Autophagy induction in atrophic muscle cells requires ULK1 activation by TRIM32 through unanchored K63-linked polyubiquitin chains.

Optimal autophagic activity is crucial to maintain muscle integrity, with either reduced or excessive levels leading to specific myopathies. LGMD2H is a muscle dystrophy caused by mutations in the ubiquitin ligase TRIM32, whose function in muscles remains not fully understood. Here, we show that TRIM32 is required for the induction of muscle autophagy in atrophic conditions using both in vitro and in vivo mouse models. Trim32 inhibition results in a defective autophagy response to muscle atrophy, associated with increased ROS and MuRF1 levels. The proautophagic function of TRIM32 relies on its ability to bind the autophagy proteins AMBRA1 and ULK1 and stimulate ULK1 activity via unanchored K63-linked polyubiquitin. LGMD2H-causative mutations impair TRIM32's ability to bind ULK1 and induce autophagy. Collectively, our study revealed a role for TRIM32 in the regulation of muscle autophagy in response to atrophic stimuli, uncovering a previously unidentified mechanism by which ubiquitin ligases activate autophagy regulators.

Methods to Study the BECN1 Interactome in the Course of Autophagic Responses.

Autophagy is an extremely dynamic process that mediates the rapid degradation of intracellular components in response to different stress conditions. The autophagic response is executed by specific protein complexes, whose function is regulated by posttranslational modifications and interactions with positive and negative regulators. A comprehensive analysis of how autophagy complexes are temporally modified upon stress stimuli is therefore particularly relevant to understand how this pathway is regulated. Here, we describe a method to define the protein-protein interaction network of a central complex involved in autophagy induction, the Beclin 1 complex. This method is based on the quantitative comparison of protein complexes immunopurified at different time points using a stable isotope labeling by amino acids in cell culture approach. Understanding how the Beclin 1 complex dynamically changes in response to different stress stimuli may provide useful insights to disclose novel molecular mechanisms responsible for the dysregulation of autophagy in pathological conditions, such as cancer, neurodegeneration, and infections.

Role of autophagy in HIV infection and pathogenesis.

The aim of autophagy is to re-establish homeostasis in response to a variety of stress conditions. By forming double-membrane vesicles, autophagy engulfs damaged or superfluous cytoplasmic material and recycles degradation products for new synthesis or energy production. Of note, the same mechanism is used to capture pathogens and has important implications in both innate and adaptive immunity. To establish a chronic infection, pathogens have therefore evolved multiple mechanisms to evade autophagy-mediated degradation. HIV infection represents one of the best characterized systems in which autophagy is disarmed by a virus using multiple strategies to prevent the sequestration and degradation of its proteins and to establish a chronic infection. HIV alters autophagy at various stages of the process in both infected and bystander cells. In particular, the HIV proteins TAT, NEF and ENV are involved in this regulation by either blocking or stimulating autophagy through direct interaction with autophagy proteins and/or modulation of the mTOR pathway. Although the roles of autophagy during HIV infection are multiple and vary amongst the different cell types, several lines of evidence point to a potential beneficial effect of stimulating autophagy-mediated lysosomal degradation to potentiate the immune response to HIV. Characterization of the molecular mechanisms regulating selective autophagy is expected to be valuable for developing new drugs able to specifically enhance the anti-HIV response.

Inhibition of autophagy in EBV-positive Burkitt's lymphoma cells enhances EBV lytic genes expression and replication.

Autophagy, an important degradation system involved in maintaining cellular homeostasis, serves also to eliminate pathogens and process their fragments for presentation to the immune system. Several viruses have been shown to interact with the host autophagic machinery to suppress or make use of this cellular catabolic pathway to enhance their survival and replication. Epstein Barr virus (EBV) is a γ-herpes virus associated with a number of malignancies of epithelial and lymphoid origin in which establishes a predominantly latent infection. Latent EBV can periodically reactivate to produce infectious particles that allow the virus to spread and can lead to the death of the infected cell. In this study, we analyzed the relationship between autophagy and EBV reactivation in Burkitt's lymphoma cells. By monitoring autophagy markers and EBV lytic genes expression, we demonstrate that autophagy is enhanced in the early phases of EBV lytic activation but decreases thereafter concomitantly with increased levels of EBV lytic proteins. In a cell line defective for late antigens expression, we found an inverse correlation between EBV early antigens expression and autophagosomes formation, suggesting that early after activation, the virus is able to suppress autophagy. We report here for the first time that inhibition of autophagy by Bafilomycin A1 or shRNA knockdown of Beclin1 gene, highly incremented EBV lytic genes expression as well as intracellular viral DNA and viral progeny yield. Taken together, these findings indicate that EBV activation induces the autophagic response, which is soon inhibited by the expression of EBV early lytic products. Moreover, our findings open the possibility that pharmacological inhibitors of autophagy may be used to enhance oncolytic viral therapy of EBV-related lymphomas.

