The antiviral adaptor proteins Cardif and Trif are processed and inactivated by caspases

The outcome of a viral infection depends on the interplay between the host's capacity to trigger potent antiviral responses and viral mechanisms that counteract them. Although Toll-like receptor (TLR)-3, which recognizes virally derived double-stranded (ds) RNA, transmits downstream antiviral signaling through the TIR adaptor Trif (TICAM-1), viral RNA-sensing RIG-like helicases (RLHs) use the mitochondrial-bound CARD protein Cardif (IPS-1/MAVS/VISA). The importance of these two antiviral signaling pathways is reflected by the fact that both adaptors are inhibited through specific cleavage triggered by the hepatitis C virus serine protease NS3-4A. Here, we show that inactivation can also occur through cellular caspases activated by various pro-apoptotic signals. Upon caspase-dependent cleavage both adaptors loose their capacity to activate the transcription factors interferon regulatory factors (IRF) and NF-kappaB. Importantly, poliovirus infection triggers a caspase-dependent cleavage of Cardif, suggesting that some viruses may activate caspases not only as a mean to facilitate shedding and replication, but also to impair antiviral responses

TRAMP, a novel apoptosis-mediating receptor with sequence homology to tumor necrosis factor receptor 1 and Fas(Apo-1/CD95).

A novel member of the tumor necrosis factor (TNF) receptor family, designated TRAMP, has been identified. The structural organization of the 393 amino acid long human TRAMP is most homologous to TNF receptor 1. TRAMP is abundantly expressed on thymocytes and lymphocytes. Its extracellular domain is composed of four cysteine-rich domains, and the cytoplasmic region contains a death domain known to signal apoptosis. Overexpression of TRAMP leads to two major responses, NF-kappaB activation and apoptosis. TRAMP-induced cell death is inhibited by an inhibitor of ICE-like proteases, but not by Bcl-2. In addition, TRAMP does not appear to interact with any of the known apoptosis-inducing ligands of the TNF family.

AMP-activated protein kinase mediates glucocorticoid-induced metabolic changes: a novel mechanism in Cushing's syndrome.

Chronic exposure to glucocorticoid hormones, resulting from either drug treatment or Cushing's syndrome, results in insulin resistance, central obesity, and symptoms similar to the metabolic syndrome. We hypothesized that the major metabolic effects of corticosteroids are mediated by changes in the key metabolic enzyme adenosine monophosphate-activated protein kinase (AMPK) activity. Activation of AMPK is known to stimulate appetite in the hypothalamus and stimulate catabolic processes in the periphery. We assessed AMPK activity and the expression of several metabolic enzymes in the hypothalamus, liver, adipose tissue, and heart of a rat glucocorticoid-excess model as well as in in vitro studies using primary human adipose and primary rat hypothalamic cell cultures, and a human hepatoma cell line treated with dexamethasone and metformin. Glucocorticoid treatment inhibited AMPK activity in rat adipose tissue and heart, while stimulating it in the liver and hypothalamus. Similar data were observed in vitro in the primary adipose and hypothalamic cells and in the liver cell line. Metformin, a known AMPK regulator, prevented the corticosteroid-induced effects on AMPK in human adipocytes and rat hypothalamic neurons. Our data suggest that glucocorticoid-induced changes in AMPK constitute a novel mechanism that could explain the increase in appetite, the deposition of lipids in visceral adipose and hepatic tissue, as well as the cardiac changes that are all characteristic of glucocorticoid excess. Our data suggest that metformin treatment could be effective in preventing the metabolic complications of chronic glucocorticoid excess.

Insulin resistance in adult cardiomyocytes undergoing dedifferentiation: role of GLUT4 expression and translocation.

