Katia Vancompernolle

Atractyloside-induced release of cathepsin B, a protease with caspase-processing activity.

Recent data show that a strong relation exists in certain cells between mitochondria and caspase activation in apoptosis. We further investigated this relation and tested whether treatment with the permeability transition (PT)‐inducing agent atractyloside of Percoll‐purified mitochondria released a caspase‐processing activity. Following detection of procaspase‐11 processing, we further purified this caspase‐processing protease and identified it as cathepsin B. The purified cathepsin B, however, was found to be derived from lysosomes which were present as minor contaminants in the mitochondrial preparation. Besides procaspase‐11, caspase‐1 is also readily processed by cathepsin B. Procaspase‐2, ‐6, ‐7, ‐14 are weak substrates and procaspase‐3 is a very poor substrate, while procaspase‐12 is no substrate at all for cathepsin B. In addition, cathepsin B induces nuclear apoptosis in digitonin‐permeabilized cells as well as in isolated nuclei. All newly described activities of cathepsin B, namely processing of caspase zymogens and induction of nuclear apoptosis, are inhibited by the synthetic peptide caspase inhibitors z‐VAD.fmk, z‐DEVD.fmk and to a lesser extent by Ac‐YVAD.cmk.

Identification of two regions in the N‐terminal domain of ActA involved in the actin comet tail formation by Listeria monocytogenes

The ActA protein of Listeria monocytogenes induces actin nucleation on the bacterial surface. The continuous process of actin filament elongation provides the driving force for bacterial propulsion in infected cells or cytoplasmic extracts. Here, by fusing the N‐terminus of ActA (residues 1–234) to the ω fragment of β‐galactosidase, we present the first evidence that this domain contains all the necessary elements for actin tail formation. A detailed analysis of ActA variants, in which small fragments of the N‐terminal region were deleted, allowed the identification of two critical regions. Both are required to initiate the actin polymerization process, but each has in addition a specific role to maintain the dynamics of the process. The first region (region T, amino acids 117–121) is critical for filament elongation, as shown by the absence of actin tail in a 117–121 deletion mutant or when motility assays are performed in the presence of anti‐region T antibodies. The second region (region C, amino acids 21–97), is more specifically involved in maintenance of the continuity of the process, probably by F‐actin binding or prevention of barbed end capping, as strongly suggested by both a deletion (21–97) leading to ‘discontinuous’ actin tail formation and in vitro experiments showing that a synthetic peptide covering residues 33–74 can interact with F‐actin. Our results provide the first insights in the molecular dissection of the actin polymerization process induced by the N‐terminal domain of ActA.

Tumor necrosis factor-induced modulation of glyoxalase I activities through phosphorylation by PKA results in cell death and is accompanied by the formation of a specific methylglyoxal-derived AGE

Tumor necrosis factor (TNF)-induced cell death in the fibrosarcoma cell line L929 is a caspase-independent process that is characterized by increased production of reactive oxygen species (ROS) in the mitochondria. To elucidate this ROS-dependent cell death pathway, a comparative study of the phosphoproteins present in TNF-treated and control cells was performed. Here we report that TNF induces an increased phosphorylation of glyoxalase I that is mediated by protein kinase A and required for cell death. We also show that TNF induces a substantial increase in intracellular levels of methylglyoxal (MG) that leads to the formation of a specific MG-derived advanced glycation end product and that this formation occurs as a consequence of increased ROS production. These data indicate that MG modification of proteins is a targeted process and that MG may thus function as a signal molecule during the regulation of cell death. Furthermore, we provide evidence that the TNF-induced phosphorylation of glyoxalase I is not involved in detoxification of MG by means of the glyoxalase system, but that phosphorylated glyoxalase I is on the pathway leading to the formation of a specific MG-derived advanced glycation end product.

Villin Function in the Organization of the Actin Cytoskeleton CORRELATION OF IN VIVO EFFECTS TO ITS BIOCHEMICAL ACTIVITIES IN VITRO

Villin is an actin-binding protein of the intestinal brush border that bundles, nucleates, caps, and severs actin in a Ca2+-dependent manner in vitro. Villin induces the growth of microvilli in transfected cells, an activity that requires a carboxyl-terminally located KKEK motif. By combining cell transfection and biochemical assays, we show that the capacity of villin to induce growth of microvilli in cells correlates with its ability to bundle F-actin in vitro but not with its nucleating activity. In agreement with its importance for microfilament bundling in cells, the KKEK motif of the carboxyl-terminal F-actin-binding site is crucial for bundling in vitro. In addition, substitutions of basic residues in a second site, located in the amino-terminal portion of villin, impaired its activity in cells and reduced its binding to F-actin in the absence of Ca2+ as well as its bundling and severing activitiesin vitro. Altogether, these findings suggest that villin participates in the organization and stabilization of the brush border core bundle but does not initiate its assembly by nucleation of actin filaments.

