Biomedical subjects

R Beyaert

Publications and source records attributed to R Beyaert.

At least 19 recordsLinked to original sources

PubMed2006

A novel link between Lck, Bak expression and chemosensitivity.

Protein kinases are critically involved in signaling pathways that regulate cell growth, differentiation, activation, and survival. Lck, a member of the Src family of protein tyrosine kinases, plays a key role in T-lymphocyte activation and differentiation. However, under certain conditions Lck is also involved in the induction of apoptosis. In this issue of Oncogene, Samraj et al. used the Lck-defective JCaM1.6 cell line to demonstrate the critical role of Lck in the apoptotic response of T-cell leukemia cells to several chemotherapeutic drugs. They further showed that Lck controls the mitochondrial death pathway by regulating proapoptotic Bak expression. This chemosensitizing effect of Lck is independent of T-cell receptor signaling and does not require the kinase activity of Lck. These findings demonstrate that Lck might be part of two independent signaling pathways leading to either cell proliferation or apoptosis, and reveal a hitherto unrecognized link between Lck, Bak, and chemosensitivity of human leukemic cells.

Animals↗

PubMed2004

Function and regulation of tumor necrosis factor receptor type 2.

TNF is a major proinflammatory cytokine, which exerts its effects through two different receptors known as TNF-R1 and TNF-R2. Whereas TNF-R1 induced signaling pathways have been very well characterized during the past years, TNF-R2 has been much less well studied. Nevertheless, the function of TNF-R2 should not be underestimated, the more because this receptor shows a much more restricted but inducible expression. Several inflammatory diseases and cancers show upregulated levels of soluble TNF-R2 or are associated with TNF-R2 polymorphisms, implicating an important role for TNF-R2 as a therapeutic target. Here we will review the mechanisms that regulate TNF-R2 expression, as well as discuss TNF-R2 induced signal transduction and cross-talk with TNF-R1.

Animals↗

PubMed2003

Role of Toll-like receptors in pathogen recognition.

The innate immune system relies on a vast array of non-clonally expressed pattern recognition receptors for the detection of pathogens. Pattern recognition receptors bind conserved molecular structures shared by large groups of pathogens, termed pathogen-associated molecular patterns. The Toll-like receptors (TLRs) are a recently discovered family of pattern recognition receptors which show homology with the Drosophila Toll protein and the human interleukin-1 receptor family. Engagement of different TLRs can induce overlapping yet distinct patterns of gene expression that contribute to an inflammatory response. The TLR family is characterized by the presence of leucine-rich repeats and a Toll/interleukin-1 receptor-like domain, which mediate ligand binding and interaction with intracellular signaling proteins, respectively. Most TLR ligands identified so far are conserved microbial products which signal the presence of an infection, but evidence for some endogenous ligands that might signal other danger conditions has also been obtained. Molecular mechanisms for pathogen-associated molecular pattern recognition still remain elusive but seem to be more complicated than initially anticipated. In most cases, direct binding of microbial ligands to TLRs still has to be demonstrated. Moreover, Drosophila TLRs bind endogenous ligands, generated through a proteolytic cascade in response to an infection. In the case of endotoxin, recognition involves a complex of TLR4 and a number of other proteins. Moreover, TLR heterodimerization further extends the spectrum of ligands and modulates the response towards specific ligands. The fact that TLR expression is regulated in both a cell type- and stimulus-dependent fashion further contributes to the complexity.

Animals↗

PubMed2002

A matrix-assisted laser desorption ionization post-source decay (MALDI-PSD) analysis of proteins released from isolated liver mitochondria treated with recombinant truncated Bid.

A crucial event in the process of apoptosis is caspase-dependent generation of truncated Bid (tBid), inducing release of cytochrome c. In an in vitro reconstitution system we combined purified recombinant tBid with isolated liver mitochondria and identified the released proteins using a proteomic matrix-assisted laser desorption ionization post-source decay (MALDI-PSD) approach. In order to meet physiological conditions, the concentration of tBid was chosen such that it was unable to induce cytochrome c release in mitochondria derived from liver-specific Bcl-2-transgenic mice. Several mitochondrial proteins were identified to be released in a tBid-dependent way, among which cytochrome c, DIABLO/Smac, adenylate kinase 2, acyl-CoA-binding protein, endonuclease G, polypyrimidine tract-binding protein, a type-I RNA helicase, a WD-40 repeat-containing protein and the serine protease Omi. Western blotting confirmed the absence of adenylate kinase 3, a matrix mitochondrial protein. These results demonstrate that a physiologically relevant concentration of tBid is sufficient to induce release of particular intermembrane mitochondrial proteins belonging to a broad molecular-mass range.