Myocardium undergoing remodeling in vivo exhibits insulin resistance that has been attributed to a shift from the insulin-sensitive glucose transporter GLUT4 to the fetal, less insulin-sensitive, isoform GLUT1. To elucidate the role of altered GLUT4 expression in myocardial insulin resistance, glucose uptake and the expression of the glucose transporter isoforms GLUT4 and GLUT1 were measured in adult rat cardiomyocytes (ARC). ARC in culture spontaneously undergo dedifferentiation, hypertrophy-like spreading, and return to a fetal-like gene expression pattern. Insulin stimulation of 2-deoxy-D-glucose uptake was completely abolished on day 2 and 3 of culture and recovered thereafter. Although GLUT4 protein level was reduced, the time-course of unresponsiveness to insulin did not correlate with altered expression of GLUT1 and GLUT4. However, translocation of GLUT4 to the sarcolemma in response to insulin was completely abolished during transient insulin resistance. Insulin-mediated phosphorylation of Akt was not reduced, indicating that activation of phosphatidylinositol 3-kinase (PI3K) was preserved. On the other hand, total and phosphorylated Cbl was reduced during insulin resistance, suggesting that activation of Cbl/CAP is essential for insulin-mediated GLUT4 translocation, in addition to activation of PI3K. Pharmacological inhibition of contraction in insulin-sensitive ARC reduced insulin sensitivity and lowered phosphorylated Cbl. The results suggest that transient insulin resistance in ARC is related to impairment of GLUT4 translocation. A defect in the PI3K-independent insulin signaling pathway involving Cbl seems to contribute to reduced insulin responsiveness and may be related to contractile arrest.

Peptides from Lactobacillus hydrolysates of bovine milk caseins inhibit prolyl-peptidases of human colon cells.

Prolyl-rich peptides derived from hydrolysates of bovine caseins have been previously shown to inhibit angiotensin converting enzyme (ACE) activity, suggesting that they may also be able to inhibit the enzymatic activities of prolyl-specific peptidases. This study shows that peptides derived from α(S1)-casein and β-casein inhibited the enzymatic activities of purified recombinant matrix metalloprotease (MMP)-2, MMP-7, and MMP-9. The inhibitory efficacy was sequence-dependent. These peptides also selectively inhibited the enzymatic activities of prolyl-amino-peptidases, prolyl-amino-dipeptidases, and prolyl-endopeptidases in extracts of HT-29 and SW480 human colon carcinoma cells, but not in intact cells. They were not cytotoxic or growth inhibitory for these cells. Thus, the prolyl-rich selected peptides were good and selective inhibitors of MMPs and post-proline-cleaving proteases, demonstrating their potential to control inadequate proteolytic activity in the human digestive tract, without inducing cytotoxic effects.

