Carme Solé

The death receptor antagonist FAIM promotes neurite outgrowth by a mechanism that depends on ERK and NF-κB signaling

Fas apoptosis inhibitory molecule (FAIM) is a protein identified as an antagonist of Fas-induced cell death. We show that FAIM overexpression fails to rescue neurons from trophic factor deprivation, but exerts a marked neurite growth–promoting action in different neuronal systems. Whereas FAIM overexpression greatly enhanced neurite outgrowth from PC12 cells and sympathetic neurons grown with nerve growth factor (NGF), reduction of endogenous FAIM levels by RNAi decreased neurite outgrowth in these cells. FAIM overexpression promoted NF-κB activation, and blocking this activation by using a super-repressor IκBα or by carrying out experiments using cortical neurons from mice that lack the p65 NF-κB subunit prevented FAIM-induced neurite outgrowth. The effect of FAIM on neurite outgrowth was also blocked by inhibition of the Ras–ERK pathway. Finally, we show that FAIM interacts with both Trk and p75 neurotrophin receptor NGF receptors in a ligand-dependent manner. These results reveal a new function of FAIM in promoting neurite outgrowth by a mechanism involving activation of the Ras–ERK pathway and NF-κB.

Met signals hepatocyte survival by preventing Fas-triggered FLIP degradation in a PI3k-Akt-dependent manner.

The FasL‐Fas couple is a general death mediator whose activated signals lead to caspase‐8 activation and apoptosis in adult hepatocytes. Suppression of caspase‐8 activation and cell death is a protective mechanism modulated by the FLICE‐Like Inhibitory Protein (FLIP). Although hepatocyte growth factor (HGF) and its receptor Met are known to mediate cell survival in developing livers, the molecular mechanisms involved in this process are poorly understood. We show here that Met activation by HGF impairs Fas‐triggered apoptosis of primary embryonic hepatocytes and cell survival correlates with inhibition of caspase‐8 and caspase‐3 activities. Furthermore, we found that HGF treatment prevents degradation of FLIPL triggered by Fas activation. In contrast to this, Met activation does not modulate FLIPL levels and its stability in untreated cells, thus showing the specificity of this regulatory mechanism for embryonic hepatocyte survival. Knocking down FLIP expression abolishes the ability of Met to inhibit Fas‐triggered hepatocyte death, demonstrating the functional requirement of FLIP in HGF anti‐apoptotic signals. By combining genetic and pharmacological approaches, we also demonstrate that the PI3K‐Akt pathway is required in embryonic hepatocytes to prevent Fas‐triggered FLIP degradation and death. Thus, Met acting on PI3K and Akt ensures high levels of FLIPL, and disruption of this pathway contributes to hepatic apoptosis and possibly to Fas‐related liver diseases. (HEPATOLOGY 2007;45:1210–1217.)

Lifeguard/neuronal membrane protein 35 regulates Fas ligand-mediated apoptosis in neurons via microdomain recruitment

Fas ligand (FasL)‐receptor system plays an essential role in regulating cell death in the developing nervous system, and it has been implicated in neurodegenerative and inflammatory responses in the CNS. Lifeguard (LFG) is a protein highly expressed in the hippocampus and the cerebellum, and it shows a particularly interesting regulation by being up‐regulated during postnatal development and in the adult. We show that over‐expression of LFG protected cortical neurons from FasL‐induced apoptosis and decreased caspase‐activation. Reduction of endogenous LFG expression by small interfering RNA sensitized cerebellar granular neurons to FasL‐induced cell death and caspase‐8 activation, and also increased sensitivity of cortical neurons. In differentiated cerebellar granular neurons, protection from FasL‐induced cell death could be attributed exclusively to LFG and appears to be independent of FLICE inhibitor protein. Thus, LFG is an endogenous inhibitor of FasL‐mediated neuronal death and it mediates the FasL resistance of CNS differentiated neurons. Finally, we also demonstrate that LFG is detected in lipid rafts microdomains, where it may interact with Fas receptor and regulate FasL‐activated signaling pathways.

