Biomedical subjects

Marcus E Peter

Publications and source records attributed to Marcus E Peter.

At least 19 recordsLinked to original sources

PubMed2006

Palmitoylation of CD95 facilitates formation of SDS-stable receptor aggregates that initiate apoptosis signaling.

Apoptosis signaling through CD95 (Fas/APO-1) involves aggregation and clustering of the receptor followed by its actin-dependent internalization. Internalization is required for efficient formation of the death-inducing signaling complex (DISC) with maximal recruitment of FADD, caspase-8/10 and c-FLIP occurring when the receptor has reached an endosomal compartment. The first detectable event during CD95 signaling is the formation of SDS-stable aggregates likely reflecting intense oligomerization of the receptor. We now demonstrate that these SDS-stable forms of CD95 correspond to very high molecular weight DISC complexes (hiDISC) and are the sites of caspase-8 activation. hiDISCs are found both inside and outside of detergent-resistant membranes. The formation of SDS-stable CD95 aggregates involves palmitoylation of the membrane proximal cysteine 199 in CD95. Cysteine 199 mutants no longer form SDS-stable aggregates, and inhibition of palmitoylation reduces internalization of CD95 and activation of caspase-8. Our data demonstrate that SDS-stable forms of CD95 are the sites of apoptosis initiation and represent an important early step in apoptosis signaling through CD95 before activation of caspases.

Amino Acid Sequence↗

PubMed2006

Assays of endogenous caspase activities: a comparison of mass spectrometry and fluorescence formats.

This paper describes a label-free assay for measuring endogenous caspase protease activities in cell lysates. The assay format, termed SAMDI-MS (self-assembled monolayers for matrix assisted laser desorption ionization time-of-flight mass spectrometry), is based on the enzymatic modification of peptides immobilized to monolayer substrates, followed by direct detection of the products with mass spectrometry. Monolayers presenting peptide substrates for either caspase-3 or -8 were treated with lysates from Jurkat cells that were stimulated with staurosporine and SKW6.4 cells that were stimulated with LzCD95L. In both cases, the SAMDI assays reported on the activation of endogenous caspase enzymes with levels of detection that are similar to those observed using the commonly employed fluorogenic assays. The use of longer peptide substrates, which are not compatible with the fluorogenic assays, provided for a better resolution of the two caspase activities. This work is significant because it demonstrates that the SAMDI assay can be used to measure endogenous enzyme activities and because it avoids the loss of activity and specificity that often accompany label-dependent assay formats.

Amino Acid Sequence↗

PubMed2006

The role of receptor internalization in CD95 signaling.

Activation of the cell surface CD95 receptor triggers a cascade of signaling events, including assembly of the death-inducing signaling complex (DISC), that culminate in cellular apoptosis. In this study, we demonstrate a general requirement of receptor internalization for CD95 ligand-mediated DISC amplification, caspase activation and apoptosis in type I cells. Recruitment of DISC components to the activated receptor predominantly occurs after the receptor has moved into an endosomal compartment and blockade of CD95 internalization impairs DISC formation and apoptosis. In contrast, CD95 ligand stimulation of cells unable to internalize CD95 results in activation of proliferative Erk and NF-kappaB signaling pathways. Hence, the subcellular localization and internalization pathways of CD95 play important roles in controlling activation of distinct signaling cascades to determine divergent cellular fates.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Serine protease inhibitor 6 protects cytotoxic T cells from self-inflicted injury by ensuring the integrity of cytotoxic granules.

How cytotoxic T lymphocytes (CTLs) kill intracellular pathogens without killing themselves has been a recurring question ever since their discovery. By using mice deficient in Serine Protease Inhibitor 6 (Spi6), we show that by inhibiting granzyme B (GrB), Spi6 protects CTLs from self-inflicted injury. Infection with either Lymphocytic Choriomeningitis virus (LCMV) or Listeria monocytogenes (LM) revealed increased apoptosis and diminished survival of Spi6 knockout (KO) CTLs, which was cell autonomous and could be corrected by GrB deficiency. Spi6 KO mice in turn were impaired in their ability to clear LCMV infection. Spi6 KO CTLs revealed a breakdown in the integrity of cytotoxic granules, increased cytoplasmic GrB, and ensuing apoptosis. We conclude that Spi6 protects CTLs from suicide caused by GrB-mediated breakdown of cytotoxic granules.