Differential phosphoproteomics of fragmenting yeast vacuoles

Many organelles exist in an equilibrium of fragmentation into smaller units and fusion into larger structures, which is coordinated with cell division, the increase in cell mass, and envi¬ronmental conditions. In yeast cells, organelle homeostasis can be studied using the yeast vacuole (lysosome) as a model system. Yeast vacuoles are the main compartment for degrada¬tion of cellular proteins and storage of nutrients, ions and metabolites. Fission and fusion of vacuoles can be induced by hyper- and hypotonic shock in vivo, respectively, and have also been reconstituted in vitro using isolated vacuoles. The conserved serine/threonine kinase TOR (target of rapamycin) is a central nutrient sensor and regulates cell growth and metabolism. In yeast, there are two TOR proteins, Torlp and Tor2p, which are part of larger protein complexes, TORCI and TORC2. Only TORCI is rapamycin-sensitive. Disregulation of TOR signaling is linked to a multitude of diseases in humans, e.g. cancer, neurodegenerative diseases and metabolic syndrome. It has been shown that TORCI localizes to the vacuole membrane, and recent findings of our laboratory demonstrated that TORCI positively regulates vacuole fragmentation. This suggests that the fragmentation machinery should contain target proteins phosphorylated by TORCI. I explored the rapamycin-and fission-dependent vacuolar phosphoproteome during frag¬mentation, using a label-free mass-spectrometry approach. I identified many vacuolar factors whose phosphorylation was downregulated in a TORCI- and fission-dependent manner. Among them were known protein complexes that are functionally linked to fission or fusion, like the HOPS, VTC and FAB1 complexes. Hence, TORCI-dependent phosphorylations might positively regulate vacuole fission. Several candidates were chosen for detailed microscopic analysis of in vivo vacuole frag-mentation, using deletion mutants. I was able to identify novel factors not previously linked to fission phenotypes, e.g. the SEA complex, Pib2, and several vacuolar amino acid transporters. Transport of neutral and basic amino acids across the membrane seems to control vacuole fission, possibly via TORCI. I analyzed vacuolar fluxes of amino acids in wildtype yeast cells and found evidence for a selective vacuolar export of basic amino acids upon hyperosmotic stress. This leads me to propose a model where vacuolar export of amino acids is necessary to reshape the organelle under salt stress. - Le nombre et la taille de certaines organelles peut être déterminé par un équilibre entre la fragmentation qui produit des unités plus petites et la fusion qui génère des structures plus larges. Cet équilibre est coordonné avec la division cellulaire, l'augmentation de la masse cellulaire, et les conditions environnementales. Dans des cellules de levure, l'homéostasie des organelles peut être étudié à l'aide d'un système modèle, la vacuole de levure (lysosome). Les vacuoles constituent le principal compartiment de la dégradation des protéines et de stockage des nutriments, des ions et des métabolites. La fragmentation et la fusion des vacuoles peuvent être respectivement induites par un traitement hyper- ou hypo-tonique dans les cellules vivantes. Ces processus ont également été reconstitués in vitro en utilisant des vacuoles isolées. La sérine/thréonine kinase conservée TOR (target of rapamycin/cible de la rapamycine) est un senseur de nutriments majeur qui régule la croissance cellulaire et le métabolisme. Chez la levure, il existe deux protéines TOR, Torlp et Tor2p, qui sont les constituants de plus grands complexes de protéines, TORCI et TORC2. TORCI est spécifiquement inhibé par la rapamycine. Une dysrégulation de la signalisation de TOR est liée à une multitude de maladies chez l'homme comme le cancer, les maladies neurodégénératives et le syndrome métabolique. Il a été montré que TORCI se localise à la membrane vacuolaire et les découvertes récentes de notre laboratoire ont montré que TORCI régule positivement la fragmentation de la vacuole. Ceci suggère que le mécanisme de fragmentation doit être contrôlé par la phosphorylation de certaines protéines cibles de TORCI. J'ai exploré le phosphoprotéome vacuolaire lors de la fragmentation, en présence ou absence de rapamycine et dans des conditions provoquant la fragmentation des organelles. La méthode choisie pour réaliser la première partie de ce projet a été la spectrométrie de masse différentielle sans marquage. J'ai ainsi identifié plusieurs facteurs vacuolaires dont la phosphorylation est régulée d'une manière dépendante de TORCI et de la fragmentation. Parmi ces facteurs, des complexes protéiques connus qui sont fonctionnellement liées à fragmentation ou la fusion, comme les complexes HOPS, VTC et FAB1 ont été mis en évidence. Par conséquent, la phosphorylation dépendante de TORCI peut réguler positivement la fragmentation des vacuoles. Plusieurs candidats ont été choisis pour une analyse microscopique détaillée de la fragmentation vacuolaire in vivo en utilisant des mutants de délétion. J'ai été en mesure d'identifier de nouveaux facteurs qui n'avaient pas été encore associés à des phénotypes de fragmentation tels que les complexes SEA, Pib2p, ainsi que plusieurs transporteurs vacuolaires d'acides aminés. Le transport des acides aminés à travers la membrane semble contrôler la fragmentation de la vacuole. Puisque ces transporteurs sont phosphorylés par TORCI, ces résultats semblent confirmer la

Intracellular ubiquitylation of the epithelial Na+ channel controls extracellular proteolytic channel activation via conformational change.