The prevention of the staurosporine‐induced apoptosis by Bcl‐XL, but not by Bcl‐2 or caspase inhibitors, allows the extensive differentiation of human neuroblastoma cells

Staurosporine is one of the best apoptotic inducers in different cell types including neuroblastomas. In this study we have compared the efficiency and final outcome of three different anti‐apoptotic strategies in staurosporine‐treated SH‐SY5Y human neuroblastoma cells. At staurosporine concentrations up to 500 nm, z‐VAD.fmk a broad‐spectrum, noncompetitive inhibitor of caspases, reduced apoptosis in SH‐SY5Y cells. At higher concentrations, z‐VAD.fmk continued to inhibit caspases and the apoptotic phenotype but not cell death which seems to result from oxidative damage. Stable over‐expression of Bcl‐2 in SH‐SY5Y protected cells from death at doses of staurosporine up to 1 µm. At higher doses, cytochrome c release from mitochondria occurred, caspases were activated and cells died by apoptosis. Therefore, we conclude that Bcl‐2 increased the threshold for apoptotic cell death commitment. Over‐expression of Bcl‐XL was far more effective than Bcl‐2. Bcl‐XL transfected cells showed a remarkable resistance staurosporine‐induced cytochrome c release and associated apoptotic changes and survived for up to 15 days in 1 µm staurosporine. In these conditions, SH‐SY5Y displayed a remarkable phenotype of neuronal differentiation as assessed by neurite outgrowth and expression of neurofilament, Tau and MAP‐2 neuronal specific proteins.

The long form of fas apoptotic inhibitory molecule is expressed specifically in neurons and protects them against death receptor-triggered apoptosis

Death receptors (DRs) and their ligands are expressed in developing nervous system. However, neurons are generally resistant to death induction through DRs and rather their activation promotes neuronal outgrowth and branching. These results suppose the existence of DRs antagonists expressed in the nervous system. Fas apoptosis inhibitory molecule (FAIMS) was first identified as a Fas antagonist in B-cells. Soon after, a longer alternative spliced isoform with unknown function was identified and named FAIML. FAIMS is widely expressed, including the nervous system, and we have shown previously that it promotes neuronal differentiation but it is not an anti-apoptotic molecule in this system. Here, we demonstrate that FAIML is expressed specifically in neurons, and its expression is regulated during the development. Expression could be induced by NGF through the extracellular regulated kinase pathway in PC12 (pheochromocytoma cell line) cells. Contrary to FAIMS, FAIML does not increase the neurite outgrowth induced by neurotrophins and does not interfere with nuclear factor κB pathway activation as FAIMS does. Cells overexpressing FAIML are resistant to apoptotic cell death induced by DRs such as Fas or tumor necrosis factor R1. Reduction of endogenous expression by small interfering RNA shows that endogenous FAIML protects primary neurons from DR-induced cell death. The detailed analysis of this antagonism shows that FAIML can bind to Fas receptor and prevent the activation of the initiator caspase-8 induced by Fas. In conclusion, our results indicate that FAIML could be responsible for maintaining initiator caspases inactive after receptor engagement protecting neurons from the cytotoxic action of death ligands.

Cooperation between the INO80 Complex and Histone Chaperones Determines Adaptation of Stress Gene Transcription in the Yeast Saccharomyces cerevisiae

ABSTRACT In yeast, environmental stresses provoke sudden and dramatic increases in gene expression at stress-inducible loci. Stress gene transcription is accompanied by the transient eviction of histones from the promoter and the transcribed regions of these genes. We found that mutants defective in subunits of the INO80 complex, as well as in several histone chaperone systems, exhibit extended expression windows that can be correlated with a distinct delay in histone redeposition during adaptation. Surprisingly, Ino80 became associated with the ORFs of stress genes in a stress-specific way, suggesting a direct function in the repression during adaptation. This recruitment required elongation by RNA polymerase (Pol) II but none of the histone modifications that are usually associated with active transcription, such as H3 K4/K36 methylation. A mutant lacking the Asf1-associated H3K56 acetyltransferase Rtt109 or Asf1 itself also showed enhanced stress-induced transcript levels. Genetic data, however, suggest that Asf1 and Rtt109 function in parallel with INO80 to restore histone homeostasis, whereas Spt6 seems to have a function that overlaps that of the chromatin remodeler. Thus, chromatin remodeling by INO80 in cooperation with Spt6 determines the shape of the expression profile under acute stress conditions, possibly by an elongation-dependent mechanism.

Hepatitis C virus reinfection among prisoners with sustained virological response after treatment for chronic hepatitis C