Animals↗

PubMed2005

Nonapoptotic functions of FADD-binding death receptors and their signaling molecules.

Death receptors (DRs) are surface receptors that when triggered have the capacity to induce apoptosis in cells by forming the death-inducing signaling complex (DISC). The first protein recruited to form the DISC is the adaptor protein FADD/Mort1. Some members of the DR family, CD95 and the TRAIL receptors DR4 and DR5, directly bind FADD, whereas others, such as TNF receptor I and DR3, initially bind another adaptor protein, TRADD, which then recruits FADD. While all DRs can activate both apoptotic and non-apoptotic pathways, it has been widely assumed that the main physiological role of FADD-binding death receptors is to trigger apoptosis. However, recent work has ascribed multiple non-apoptotic activities to these receptors and/or the signaling components of the DISC.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Local gene density predicts the spatial position of genetic loci in the interphase nucleus.

Specific chromosomal translocations are hallmarks of many human leukemias. The basis for these translocation events is poorly understood, but it has been assumed that spatial positioning of genes in the nucleus of hematopoietic cells is a contributing factor. Analysis of the nuclear 3D position of the gene MLL, frequently involved in chromosomal translocations and five of its translocation partners (AF4, AF6, AF9, ENL and ELL), and two control loci revealed a characteristic radial distribution pattern in all hematopoietic cells studied. Genes in areas of high local gene density were found positioned towards the nuclear center, whereas genes in regions of low gene density were detected closer to the nuclear periphery. The gene density within a 2 Mbp window was found to be a better predictor for the relative positioning of a genomic locus within the cell nucleus than the gene density of entire chromosomes. Analysis of the position of MLL, AF4, AF6 and AF9 in cell lines carrying chromosomal translocations involving these genes revealed that the position of the normal genes was different from that of the fusion genes, and this was again consistent with the changes in local gene density within a 2 Mbp window. Thus, alterations in gene density directly at translocation junctions could explain the change in the position of affected genes in leukemia cells.

Animals↗

PubMed2005

Phosphorylation of FADD at serine 194 by CKIalpha regulates its nonapoptotic activities.

FADD is essential for death receptor (DR)-induced apoptosis. However, it is also critical for cell cycle progression and proliferation, activities that are regulated by phosphorylation of its C-terminal Ser194, which has also been implicated in sensitizing cancer cells to chemotherapeutic drugs and in regulating FADD's intracellular localization. We now demonstrate that casein kinase Ialpha (CKIalpha) phosphorylates FADD at Ser194 both in vitro and in vivo. FADD-CKIalpha association regulates the subcellular localization of FADD, and phosphorylated FADD was found to colocalize with CKIalpha on the spindle poles in metaphase. Inhibition of CKIalpha diminished FADD phosphorylation, prevented the ability of Taxol to arrest cells in mitosis, and blocked mitogen-induced proliferation of mouse splenocytes. In contrast, a low level of cycling splenocytes from mice expressing FADD with a mutated phosphorylation site was insensitive to CKI inhibition. These data suggest that phosphorylation of FADD by CKI is a crucial event during mitosis.

Adaptor Proteins, Signal Transducing↗

PubMed2005

A role for caspase-8 and c-FLIPL in proliferation and cell-cycle progression of primary hepatocytes.

Growth factors are known to favor both proliferation and survival of hepatocytes. In the present study, we investigated if c-FLIP(L) (cellular FLICE-inhibitory protein, long isoform) could be involved in epidermal growth factor (EGF)-stimulated proliferation of rat hepatocytes since c-FLIP(L) regulates both cell proliferation and procaspase-8 maturation. Treatment with MEK inhibitors prevented induction of c-FLIP(L) by EGF along with total inhibition of DNA replication. However, EGF failed to inhibit processing of procaspase-8 in the presence of EGF suggesting that c-FLIP(L) does not play its canonical anti-apoptotic role in this model. Downregulation of c-FLIP expression using siRNA oligonucleotides strongly reduced DNA replication but did not result in enhanced apoptosis. Moreover, intermediate cleavage products of c-FLIP(L) and caspase-8 were found in EGF-treated hepatocytes in the absence of caspase-3 maturation and cell death. To determine whether the Fas/FADD/caspase-8/c-FLIP(L) complex was required for this activity, Fas, procaspase-8 and Fas-associated death domain protein (FADD) expression or function was inhibited using siRNA or constructs encoding dominant negative mutant proteins. Inhibition of any of these components of the Fas/FADD/caspase-8 pathway decreased DNA replication suggesting a function of these proteins in cell-cycle arrest. Similar results were obtained when the IETD-like caspase activity detectable in EGF-treated hepatocytes was inhibited by the pan-caspase inhibitor, z-ASP. Finally, we demonstrated co-immunoprecipitation between EGFR and Fas within 15 min following EGF stimulation. In conclusion, our results indicate that the Fas/FADD/c-FLIP(L)/caspase-8 pathway positively controls the G(1)/S transition in EGF-stimulated hepatocytes. Our data provide new insights into the mechanisms by which apoptotic proteins participate to mitogenic signals during the G(1) phase.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Does CD95 have tumor promoting activities?