The epithelial Na(+) channel ENaC is a key player in the maintenance of whole body Na(+) balance, and consequently of blood pressure. It is tightly regulated by numerous signaling pathways including ubiquitylation via the ubiquitin-protein ligase Nedd4-2. This mechanism is itself under the control of several kinases, which phosphorylate Nedd4-2, thereby interfering with ENaC/Nedd4-2 interaction, or by Usp2-45, which binds to and deubiquitylates ENaC. Another, different regulatory mechanism concerns the proteolytic activation of ENaC, during which the channel is cleaved on its luminal side by intracellular convertases such as furin, and further activated by extracellular proteases such as CAP-1. This process is regulated as well but the underlying mechanisms are not understood. Previously, evidence was provided that the ubiquitylation status of ENaC may affect the cleavage of the channel. When ubiquitylation of ENaC was reduced, either by co-expressing Usp2-45, or mutating either the ENaC PY-motifs (i.e. the binding sites for Nedd4-2) or intracellular lysines (i.e. ubiquitylation sites), the level of channel cleavage was increased. Here we demonstrate that lysine-mutated ENaC channels are not ubiquitylated at the cell surface, are preferentially cleaved, and Usp2-45 does not affect their cleavage efficiency. We further show by limited proteolysis that the intracellular ubiquitylation status of ENaC affects the extracellular conformation of αENaC, by demonstrating that non-ubiquitylated channels are more efficiently cleaved when treated with extracellularly added trypsin or chymotrypsin. These results present a new paradigm in which an intracellular, post-translational modification (e.g. ubiquitylation) of a transmembrane protein can affect its extracellular conformation.

Sorafenib fourth-line treatment in imatinib-, sunitinib-, and nilotinib-resistant metastatic GIST : a retrospective analysis : 10564

Background: Gastrointestinal stromal tumors (GISTs) are rare mesenchymal tumors usually caused by mutations in the KIT or PDGFRA gene. Advanced disease generally cannot be cured by surgery nor by tyrosine kinase inhibitors (TKI), but TKIs have considerably improved outcome for patients (pts) with advanced GIST. Patients failing TKI treatment with imatinib (IM), sunitinib (SU) or nilotinib (NI) have a poor prognosis. Sorafenib is a multi kinase inhibitor that blocks not only receptor tyrosine kinases such as KIT, VEGFR and PDGFR but also serine/threonine kinases along the RAS/RAF/MEK/ERK pathway. Recently, clinical activity of sorafenib in third-line treatment in patients with GIST after IM and SU failure has been shown (Wiebe et al. ASCO 2008, #10502). Methods: We report herein preliminary data of 32 pts treated with sorafenib in nine European centers. Centers were selected based on their previous and known experience in GIST and reported all pts treated. Pts received sorafenib after failure of IM, SU and NI in fourth-line treatment. Baseline characteristics and treatment details have been retrieved via questionary. Results: Median age at sorafenib treatment start was 62 years (range 33-81 y), and the majority of pts were male (63 %). Primary tumor site was gastric or small intestine in 25% and 41% of pts, respectively. All pts had failed IM, SU, NI. 19 % of pts achieved partial remission and 44% disease stabilization. Approximately half of the pts had an improvement of symptoms and/or performance. Half of the pts were on treatment longer than 4 months (actuarial data) and 41% of pts continue to receive sorafenib. Median progression-free survival is 20 weeks and median overall survival 42 weeks (Kaplan-Meier), at a median follow-up of 22 weeks (range 3-54). Conclusions: This is the largest series assessing efficacy of sorafenib fourth-line treatment for IM, SU and NI refractory GIST reported yet. Sorafenib displays significant clinical activity in this heavily pretreated group of patients.