BACKGROUND & AIMS We estimated HCV reinfection rate and its associated risk factors in inmates with chronic hepatitis C who had achieved sustained virological response (SVR) after completing combination therapy while in prison. METHODS Individuals who had achieved an SVR after treatment provided from January 2003 to December 2009 at four prisons in Catalonia, had been tested annually for HCV RNA and were in prison during 2010, were invited to complete a questionnaire regarding risk factors for reinfection. Incidence rate was calculated as 100 person-years of follow-up. Risk factors potentially associated with reinfection were evaluated by bivariate log-rank test and multivariate Cox regression analysis. RESULTS One hundred and nineteen subjects who had achieved an SVR agreed to participate. 98% were male, with a median age of 33.3 ± 6.3 years and 81% had a history of injection drug use (IDU). After a mean follow-up of 1.4 years, HCV reinfection was identified in nine former IDUs, seven with HCV genotype switch, for an overall reinfection rate of 5.27 cases per 100 person-years. Reinfection incidence was significantly higher among active drug users (HR=12.47; 95% CI: 2.90-53.71), HIV co-infected (HR=9.95; 95% CI: 1.73-57.34), and those engaging in more than one risk behaviors after treatment (HR=7.47; 95% CI: 1.19-46.89). CONCLUSIONS HCV reinfection among inmates after successful treatment is high especially in those with ongoing IDU. Preventative interventions at diagnosis and during and after HCV treatment should be strongly reinforced.

Control of Ubp3 ubiquitin protease activity by the Hog1 SAPK modulates transcription upon osmostress

Protein ubiquitylation is a key process in the regulation of many cellular processes. The balance between the activity of ubiquitin ligases and that of proteases controls the level of ubiquitylation. In response to extracellular stimuli, stress‐activated protein kinases (SAPK) modulate gene expression to maximize cell survival. In yeast, the Hog1 SAPK has a key role in reprogramming the gene expression pattern required for cell survival upon osmostress. Here, we show that the Ubp3 ubiquitin protease is a target for the Hog1 SAPK to modulate gene expression. ubp3 mutant cells are defective in expression of osmoresponsive genes. Hog1 interacts with and phosphorylates Ubp3 at serine 695, which is essential to determine the extent of transcriptional activation in response to osmostress. Furthermore, Ubp3 is recruited to osmoresponsive genes to modulate transcriptional initiation as well as elongation. Therefore, Ubp3 activity responds to external stimuli and is required for transcriptional activation upon osmostress.

BCL-XL regulates TNF-α-mediated cell death independently of NF-κB, FLIP and IAPs

Upon activation, tumor necrosis factor alpha (TNF-α) receptor can engage apoptotic or survival pathways. Inhibition of macromolecular synthesis is known to sensitize cells to TNF-α-induced cell death. It is believed that this sensitization is due to the transcriptional blockade of genes regulated by NF-κB. Nevertheless, such evidence has remained elusive in the nervous system. Here, we show that TNF-α cannot normally induce apoptosis in PC12 cells or cortical neurons. However, cells treated with Actinomycin D (ActD) become susceptible to TNF-α-induced cell death through the activation of caspase-8, generation of tBid and activation of caspase-9 and -3. Analysis of several proteins involved in TNF-α receptor signaling showed no significant downregulation of NF-κB target genes, such as IAPs or FLIP, under such conditions. However, Bcl-xL protein levels, but not those of Bcl-2, Bax and Bak, are reduced by ActD or TNF-α/ActD treatments. Moreover, Bcl-xL overexpression fully protects cells against TNF-α/ActD-induced cell death. When endogenous levels of Bcl-xL are specifically downregulated by lentiviral-based RNAi, cells no longer require ActD to be sensitive to TNF-α-triggered apoptosis. Furthermore, Bcl-xL downregulation does not affect TNF-α-mediated NF-κB activation. Altogether, our results demonstrate that Bcl-xL, and not Bcl-2, FLIP or IAPs, acts as the endogenous regulator of neuronal resistance/sensitivity to TNF-α-induced apoptosis in an NF-κB-independent manner.

Evolution of protein phosphorylation across 18 fungal species

Phosphorylation and fungal evolution Phosphorylation after transcription modifies the activity of proteins. To understand how phosphorylation sites have evolved, Studer et al. studied a range of fungal species (see the Perspective by Matalon et al.). Only a few sites were apparently present in the common ancestor of all 18 species investigated. Evolutionary age appeared to predict the potential functional importance of specific conserved phosphosites. Science, this issue p. 229; see also p. 176 Phosphorylation in fungal proteins offers an understanding of evolutionary constraints acting on posttranscriptional modification. Living organisms have evolved protein phosphorylation, a rapid and versatile mechanism that drives signaling and regulates protein function. We report the phosphoproteomes of 18 fungal species and a phylogenetic-based approach to study phosphosite evolution. We observe rapid divergence, with only a small fraction of phosphosites conserved over hundreds of millions of years. Relative to recently acquired phosphosites, ancient sites are enriched at protein interfaces and are more likely to be functionally important, as we show for sites on H2A1 and eIF4E. We also observe a change in phosphorylation motif frequencies and kinase activities that coincides with the whole-genome duplication event. Our results provide an evolutionary history for phosphosites and suggest that rapid evolution of phosphorylation can contribute strongly to phenotypic diversity.