Adenylate Kinase↗

PubMed2002

Caspases are not localized in mitochondria during life or death.

Caspases are crucial for the initiation, propagation and execution of apoptosis. They normally exist as proenzymes, which can be activated through recruitment into activating complexes and by proteolytic cleavage by other caspases or proteases. Perturbation of organelles such as nuclei, endoplasmatic reticulum and mitochondria results in the activation of caspases. A number of caspases (-2, -3, -8 and -9) were published as being localized in the intermembrane space of mitochondria. However, in three different models of apoptosis (anti-Fas-induced cell death in murine hepatocytes, Fas ligand-induced apoptosis in Jurkat cells and apoptosis induced by growth factor withdrawal in Ba/F3 cells) we could not identify a mitochondrial location of caspases, neither under control nor under apoptotic conditions. In all three apoptotic models caspases were found in the cytosolic (caspases-2, -3, -6, -7, -8, -9) and nuclear subcellular fractions (caspases-2, -3). In another approach we treated isolated liver mitochondria with truncated Bid. Although tBid-dependent release of Cytochrome c, AIF, adenylate kinase, Smac/DIABLO and Omi/HtrA2 could be demonstrated, none of the caspases were detectable both in the supernatant and the mitochondrial fraction after treatment. Our results demonstrate that, in contrast to previous studies, no caspases-2, -3, -8 and -9 are associated with the mitochondrial fraction. These findings support the concept of a separate compartmentalization between proapoptotic cofactors in the mitochondria and silent precursor caspases in the cytosol.

Animals↗

PubMed2001

Isolation and characterization of two novel A20-like proteins.

The transcription factor nuclear factor kappa B (NF-kappa B) plays a pivotal role in inflammatory processes through induction of adhesion molecules and chemokines. The zinc finger molecule A20 is an important negative regulator of NF-kappa B. The mechanism utilized by A20 is not fully understood, but A20 has been shown to bind to tumour-necrosis-factor-receptor-associated factor (TRAF) molecules, which are necessary for pro-inflammatory cytokine signalling. We report two novel genes, Cezanne (cellular zinc finger anti-NF-kappa B) and TRABID (TRAF-binding domain), with sequence similarity to A20. Co-immunoprecipitation studies indicated that TRAF6 was able to interact with both Cezanne and TRABID. In contrast, reporter gene experiments revealed a specific ability of Cezanne to down-regulate NF-kappa B. It is likely, therefore, that Cezanne participates in the regulation of inflammatory processes.

Amino Acid Sequence↗

PubMed2001

Identification of a novel A20-binding inhibitor of nuclear factor-kappa B activation termed ABIN-2.

The nuclear factor kappaB (NF-kappaB) plays a central role in the regulation of genes implicated in immune responses, inflammatory processes, and apoptotic cell death. The zinc finger protein A20 is a cellular inhibitor of NF-kappaB activation by various stimuli and plays a critical role in terminating NF-kappaB responses. The underlying mechanism for NF-kappaB inhibition by A20 is still unknown. A20 has been shown to interact with several proteins including tumor necrosis factor (TNF) receptor-associated factors 2 and 6, as well as the inhibitory protein of kappaB kinase (IKK) gamma protein. Here we report the cloning and characterization of ABIN-2, a previously unknown protein that binds to the COOH-terminal zinc finger domain of A20. NF-kappaB activation induced by TNF and interleukin-1 is inhibited by overexpression of ABIN-2. The latter also inhibits NF-kappaB activation induced by overexpression of receptor-interacting protein or TNF receptor-associated factor 2. In contrast, NF-kappaB activation by overexpression of IKKbeta or direct activators of the IKK complex, such as Tax, cannot be inhibited by ABIN-2. These results indicate that ABIN-2 interferes with NF-kappaB activation upstream of the IKK complex and that it might contribute to the NF-kappaB-inhibitory function of A20.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Functional redundancy of the zinc fingers of A20 for inhibition of NF-kappaB activation and protein-protein interactions.

