cytotoxicity. Thus, our experiments demonstrate that the C‐terminal region of HIV‐1 Tat is required to suppress Mn‐SOD expression and to induce pro‐oxidative conditions reflected by a drop in reduced glutathione (GSH) and the GSH:oxidized GSH (GSSG) ratio.(ABSTRACT TRUNCATED AT 250 WORDS)

Sesquiterpene Lactones Specifically Inhibit Activation of NF-κB by Preventing the Degradation of IκB-α and IκB-β

Extracts from certain Mexican Indian medicinal plants used in traditional indigenous medicine for the treatment of inflammations contain sequiterpene lactones (SLs), which specifically inhibit the transcription factor NF-κB (Bork, P. M., Schmitz, M. L., Kuhnt, M., Escher, C., and Heinrich, M. (1997) FEBS Lett. 402, 85–90). Here we show that SLs prevented the activation of NF-κB by different stimuli such as phorbol esters, tumor necrosis factor-α, ligation of the T-cell receptor, and hydrogen peroxide in various cell types. Treatment of cells with SLs prevented the induced degradation of IκB-α and IκB-β by all these stimuli, suggesting that they interfere with a rather common step in the activation of NF-κB. SLs did neither interfere with DNA binding activity of activated NF-κB nor with the activity of the protein tyrosine kinases p59 fyn and p60 src . Micromolar amounts of SLs prevented the induced expression of the NF-κB target gene intracellular adhesion molecule 1. Inhibition of NF-κB by SLs resulted in an enhanced cell killing of murine fibroblast cells by tumor necrosis factor-α. SLs lacking an exomethylene group in conjugation with the lactone function displayed no inhibitory activity on NF-κB. The analysis of the cellular redox state by fluorescence-activated cell sorter showed that the SLs had no direct or indirect anti-oxidant properties.

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

T-helper-1-cell cytokines drive cancer into senescence

Cancer control by adaptive immunity involves a number of defined death and clearance mechanisms. However, efficient inhibition of exponential cancer growth by T cells and interferon-γ (IFN-γ) requires additional undefined mechanisms that arrest cancer cell proliferation. Here we show that the combined action of the T-helper-1-cell cytokines IFN-γ and tumour necrosis factor (TNF) directly induces permanent growth arrest in cancers. To safely separate senescence induced by tumour immunity from oncogene-induced senescence, we used a mouse model in which the Simian virus 40 large T antigen (Tag) expressed under the control of the rat insulin promoter creates tumours by attenuating p53- and Rb-mediated cell cycle control. When combined, IFN-γ and TNF drive Tag-expressing cancers into senescence by inducing permanent growth arrest in G1/G0, activation of p16INK4a (also known as CDKN2A), and downstream Rb hypophosphorylation at serine 795. This cytokine-induced senescence strictly requires STAT1 and TNFR1 (also known as TNFRSF1A) signalling in addition to p16INK4a. In vivo, Tag-specific T-helper 1 cells permanently arrest Tag-expressing cancers by inducing IFN-γ- and TNFR1-dependent senescence. Conversely, Tnfr1−/− Tag-expressing cancers resist cytokine-induced senescence and grow aggressively, even in TNFR1-expressing hosts. Finally, as IFN-γ and TNF induce senescence in numerous murine and human cancers, this may be a general mechanism for arresting cancer progression.