CD95 (APO-1/Fas) is an important inducer of the extrinsic apoptosis signaling pathway and therapy induced apoptosis of many tumor cells has been linked to the activity of CD95. Changes in the expression of CD95 and/or its ligand CD95L are frequently found in human cancer. The downregulation or mutation of CD95 has been proposed as a mechanism by which cancer cells avoid destruction by the immune system through reduced apoptosis sensitivity. CD95 has therefore been viewed as a tumor suppressor. Furthermore, increased CD95L concentration in tumor patients has been linked to tumor cells killing infiltrating lymphocytes in a process called "the tumor counter-attack". Recent data have illuminated unknown activities of CD95 in tumor cells with downregulated or mutated CD95 in the presence of increased CD95L. Under these conditions the stimulation of CD95 signals nonapoptotic pathways, activating NF-kappaB and MAP kinases for example, which may result in the induction of tumorigenic or prosurvival genes. A new model of CD95 functions is proposed in which CD95 is converted from a tumor suppressor to a tumor promotor by a single point mutation in one of the CD95 alleles, a situation frequently found in advanced human cancer, resulting in apoptosis resistance and activation of tumorigenic pathways.

Animals↗

PubMed2004

The relevance of NF-kappaB for CD95 signaling in tumor cells.

Most members of the death receptor family including CD95 (APO-1/Fas) have been shown to induce both apoptosis as well as non-apoptotic pathways depending on the tissue and the circumstances. One of the non-apoptotic pathways emanating from CD95, activation of NF-kappaB, has recently been demonstrated to regulate invasiveness of apoptosis resistant tumor cells. In contrast, activation of NF-kappaB in apoptosing cells is believed to be suppressed due to cleavage of various NF-kappaB pathway components by active caspases that execute apoptosis. We now present data demonstrating that in certain highly CD95 apoptosis sensitive cells NF-kappaB is robustly activated. In fact overexpression of apoptosis inhibitors such as Bcl-2 or c-FLIPL in these cells results in decreased activation of NF-kappaB through CD95. We propose a model in which NF-kappaB is generally activated in certain cells but may have different functions depending on whether cells are programmed to die or to survive.

CASP8 and FADD-Like Apoptosis Regulating Protein↗

PubMed2004

Identification of SNF1/AMP kinase-related kinase as an NF-kappaB-regulated anti-apoptotic kinase involved in CD95-induced motility and invasiveness.

The death receptor CD95 (APO-1/Fas) induces apoptosis in many tissues. However, in apoptosis-resistant tumor cells, stimulation of CD95 induces up-regulation of a defined number of mostly anti-apoptotic genes, resulting in increased motility and invasiveness of tumor cells. The majority of these genes are known NF-kappaB target genes. We have identified one of the CD95-regulated genes as the serine/threonine kinase (SNF1/AMP kinase-related kinase (SNARK)), which is induced in response to various forms of metabolic stress. We demonstrate that up-regulation of SNARK in response to CD95 ligand and tumor necrosis factor alpha depends on activation of NF-kappaB. Overexpression of SNARK rendered tumor cells more resistant, whereas a kinase-inactive mutant of SNARK sensitized cells to CD95-mediated apoptosis. Furthermore, small interfering RNA-mediated knockdown of SNARK increased the sensitivity of tumor cells to CD95 ligand- and TRAIL-induced apoptosis. Importantly, cells with reduced expression of SNARK also showed reduced motility and invasiveness in response to CD95 engagement. SNARK therefore represents an NF-kappaB-regulated anti-apoptotic gene that contributes to the tumor-promoting activity of CD95 in apoptosis-resistant tumor cells.