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Protein destabilization by mutations or external stresses may lead to misfolding and aggregation in the cell. Often, damage is not limited to a simple loss of function, but the hydrophobic exposure of aggregate surfaces may impair membrane functions and promote the aggregation of other proteins. Such a "proteinacious infectious" behavior is not limited to prion diseases. It is associated to most protein-misfolding neurodegenerative diseases and to aging in general. With the molecular chaperones and proteases, cells have evolved powerful tools that can specifically recognize and act upon misfolded and aggregated proteins. Whereas some chaperones passively prevent aggregate formation and propagation, others actively unfold and solubilize stable aggregates. In particular, ATPase chaperones and proteases serve as an intracellular defense network that can specifically identify and actively remove by refolding or degradation potentially infectious cytotoxic aggregates. Here we discuss two types of molecular mechanisms by which ATPase chaperones may actively solubilize stable aggregates: (1) unfolding by power strokes, using the Hsp100 ring chaperones, and (2) unfolding by random movements of individual Hsp70 molecules. In bacteria, fungi, and plants, the two mechanisms are key for reducing protein damages from abiotic stresses. In animals devoid of Hsp100, Hsp70 appears as the core element of the chaperone network, preventing the formation and actively removing disease-causing protein aggregates.

Induction of oxidative stress, lysosome activation and autophagy by nanoparticles in human brain-derived endothelial cells.

Different types of NPs (nanoparticles) are currently under development for diagnostic and therapeutic applications in the biomedical field, yet our knowledge about their possible effects and fate in living cells is still limited. In the present study, we examined the cellular response of human brain-derived endothelial cells to NPs of different size and structure: uncoated and oleic acid-coated iron oxide NPs (8-9 nm core), fluorescent 25 and 50 nm silica NPs, TiO2 NPs (21 nm mean core diameter) and PLGA [poly(lactic-co-glycolic acid)]-PEO [poly(ethylene oxide)] polymeric NPs (150 nm). We evaluated their uptake by the cells, and their localization, generation of oxidative stress and DNA-damaging effects in exposed cells. We show that NPs are internalized by human brain-derived endothelial cells; however, the extent of their intracellular uptake is dependent on the characteristics of the NPs. After their uptake by human brain-derived endothelial cells NPs are transported into the lysosomes of these cells, where they enhance the activation of lysosomal proteases. In brain-derived endothelial cells, NPs induce the production of an oxidative stress after exposure to iron oxide and TiO2 NPs, which is correlated with an increase in DNA strand breaks and defensive mechanisms that ultimately induce an autophagy process in the cells.

LKB1 regulates polarity remodeling and adherens junction formation in the Drosophila eye.

The serine-threonine kinase LKB1 regulates cell polarity from Caenorhabditis elegans to man. Loss of lkb1 leads to a cancer predisposition, known as Peutz-Jeghers Syndrome. Biochemical analysis indicates that LKB1 can phosphorylate and activate a family of AMPK- like kinases, however, the precise contribution of these kinases to the establishment and maintenance of cell polarity is still unclear. Recent studies propose that LKB1 acts primarily through the AMP kinase to establish and/or maintain cell polarity. To determine whether this simple model of how LKB1 regulates cell polarity has relevance to complex tissues, we examined lkb1 mutants in the Drosophila eye. We show that adherens junctions expand and apical, junctional, and basolateral domains mix in lkb1 mutants. Surprisingly, we find LKB1 does not act primarily through AMPK to regulate cell polarity in the retina. Unlike lkb1 mutants, ampk retinas do not show elongated rhabdomeres or expansion of apical and junctional markers into the basolateral domain. In addition, nutrient deprivation does not reveal a more dramatic polarity phenotype in lkb1 photoreceptors. These data suggest that AMPK is not the primary target of LKB1 during eye development. Instead, we find that a number of other AMPK-like kinase, such as SIK, NUAK, Par-1, KP78a, and KP78b show phenotypes similar to weak lkb1 loss of function in the eye. These data suggest that in complex tissues, LKB1 acts on an array of targets to regulate cell polarity.

Genome sequencing of the plant pathogen Taphrina deformans, the causal agent of peach leaf curl.