The tumor necrosis factor (TNF) inducible protein A20 is a potent inhibitor of nuclear factor-kappaB (IkappaB)-mediated gene expression in response to TNF and several other stimuli. The C-terminal domain of A20 is characterized by seven zinc finger structures. Here, we show that a minimum of four zinc fingers is required to inhibit TNF-induced nuclear factor-kappaB (NF-kappaB) activation to a level that is comparable to that obtained with the wild-type A20 protein. However, there was no strict requirement for a particular zinc finger structure, since a mutant A20 protein containing only the first four zinc fingers was as potent as a mutant protein containing only the last four zinc fingers. A similar functional redundancy of the A20 zinc fingers was also observed for binding of A20 to a number of other proteins, including two novel NF-kappaB inhibitory proteins (ABIN-1, ABIN-2), A20 itself, the anti-apoptotic protein TXBP151, and a regulatory component of the IkappaB kinase complex, IKKgamma. Moreover, we demonstrate that complete loss of binding of any of these proteins correlates with complete loss of A20's ability to inhibit TNF-induced NF-kappaB activation. However, binding of IKKgamma as such is not sufficient for inhibition of NF-kappaB dependent gene expression in response to TNF.

Amino Acid Sequence↗

PubMed2001

Lithium sensitizes tumor cells in an NF-kappa B-independent way to caspase activation and apoptosis induced by tumor necrosis factor (TNF). Evidence for a role of the TNF receptor-associated death domain protein.

We have previously shown that lithium salts can considerably increase the direct cytotoxic effect of tumor necrosis factor (TNF) on various tumor cells in vitro and in vivo. However, the underlying mechanism has remained largely unknown. Here we show that the TNF-sensitizing effect of lithium chloride (LiCl) is independent of the type of cell death, either necrosis or apoptosis. In the case of apoptosis, TNF/lithium synergism is associated with an enhanced activation of caspases and mitochondrial cytochrome c release. Sensitization to apoptosis is specific for TNF-induced apoptosis, whereas Fas-mediated or etoposide-induced apoptosis remains unaffected. LiCl also potentiates cell death induced by artificial oligomerization of a fusion protein between FKBP and the TNF receptor-associated death domain protein. TNF-induced activation of NF-kappa B-dependent gene expression is not modulated by LiCl treatment. These results indicate that LiCl enhances TNF-induced cell death in an NF-kappa B-independent way, and suggest that the TNF receptor-associated death domain protein plays a crucial role in the TNF-sensitizing effect of LiCl.

Adjuvants, Immunologic↗

PubMed2001

The cathepsin B inhibitor z-FA.fmk inhibits cytokine production in macrophages stimulated by lipopolysaccharide.

Cathepsin B has previously been shown to proteolytically activate the proinflammatory caspase-11 in vitro. Here we show that cathepsin B is not involved in activation of caspase-11 induced by lipopolysaccharide (LPS) and subsequent maturation of interleukin (IL)-1beta in macrophages. Nevertheless, we found that the cathepsin B inhibitor benzyloxycarbonyl-Phe-Ala-fluoromethylketone (z-FA.fmk) prevents LPS-induced production of IL-1alpha, IL-1beta, and tumor necrosis factor at the transcriptional level. The latter was not because of cathepsin B inhibition, but was mediated by inhibition of the transactivation potential of the nuclear factor kappaB (NF-kappaB). z-FA.fmk did not prevent LPS-induced activation of p38 mitogen-activated protein kinase, which was shown to be involved in NF-kappaB transactivation in response to LPS. These results suggest that the previously described therapeutic effect of z-FA.fmk in the treatment of rheumatoid arthritis might not only result from inhibition of cathepsin B but also implicates an important contribution from the inhibition of NF-kappaB-dependent gene expression.

Animals↗

PubMed2001

Activation of caspase-8 in drug-induced apoptosis of B-lymphoid cells is independent of CD95/Fas receptor-ligand interaction and occurs downstream of caspase-3.

The activation of caspase-8, a crucial upstream mediator of death receptor signaling, was investigated in epirubicin- and Taxol-induced apoptosis of B-lymphoma cells. This study was performed because the CD95/Fas receptor-ligand interaction, recruitment of the Fas-associated death domain (FADD) adaptor protein, and subsequent activation of procaspase-8 have been implicated in the execution of drug-induced apoptosis in other cell types. Indeed, active caspase-8 was readily detected after treatment of mature and immature B-lymphoid cells with epirubicin or Taxol. However, neither constitutive nor drug-induced expression of the CD95/Fas ligand was detectable in B-lymphoma cells. Furthermore, overexpression of a dominant-negative FADD mutant (FADDdn) did not block caspase-8 processing and subsequent DNA fragmentation, indicating that drug-induced caspase-8 activation was mediated by a CD95/Fas-independent mechanism. Instead, caspase-8 cleavage was slightly preceded by activation of caspase-3, suggesting that drug-induced caspase-8 activation in B-lymphoma cells is a downstream event mediated by other caspases. This assumption was confirmed in 2 experimental systems-zDEVD-fmk, a cell-permeable inhibitor of caspase-3-like activity, blocked drug-induced caspase-8 cleavage, and depletion of caspase-3 from cell extracts impaired caspase-8 cleavage after in vitro activation with dATP and cytochrome c. Thus, these data indicate that drug-induced caspase-8 activation in B-lymphoma cells is independent of death receptor signaling and is mediated by postmitochondrial caspase-3 activation.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Crosstalk between NF-kappaB-activating and apoptosis-inducing proteins of the TNF-receptor complex.