Apoptosis↗

PubMed2004

The flip side of FLIP.

Two major pathways regulate apoptosis induction in mammalian cells. In the extrinsic pathway, apoptosis is induced through specialized surface receptors, whereas in the intrinsic pathway, apoptosis is induced from within the cell, mainly through activation of mitochondria. Shortly after the major mediators of the extrinsic apoptosis pathway, the initiator caspases-8 and -10, were identified, c-FLIP [FLICE-like inhibitory protein; FLICE is FADD (Fas-associated death domain protein)-like interleukin-1beta-converting enzyme], a catalytically inactive caspase-8/-10 homologue, was reported. Whether this structure acts as an inhibitor or promoter of the extrinsic apoptosis pathway has been the subject of much debate. In this issue of the Biochemical Journal, Boatright et al. provide further evidence for the long splice form of c-FLIP (c-FLIP(L)) being an activator of caspase-8/-10, and demonstrate that the resulting heterodimer is enzymically active with a substrate specificity identical with that of the caspase-8 homodimer. Our understanding of the regulators of the extrinsic apoptosis signalling pathway biochemically may provide the means to design drugs to correct the imbalance between apoptosis and proliferation, as found in many diseases.

CASP8 and FADD-Like Apoptosis Regulating Protein↗

PubMed2004

CD95 ligand induces motility and invasiveness of apoptosis-resistant tumor cells.

The apoptosis-inducing death receptor CD95 (APO-1/Fas) controls the homeostasis of many tissues. Despite its apoptotic potential, most human tumors are refractory to the cytotoxic effects of CD95 ligand. We now show that CD95 stimulation of multiple apoptosis-resistant tumor cells by CD95 ligand induces increased motility and invasiveness, a response much less efficiently triggered by TNFalpha or TRAIL. Three signaling pathways resulting in activation of NF-kappaB, Erk1/2 and caspase-8 were found to be important to this novel activity of CD95. Gene chip analyses of a CD95-stimulated tumor cell line identified a number of potential survival genes and genes that are known to regulate increased motility and invasiveness of tumor cells to be induced. Among these genes, urokinase plasminogen activator was found to be required for the CD95 ligand-induced motility and invasiveness. Our data suggest that CD95L, which is found elevated in many human cancer patients, has tumorigenic activities on human cancer cells. This could become highly relevant during chemotherapy, which can cause upregulation of CD95 ligand by both tumor and nontumor cells.

Apoptosis↗

PubMed2004

Intermediate filaments control the intracellular distribution of caspases during apoptosis.

Caspases are responsible for a cascade of events controlling the disassembly of apoptotic cells. We now demonstrate that caspase-9 is activated at an early stage of apoptosis in epithelial cells and all its detectable, catalytically active large subunits (both the p35 and p37) are concentrated on cytokeratin fibrils. Immunolabeling of distinctive neoepitopes, exposed by cleavage of procaspase-9 at either Asp315 or Asp330, was co-localized on these fibrils with active caspase-3, caspase-cleaved cytokeratin-18, death-effector-domain containing DNA-binding protein and ubiquitin. Cytokeratin filaments may thus provide a scaffold whereby active subunits of caspase-9 can activate caspase-3 which, in turn, can activate more caspase-9 so forming an amplification loop to facilitate cleavage of cytokeratin-18, disruption of the cytoskeleton and the ensuing formation of cytoplasmic inclusions. These inclusions, formed from the collapse of fibrils, together with their associated components, also contain ubiquitinated proteins, vimentin, heat-shock protein 72, and tumor necrosis factor receptor type-1-associated death domain protein. Many of their constituents, including active caspases, remain sequestered within these inclusions, even after detergent treatment and isolation. Thus, such inclusions do not merely accumulate disrupted cytokeratins but also sequestrate potentially noxious proteins that could injure healthy neighboring cells.

Apoptosis↗

PubMed2004

Induction of apoptosis and activation of NF-kappaB by CD95 require different signalling thresholds.