Oncogenic BRAF induces chronic ER stress condition resulting in increased basal autophagy and apoptotic resistance of cutaneous melanoma.

The notorious unresponsiveness of metastatic cutaneous melanoma to current treatment strategies coupled with its increasing incidence constitutes a serious worldwide clinical problem. Moreover, despite recent advances in targeted therapies for patients with BRAF(V600E) mutant melanomas, acquired resistance remains a limiting factor and hence emphasises the acute need for comprehensive pre-clinical studies to increase the biological understanding of such tumours in order to develop novel effective and longlasting therapeutic strategies. Autophagy and ER stress both have a role in melanoma development/progression and chemoresistance although their real impact is still unclear. Here, we show that BRAF(V600E) induces a chronic ER stress status directly increasing basal cell autophagy. BRAF(V600E)-mediated p38 activation stimulates both the IRE1/ASK1/JNK and TRB3 pathways. Bcl-XL/Bcl-2 phosphorylation by active JNK releases Beclin1 whereas TRB3 inhibits the Akt/mTor axes, together resulting in an increase in basal autophagy. Furthermore, we demonstrate chemical chaperones relieve the BRAF(V600E)-mediated chronic ER stress status, consequently reducing basal autophagic activity and increasing the sensitivity of melanoma cells to apoptosis. Taken together, these results suggest enhanced basal autophagy, typically observed in BRAF(V600E) melanomas, is a consequence of a chronic ER stress status, which ultimately results in the chemoresistance of such tumours. Targeted therapies that attenuate ER stress may therefore represent a novel and more effective therapeutic strategy for BRAF mutant melanoma.

The transcriptional co-activator SND1 is a novel regulator of alternative splicing in prostate cancer cells.

Splicing abnormalities have profound impact in human cancer. Several splicing factors, including SAM68, have pro-oncogenic functions, and their increased expression often correlates with human cancer development and progression. Herein, we have identified using mass spectrometry proteins that interact with endogenous SAM68 in prostate cancer (PCa) cells. Among other interesting proteins, we have characterized the interaction of SAM68 with SND1, a transcriptional co-activator that binds spliceosome components, thus coupling transcription and splicing. We found that both SAM68 and SND1 are upregulated in PCa cells with respect to benign prostate cells. Upregulation of SND1 exerts a synergic effect with SAM68 on exon v5 inclusion in the CD44 mRNA. The effect of SND1 on CD44 splicing required SAM68, as it was compromised after knockdown of this protein or mutation of the SAM68-binding sites in the CD44 pre-mRNA. More generally, we found that SND1 promotes the inclusion of CD44 variable exons by recruiting SAM68 and spliceosomal components on CD44 pre-mRNA. Inclusion of the variable exons in CD44 correlates with increased proliferation, motility and invasiveness of cancer cells. Strikingly, we found that knockdown of SND1, or SAM68, reduced proliferation and migration of PCa cells. Thus, our findings strongly suggest that SND1 is a novel regulator of alternative splicing that promotes PCa cell growth and survival.

Caspase-2 promotes cytoskeleton protein degradation during apoptotic cell death.

The caspase family of proteases cleaves large number of proteins resulting in major morphological and biochemical changes during apoptosis. Yet, only a few of these proteins have been reported to selectively cleaved by caspase-2. Numerous observations link caspase-2 to the disruption of the cytoskeleton, although it remains elusive whether any of the cytoskeleton proteins serve as bona fide substrates for caspase-2. Here, we undertook an unbiased proteomic approach to address this question. By differential proteome analysis using two-dimensional gel electrophoresis, we identified four cytoskeleton proteins that were degraded upon treatment with active recombinant caspase-2 in vitro. These proteins were degraded in a caspase-2-dependent manner during apoptosis induced by DNA damage, cytoskeleton disruption or endoplasmic reticulum stress. Hence, degradation of these cytoskeleton proteins was blunted by siRNA targeting of caspase-2 and when caspase-2 activity was pharmacologically inhibited. However, none of these proteins was cleaved directly by caspase-2. Instead, we provide evidence that in cells exposed to apoptotic stimuli, caspase-2 probed these proteins for proteasomal degradation. Taken together, our results depict a new role for caspase-2 in the regulation of the level of cytoskeleton proteins during apoptosis.

Applying proteomic technology to clinical virology.