G- to F-actin modulation by a single amino acid substitution in the actin binding site of actobindin and thymosin beta 4.

The actin binding sites of actobindin and thymosin beta 4, two small polypeptides that inhibit actin polymerization by interacting with monomeric actin, have been localized using peptide mimetics. Both sites are functionally similar and extend over 20 residues and are located in the NH2‐terminus of the polypeptides. They can be dissected into two functional entities: a conserved hexapeptide motif (LKHAET or LKKTET), which forms the major contact site through electrostatic interactions with actin, and a non‐conserved NH2‐terminal segment preceding the motif, which exerts the inhibitory activity on actin polymerization probably by steric hindrance. The introduction of a glutamic acid at the third position in the motif, creating LKEAET or LKETET sequences, which are similar to those found in some F‐actin binding proteins, converts the peptides inhibitory phenotype into an F‐actin stimulatory property. These results allow the proposal of a simple model for G‐ to F‐actin modulation.

Isolation and sequence of a tropomyosin-binding fragment of turkey gizzard calponin

Limited chymotryptic cleavage of turkey gizzard calponin yields a 13 kDa fragment which could be purified by its ability to bind to Sepharose‐immobilized tropomyosin. This 13 kD polypcptide is shown to be derived from a 22 kDa fragment. Complete amino acid sequence analysis of the 13 kD and 22 kD fragments reveals high homology with the formerly characterized smooth muscle‐specific protein SM222 (Pearlstone. J.R., Weber. M., Lees‐Miller, J.P.. Carpenter, M.R. and Smillie L.B., 1987. J. Biol. Chem. 262, 5985‐5991) and the product of gene mp20 of Drosophila (Ayme‐Southqate. A., Lasko. P., French. C. and Pardue, M.L. [(1989) J. Cell Biol. 108, 521 531]. Furthermore we recognize sequence elements of a putative actin‐binding domain of α‐actinin, the calpactin I or p 36 sequence, and a consensus motif present in the repeats of the gene product of the candidate unc‐87 gene of C. elegants (S.D. Goetinck and R.H. Waterston, personal communication).

Tumor necrosis factor-induced microtubule stabilization mediated by hyperphosphorylated oncoprotein 18 promotes cell death

Tumor necrosis factor (TNF)-induced cell death in the fibrosarcoma cell line L929 occurs independently of caspase activation and cytochrome c release. However, it is dependent on mitochondria and is characterized by increased production of reactive oxygen intermediates that are essential to the death process. To identify signaling molecules involved in this TNF-induced, reactive oxygen intermediate-dependent cell death pathway, we performed a comparative study by two-dimensional gel electrophoresis of phosphoproteins from a mitochondria-enriched fraction derived from TNF-treated and control cells. TNF induced rapid and persistent phosphorylation of the phosphorylation-responsive regulator of the microtubule (MT) dynamics, oncoprotein 18 (Op18). By using induced overexpression of wild type Op18 and phosphorylation site-deficient mutants S25A/S38A and S16A/S63A in L929 cells, we show that TNF-induced phosphorylation on each of the four Ser residues of Op18 promotes cell death and that Ser16 and Ser63are the primary sites. This hyperphosphorylation of Op18 is known to completely turn off its MT-destabilizing activity. As a result, TNF treatment of L929 cells induced elongated and extremely tangled microtubules. These TNF-induced changes to the MT network were also observed in cells overexpressing wild type Op18 and, to a lesser extent, in cells overexpressing the S25A/S38A mutant. No changes in the MT network were observed upon TNF treatment of cells overexpressing the S16A/S63A mutant, and these cells were desensitized to TNF-induced cell death. These findings indicate that TNF-induced MT stabilization is mediated by hyperphosphorylation of Op18 and that this promotes cell death. The data suggest that Op18 and the MT network play a functional role in transduction of the cell death signal to the mitochondria.