Mutations in the death domain of the death receptor CD95 (APO-1/Fas) cause lymphoproliferation and autoimmune disease in both lpr(cg) mice and in patients with autoimmune lymphoproliferative syndrome (ALPS) type Ia. By testing lymphocytes from ALPS type Ia patients, comparing heterozygous with homozygous lpr(cg) mice and coexpressing wild-type and mutant CD95 receptors, we demonstrate that induction of apoptosis requires two wild-type alleles of CD95. By contrast, nuclear factor-kappaB (NF-kappaB) can be fully activated in cells expressing both a mutant and a wild-type CD95 allele, suggesting different thresholds to activate the two signalling pathways. This was confirmed by testing lymphocytes from heterozygous lpr mice, which showed reduced sensitivity to CD95-mediated apoptosis but normal activation of NF-kappaB when compared with wild-type mice. Mutations in CD95 may eliminate the tumour-suppressive function of CD95, at the same time allowing induction of survival or proliferative pathways, which could contribute to the increased risk for lymphoma seen in ALPS type Ia patients.

Alleles↗

PubMed2003

The death effector domain protein family.

Apoptosis signaling is regulated and executed by specialized proteins that often carry protein/protein interaction domains. One of these domains is the death effector domain (DED) that is predominantly found in components of the death-inducing signaling complex, which forms at the members of the death receptor family following their ligation. Both proapoptotic- and antiapoptotic-DED-containing proteins have been identified, which makes these proteins exquisitely suited to the regulation of apoptosis. Aside from their pivotal role in the control of the apoptotic program, DED-containing proteins have recently been demonstrated to exert their influence on other cellular processes as well, including cell proliferation. These data highlight the multiple roles for the members of this family, suggesting that they are suited to control both life and death decisions of cells. Additionally, because they can act proapoptotically, antiapoptotically, or in the regulation of the cell cycle, this family of proteins may be excellent candidates for cancer therapy targets. Oncogene (2003) 22, 8634-8644. doi:10.1038/sj.onc.1207103

Adaptor Proteins, Signal Transducing↗

PubMed2003

NF-kappaB protects from the lysosomal pathway of cell death.

The programme of gene expression induced by RelA/NF-kappaB transcription factors is critical to the control of cell survival. Ligation of 'death receptors' such as tumor necrosis factor receptor 1 (TNF-R1) triggers apoptosis, as well as NF-kappaB, which counteracts this process by activating the transcription of anti-apoptotic genes. In addition to activating caspases, TNF-R1 stimulation causes the release of cathepsins, most notably cathepsin B, from the lysosome into the cytoplasm where they induce apoptosis. Here we report a mechanism by which NF-kappaB protects cells against TNF-alpha-induced apoptosis: inhibition of the lysosomal pathway of apoptosis. NF-kappaB can protect cells from death after TNF-R1 stimulation, by extinguishing cathepsin B activity in the cytosol. This activity of NF-kappaB is mediated, at least in part, by the upregulation of Serine protease inhibitor 2A (Spi2A), a potent inhibitor of cathepsin B. Indeed, Spi2A can substitute for NF-kappaB in suppressing the induction of cathepsin B activity in the cytosol. Thus, inhibition of cathepsin B by Spi2A is a mechanism by which NF-kappaB protects cells from lysosome-mediated apoptosis.

Animals↗

PubMed2003

Two CD95 tumor classes with different sensitivities to antitumor drugs.

CD95 type I and II cells differ in their dependence on mitochondria to execute apoptosis, because antiapoptotic members of the Bcl-2 family render only type II cells resistant to death receptor-induced apoptosis. They can also be distinguished by a more efficient formation of the death-inducing signaling complex in type I cells. We have identified a soluble form of CD95 ligand (S2) that is cytotoxic to type II cells but does not kill type I cells. By testing 58 tumor cell lines of the National Cancer Institute's anticancer drug-screening panel for apoptosis sensitivity to S2 and performing death-inducing signaling complex analyses, we determined that half of the CD95-sensitive cells are type I and half are type II. Most of the type I cell lines fall into a distinct class of tumor cells expressing mesenchymal-like genes, whereas the type II cell lines preferentially express epithelium-like markers. This suggests that type I and II tumor cells represent different stages of carcinogenesis that resemble the epithelial-mesenchymal transition. We then screened the National Cancer Institute database of >42,000 compounds for reagents with patterns of growth inhibition that correlated with either type I or type II cell lines and found that actin-binding compounds selectively inhibited growth of type I cells, whereas tubulin-interacting compounds inhibited growth of type II cells. Our analysis reveals fundamental differences in programs of gene expression between type I and type II cells and could impact the way actin- and microtubule-disrupting antitumor agents are used in tumor therapy.

Antineoplastic Agents↗