Death by a thousand cuts: an ever increasing list of caspase substrates

At present, mammalian caspases comprise a group of at least 13 protease members which either generate mature pro-inflammatory cytokines or promote apoptosis (Cohen, 1997; Nicholson and Thornberry, 1997; Van de Craen et al, 1997; Humke et al, 1998; Schulze-Osthoff et al, 1998). Based on phylogenetic analysis and positional scanning studies of their peptide substrates, caspases can be divided into three subfamilies: The ICE-like protease family includes caspase-1, -4, -5 and -13 as well as murine caspase-11 and -12, for which no human equivalents have yet been identified. The Ced-3 subfamily includes caspase-3, -6, -7, -8, -9 and -10, whereas the third subfamily consists of only one member, caspase-2. Within each subfamily, the peptide sequence preferences in the substrates are remarkably similar or even identical. This demonstrates that, at least in some cases, different caspases can cleave the same substrates, suggesting some degree of functional redundancy within the caspase family. Central to the understanding of the molecular mechanism of cell death is the identification of caspase targets and the elucidation of the consequences of proteolytic cleavage. Thus far, more than 60 proteins have been found to be cleaved by caspases, and new substrates are continuously being identified (Table 1). Given the great number of different caspases, the list of substrates is still relatively small. For most proteins, the consequences of cleavage are poorly understood. In a few cases, however, proteolysis of certain components can be linked to discrete morphological changes of cell death. Which requirements should an apoptosis-relevant caspase substrate meet? Because apoptosis is an ordered sequence of rather stereotypical alterations in every cell type, one would predict that caspase substrates should be ubiquitously expressed and evolutionary conserved, at least in their aspartate cleavage site. The known substrates of caspases can be loosely categorized into a few functional groups including proteins involved in scaffolding of the cytoplasm and cell nucleus, signal transduction and transcription-regulatory proteins, cell-cycle controlling components and proteins involved in DNA replication and repair. In addition, activation of members of the first subfamily of caspases, caspase-1 and presumably caspase-4 and -5, results in the processing of cytokine precursors, which are presumably not directly involved in cell death. While some substrates are functionally inactivated upon caspase-mediated cleavage, other proteins and enzymes can be activated, mostly by cleavage of an inhibitory or regulatory domain within the caspase target. In most cases the physiological consequence of this gain-of-function cleavage for apoptosis remains unclear. Caspasemediated cleavage should result in different net effects: (i) a halt of cell cycle progression, (ii) disabling of repair mechanisms, (iii) disassembly of molecular structures, (iv) cell detachment, and (v) tagging of the apoptotic cell for engulfment by phagocytes. A number of structural proteins in the cell nucleus and cytoplasm have been identified to be cleaved by caspases, such as actin, fodrin, catenins, keratins, Gas2 and lamins (for references see Cohen, 1997; Nicholson and Thornberry, 1997; Porter et al, 1997; Tan and Wang, 1998; Cryns and Yuan, 1998). Degradation of lamin B which is predominantly mediated by caspase-6 may lead to the disassembly of the nuclear envelope and the final collapse of the cell nucleus (Rao et al, 1996). In contrast, cleavage of gelsolin, a cytoplasmic actin-severing protein, may contribute to membrane blebbing and other morphological features of the apoptotic phenotype. Gelsolin is cleaved by caspase-3 to generate a constitutively active fragment that can depolymerize F-actin (Kothakota et al, 1997). Interestingly, gelsolin-deficient cells show a strong delay in membrane blebbing when exposed to apoptotic stimuli. It has been also reported that actin can be directly cleaved by caspases in pheochromocytoma and ovarian carcinoma cells (Kayalar et al, 1996; Chen et al, 1996), whereas in many other cell types no cleavage could be detected (Song et al, 1997). Thus, it is possible that certain protein cleavages may be cell typespecific which may also due to variations in the expression of individual caspases in different cell types. Activation of caspases may be not only required for destruction of the cells architecture, but also necessary for the detachment and clearance of an apoptotic cell from the embedding tissue. Indeed, some caspase substrates participate in cell adhesion, such as b-catenin, plakoglobin and focal adhesion kinase (Brancolini et al, 1997; 1998; Herren et al, 1998; Crouch et al, 1996; Levkau et al, 1998a). A strikingly large number of caspase targets are involved in cell cycle regulation and DNA repair mechanisms. One of the first death substrates found to be cleaved by caspases was poly(ADP-ribose)polymerase (PARP), which catalyzes the transfer of ADP-ribose polymers to nuclear proteins (Tewari et al, 1995). As DNA strand breaks activate the enzyme, PARP has been proposed to trigger DNA damageinduced apoptosis by depleting NAD stores. On the other hand, due to its role in DNA repair, cleavage of PARP may Cell Death and Differentiation (1998) 5, 997 ± 1000 a 1998 Stockton Press All rights reserved 13509047/98