The cytokine tumor necrosis factor (TNF) elicits a wide range of biological responses, including inflammation, cell proliferation, differentiation, and apoptosis. Although the molecular mechanisms of TNF signaling have been largely elucidated, the principle that regulates the balance of life and death is still unknown. This review will focus on the crosstalk that exists between proteins of the TNF receptor (TNF-R) signalosome, and which are involved in the initiation of nuclear factor kappa B (NF-kappaB) activation or apoptosis. At least four different mechanisms of regulation can be distinguished: (i) NF-kappaB-mediated induction of proteins of the TNF-R complex; (ii) NF-kappaB-independent protection against apoptosis by the TNF-R-associating factor 2 (TRAF2)-mediated recruitment of antiapoptotic proteins; (iii) dual activation of apoptosis and NF-kappaB by a single molecule; and (iv) amplification of the death signal by proteolytic inactivation of signaling proteins that are involved in NF-kappaB activation or cell survival.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Endonuclease G: a mitochondrial protein released in apoptosis and involved in caspase-independent DNA degradation.

A hallmark of apoptosis is the fragmentation of nuclear DNA. Although this activity involves the caspase-3-dependent DNAse CAD (caspase-activated DNAse), evidence exists that DNA fragmentation can occur independently of caspase activity. Here we report on the ability of truncated Bid (tBid) to induce the release of a DNAse activity from mitochondria. This DNAse activity was identified by mass spectrometry as endonuclease G, an abundant 30 kDa protein released from mitochondria under apoptotic conditions. No tBid-induced endonuclease G release could be observed in mitochondria from Bcl-2-transgenic mice. The in vivo occurrence of endonuclease G release from mitochondria during apoptosis was confirmed in the liver from mice injected with agonistic anti-Fas antibody and is completely prevented in Bcl-2 transgenic mice. These data indicate that endonuclease G may be involved in CAD-independent DNA fragmentation during cell death pathways in which truncated Bid is generated.

Animals↗

PubMed2001

Dimethylfumarate is an inhibitor of cytokine-induced nuclear translocation of NF-kappa B1, but not RelA in normal human dermal fibroblast cells.

We previously demonstrated that the oral antipsoriatic dimethylfumarate is an inhibitor of cytokine-induced adhesion molecule expression in endothelial HUVEC cells. We now report the inhibitory effect of dimethylfumarate on tumor-necrosis-factor-alpha- or interleukin-1 alpha-induced intercellular adhesion molecule 1 expression in normal human dermal fibroblasts. Western blots of normal human dermal fibroblast cytoplasmic extracts showed that dimethylfumarate has minor effects on the I kappa B alpha, beta and epsilon proteins: their cytokine-induced degradation and resynthesis is only slowed down, an effect most prominently observed for I kappa B beta. No inhibitory effect of dimethylfumarate was observed on cytokine-induced RelA/p65 or c-Rel accumulation in nuclear extracts of cytokine-treated normal human dermal fibroblast cells. In contrast, cytokine-induced nuclear factor kappa B1/p50 nuclear accumulation was specifically inhibited by dimethylfumarate. This inhibitory effect on nuclear factor kappa B1 nuclear localization in normal human dermal fibroblasts proved sufficient to inhibit nuclear factor kappa B1-RelA binding to nuclear factor kappa B consensus oligonucleotides in DNA binding assays. Likewise, cytokine-induced activation of a pNF kappa B::luciferase reporter construct in transiently transfected normal human dermal fibroblasts was inhibited by dimethylfumarate. The observations support a mechanistic model for the oral antipsoriatic dimethylfumarate in which lowering of nuclear factor kappa B1 leads to changes in the nuclear factor kappa B1-RelA nuclear balance and inhibition of cytokine-induced adhesion molecule expression in normal human dermal fibroblasts.

Active Transport, Cell Nucleus↗

PubMed2000

A20 and A20-binding proteins as cellular inhibitors of nuclear factor-kappa B-dependent gene expression and apoptosis.