Taphrina deformans is a fungus responsible for peach leaf curl, an important plant disease. It is phylogenetically assigned to the Taphrinomycotina subphylum, which includes the fission yeast and the mammalian pathogens of the genus Pneumocystis. We describe here the genome of T. deformans in the light of its dual plant-saprophytic/plant-parasitic lifestyle. The 13.3-Mb genome contains few identifiable repeated elements (ca. 1.5%) and a relatively high GC content (49.5%). A total of 5,735 protein-coding genes were identified, among which 83% share similarities with other fungi. Adaptation to the plant host seems reflected in the genome, since the genome carries genes involved in plant cell wall degradation (e.g., cellulases and cutinases), secondary metabolism, the hallmark glyoxylate cycle, detoxification, and sterol biosynthesis, as well as genes involved in the biosynthesis of plant hormones. Genes involved in lipid metabolism may play a role in its virulence. Several locus candidates for putative MAT cassettes and sex-related genes akin to those of Schizosaccharomyces pombe were identified. A mating-type-switching mechanism similar to that found in ascomycetous yeasts could be in effect. Taken together, the findings are consistent with the alternate saprophytic and parasitic-pathogenic lifestyles of T. deformans. IMPORTANCE: Peach leaf curl is an important plant disease which causes significant losses of fruit production. We report here the genome sequence of the causative agent of the disease, the fungus Taphrina deformans. The genome carries characteristic genes that are important for the plant infection process. These include (i) proteases that allow degradation of the plant tissues; (ii) secondary metabolites which are products favoring interaction of the fungus with the environment, including the host; (iii) hormones that are responsible for the symptom of severely distorted leaves on the host; and (iv) drug detoxification enzymes that confer resistance to fungicides. The availability of the genome allows the design of new drug targets as well as the elaboration of specific management strategies to fight the disease.

Role of salt-inducible kinase 1 in the activation of MEF2-dependent transcription by BDNF.

Substantial evidence supports a role for myocyte enhancer factor 2 (MEF2)-mediated transcription in neuronal survival, differentiation and synaptic function. In developing neurons, it has been shown that MEF2-dependent transcription is regulated by neurotrophins. Despite these observations, little is known about the cellular mechanisms by which neurotrophins activate MEF2 transcriptional activity. In this study, we examined the role of salt-inducible kinase 1 (SIK1), a member of the AMP-activated protein kinase (AMPK) family, in the regulation of MEF2-mediated transcription by the neurotrophin brain-derived neurotrophic factor (BDNF). We show that BDNF increases the expression of SIK1 in primary cultures of rat cortical neurons through the extracellular signal-regulated kinase 1/2 (ERK1/2)-signaling pathway. In addition to inducing SIK1 expression, BDNF triggers the phosphorylation of SIK1 at Thr182 and its translocation from the cytoplasm to the nucleus of cortical neurons. The effects of BDNF on the expression, phosphorylation and, translocation of SIK1 are followed by the phosphorylation and nuclear export of histone deacetylase 5 (HDAC5). Blockade of SIK activity with a low concentration of staurosporine abolished BDNF-induced phosphorylation and nuclear export of HDAC5 in cortical neurons. Importantly, stimulation of HDAC5 phosphorylation and nuclear export by BDNF is accompanied by the activation of MEF2-mediated transcription, an effect that is suppressed by staurosporine. Consistent with these data, BDNF induces the expression of the MEF2 target genes Arc and Nur77, in a staurosporine-sensitive manner. In further support of the role of SIK1 in the regulation of MEF2-dependent transcription by BDNF, we found that expression of wild-type SIK1 or S577A SIK1, a mutated form of SIK1 which is retained in the nucleus of transfected cells, is sufficient to enhance MEF2 transcriptional activity in cortical neurons. Together, these data identify a previously unrecognized mechanism by which SIK1 mediates the activation of MEF2-dependent transcription by BDNF.