Phosphorylation on Thr-106 and NO-modification of glyoxalase I suppress the TNF-induced transcriptional activity of NF-κB

Glyoxalase I (GLO1), together with glyoxalase II and the co-factor GSH, comprise the glyoxalase system, which is responsible for the detoxification of the cytotoxic glycolytic-derived metabolite methylglyoxal (MG). We, and others, have previously reported that GLO1 is subjected to several post-translational modifications, including a NO-mediated modification and phosphorylation. In this study, we demonstrate that GLO1 is a substrate for calcium, calmodulin-dependent protein kinase II (CaMKII). Site-directed mutagenesis of several serine and threonine residues revealed that CaMKII induced phosphorylation of GLO1 at a single site Thr-106. Mutagenesis of Thr-106 to Ala in GLO1 completely abolished the CaMKII-mediated phosphorylation. A phosphopeptide bracketing phosphothreonine-106 in GLO1 was used as an antigen to generate polyclonal antibodies against phosphothreonine-106. By using this phospho-specific antibody, we demonstrated that TNF induces phosphorylation of GLO1 on Thr-106. Furthermore, we investigated the role of NO-mediated modification and phosphorylation of GLO1 in the TNF-induced transcriptional activity of NF-κB. Overexpression of WT GLO1 suppressed TNF-induced NF-κB-dependent reporter gene expression. Suppression of the basal and TNF-induced NF-κB activity was significantly stronger upon expression of a GLO1 mutant that was either deficient for the NO-mediated modification or phosphorylation on Thr-106. However, upon overexpression of a GLO1 mutant that was deficient for both types of modification, the suppressive effect of GLO1 on TNF-induced NF-κB activity was completely abolished. These results suggest that NO-modification and phosphorylation of GLO1 contribute to the suppression of TNF-induced NF-κB-dependent reporter gene expression. In line with this, knock-down of GLO1 by siRNA significantly increased TNF-induced NF-κB-dependent reporter gene expression. These findings suggest that phosphorylation and NO-modification of glyoxalase I provides another control mechanism for modulating the basal and TNF-induced expression of NF-kappaB-responsive genes.

Tumour necrosis factor induces phosphorylation primarily of the nitric-oxide-responsive form of glyoxalase I

We have previously shown that TNF (tumour necrosis factor) induces phosphorylation of GLO1 (glyoxalase I), which is required for cell death in L929 cells. In the present paper, we show that the TNF-induced phosphorylation of GLO1 occurs primarily on the NO (nitric oxide)-responsive form of GLO1. In addition, analysis of several cysteine mutants of GLO1 indicated that Cys-138, in combination with either Cys-18 or Cys-19, is a crucial target residue for the NO-mediated modification of GLO1. Furthermore, the NO-donor GSNO (S-nitrosogluthathione) induces NO-mediated modification of GLO1 and enhances the TNF-induced phosphorylation of this NO-responsive form. GSNO also strongly promotes TNF-induced cell death. By the use of pharmacological inhibition of iNOS (inducible NO synthase) and overexpression of mutants of GLO1 that are deficient for the NO-mediated modification, we have shown that the NO-mediated modification of GLO1 is not a requirement for TNF-induced phosphorylation or TNF-induced cell death respectively. In summary, these data suggest that the TNF-induced phosphorylation of GLO1 is the dominant factor for cell death.

11 – Role of Reactive Oxygen Species in Tumor Necrosis Factor Toxicity

This chapter deals with the cytotoxic activity of the pleiotropic cytokine Tumor Necrosis Factor (TNF) on transformed cell lines. The initial emphasis on the direct cell-killing activity of TNF on tumor cells and its application in therapeutic strategies against cancer is evolved to the use of this cytokine as a model system for molecular mechanisms of cell death. Many tumor-derived established cell lines are susceptible to cell killing by TNF. The molecular mechanisms underlying TNF cytotoxicity vary between cell types, ranging from typical apoptosis characterized by membrane blebbing, chromatin condensation, and formation of apoptotic cell bodies to necrosis-like cell death. Therefore, the elucidation of the signaling cascades activated by TNF in these different cell types contribute in providing information to molecular execution pathways of apoptosis and necrosis, as well as of mechanisms affecting these cell death pathways. The chapter mainly focuses on how oxidative stress participates in various stages of these cell death processes.