Proper gene expression and cell growth are critical for the survival of all organisms. Nuclear factor-kappa B (NF-kappa B)-dependent gene expression and apoptosis play crucial roles in numerous cellular processes, and defects in their regulation may contribute to a variety of diseases including inflammation and cancer. Although there has recently been tremendous progress in our understanding of the signaling pathways that lead to NF-kappa B activation and apoptosis, signaling mechanisms that negatively regulate these processes are only partially understood. This review deals with the zinc finger protein A20, which has been characterized as a dual inhibitor of NF-kappa B activation and apoptosis. Its inducible expression by a wide variety of stimuli, including cytokines such as tumor necrosis factor, interleukin-1, and CD40, as well as bacterial and viral products such as lipopolysaccharide, Epstein-Barr virus latent membrane protein 1, and human T-cell leukemia virus type I Tax, suggests that it is involved in the negative feedback regulation of signaling. We will discuss the possible underlying mechanisms, placing emphasis on the role of several A20-binding proteins that have recently been described. Moreover, evidence is presented that A20 and A20-binding proteins are potential novel therapeutic tools in the treatment of a variety of diseases.

14-3-3 Proteins↗

PubMed2000

Identification and characterization of a novel cell cycle-regulated internal ribosome entry site.

PITSLRE protein kinases are related to the large family of cyclin-dependent kinases. They have been proposed to act as tumor suppressor genes and have been shown to play a role in cell cycle progression. We report that two PITSLRE protein kinase isoforms, namely p11O(PITSLRE) and p58(PITSLRE), are translated from a single transcript by initiation at alternative in-frame AUG codons. p110(PITSLRE) is produced by classical cap-dependent translation, whereas p58(PITSLRE) results from internal initiation of translation controlled by an internal ribosome entry site (IRES) with unique properties. The IRES element is localized to the mRNA coding region, and its activity is cell cycle regulated, which permits translation of p58(PITSLRE) in G2/M.

Animals↗

PubMed2000

Ultrastructural localization of cytochrome c in apoptosis demonstrates mitochondrial heterogeneity.

Release of apoptogenic factors into the cytosol including cytochrome c is triggering the execution phase of apoptosis through activation of cytoplasmic effector caspases. How loss of function of the electron transport chain can be reconciled with an adequate energy supply necessary for executing the apoptotic program was studied in granulosa cell (GC) sheets cultured up to 72 h without gonadotrophic support. Cytochrome c was localized ultrastructurally by oxidation of diaminobenzidine tetrahydrochloride both in living and fixed cells. In uncultured GC sheets all cells show staining over their entire mitochondrial population. In 72 h cultured sheets in the absence of FSH pre-apoptotic GC's display two subsets of mitochondria: normal sized stained mitochondria and small orthodox mitochondria without demonstrable cytochrome function. Apoptotic cells contain several mitochondria with preservation of respiratory function besides unstained orthodox mitochondria. The cytochrome c containing mitochondria typically display dilated intracristal spaces, a mitochondrial conformation related to increased ATP production. Cytochrome c release was confirmed by Western blotting. In 72 h cultures supplemented with FSH, GC's displayed staining over their entire mitochondrial population. In cultures lacking FSH, but partially protected from apoptosis through caspase inhibition, the cytochrome c release was not inhibited. Thus in the present studied model dysfunction of only a subset of mitochondria is instrumental to initiate the apoptotic program while a functional electron transport chain is maintained until the degradation phase in a subset of respiring mitochondria.

Animals↗

PubMed1999

More than one way to die: apoptosis, necrosis and reactive oxygen damage.

Cell death is an essential phenomenon in normal development and homeostasis, but also plays a crucial role in various pathologies. Our understanding of the molecular mechanisms involved has increased exponentially, although it is still far from complete. The morphological features of a cell dying either by apoptosis or by necrosis are remarkably conserved for quite different cell types derived from lower or higher organisms. At the molecular level, several gene products play a similar, crucial role in a major cell death pathway in a worm and in man. However, one should not oversimplify. It is now evident that there are multiple pathways leading to cell death, and some cells may have the required components for one pathway, but not for another, or contain endogenous inhibitors which preclude a particular pathway. Furthermore, different pathways can co-exist in the same cell and are switched on by specific stimuli. Apoptotic cell death, reported to be non-inflammatory, and necrotic cell death, which may be inflammatory, are two extremes, while the real situation is usually more complex. We here review the distinguishing features of the various cell death pathways: caspases (cysteine proteases cleaving after particular aspartate residues), mitochondria and/or reactive oxygen species are often, but not always, key components. As these various caspase-dependent and caspase-independent cell death pathways are becoming better characterized, we may learn to differentiate them, fill in the many gaps in our understanding, and perhaps exploit the knowledge acquired for clinical benefit.

Animals↗