Developing antiviral drugs, vaccines and diagnostic markers is still the most ambitious challenge in clinical virology. In the past few decades, data from high-throughput technologies have allowed for the rapid development of new antiviral therapeutic strategies, thus making a profound impact on translational research. Most of the current preclinical studies in virology are aimed at evaluating the dynamic composition and localization of the protein platforms involved in various host-virus interactions. Among the different possible approaches, mass spectrometry-based proteomics is increasingly being used to define the protein composition in subcellular compartments, quantify differential protein expression among samples, characterize protein complexes, and analyse protein post-translational modifications. Here, we review the current knowledge of the most useful proteomic approaches in the study of viral persistence and pathogenicity, with a particular focus on recent advances in hepatitis C research.

Proteolysis of Ambra1 during apoptosis has a role in the inhibition of the autophagic pro-survival response.

Under stress conditions, pro-survival and pro-death processes are concomitantly activated and the final outcome depends on the complex crosstalk between these pathways. In most cases, autophagy functions as an early-induced cytoprotective response, favoring stress adaptation by removing damaged subcellular constituents. Moreover, several lines of evidence suggest that autophagy inactivation by the apoptotic machinery is a crucial event for cell death execution. Here we show that apoptotic stimuli induce a rapid decrease in the level of the autophagic factor Activating Molecule in Beclin1-Regulated Autophagy (Ambra1). Ambra1 degradation is prevented by concomitant inhibition of caspases and calpains. By both in vitro and in vivo approaches, we demonstrate that caspases are responsible for Ambra1 cleavage at the D482 site, whereas calpains are involved in complete Ambra1 degradation. Finally, we show that Ambra1 levels are critical for the rate of apoptosis induction. RNA interference-mediated Ambra1 downregulation further sensitizes cells to apoptotic stimuli, while Ambra1 overexpression and, more efficiently, a caspase non-cleavable mutant counteract cell death by prolonging autophagy induction. We conclude that Ambra1 is an important target of apoptotic proteases resulting in the dismantling of the autophagic machinery and the accomplishment of the cell death program.

The DNA repair complex Ku70/86 modulates Apaf1 expression upon DNA damage.

Apaf1 is a key regulator of the mitochondrial intrinsic pathway of apoptosis, as it activates executioner caspases by forming the apoptotic machinery apoptosome. Its genetic regulation and its post-translational modification are crucial under the various conditions where apoptosis occurs. Here we describe Ku70/86, a mediator of non-homologous end-joining pathway of DNA repair, as a novel regulator of Apaf1 transcription. Through analysing different Apaf1 promoter mutants, we identified an element repressing the Apaf1 promoter. We demonstrated that Ku70/86 is a nuclear factor able to bind this repressing element and downregulating Apaf1 transcription. We also found that Ku70/86 interaction with Apaf1 promoter is dynamically modulated upon DNA damage. The effect of this binding is a downregulation of Apaf1 expression immediately following the damage to DNA; conversely, we observed Apaf1 upregulation and apoptosis activation when Ku70/86 unleashes the Apaf1-repressing element. Therefore, besides regulating DNA repair, our results suggest that Ku70/86 binds to the Apaf1 promoter and represses its activity. This may help to inhibit the apoptosome pathway of cell death and contribute to regulate cell survival.

Bcl-2 inhibits the caspase-dependent apoptosis induced by SARS-CoV without affecting virus replication kinetics.

Vero cells transfected with either neo- or bcl-2-plasmid were infected with SARS-CoV at a high multiplicity of infection. Apoptosis appeared after the onset of CPE and completion of virus replication, and could be prevented by Bcl-2 expression. Apoptosis is likely mediated by the mitochondrial pathway, as demonstrated by its inhibition using Bcl-2, and by the activation of the caspase cascade, resulting in PARP cleavage. Prevention of apoptosis did not affect susceptibility to infection, kinetics and extent of viral replication and release, thus implying that apoptosis is not involved in facilitating release and/or dissemination of SARS-CoV in Vero cells.

Expression of Werner and Bloom syndrome genes is differentially regulated by in vitro HIV-1 infection of peripheral blood mononuclear cells.

In HIV infection, continuous immune activation leads to accelerated ageing of the adaptive immune system, similar to that observed in elderly people. We investigated the expression of WRN and BLM (genes involved in disorders characterized by premature ageing, genomic instability and cancer predisposition) in peripheral blood mononuclear cells (PBMC) activated in vitro with phytohaemagglutinin (PHA) and infected with different HIV-1 strains. The steady state levels of mRNA were analysed by reverse transcription-polymerase chain reaction (RT-PCR), and protein expression was assayed using immunocytochemistry and Western blot techniques. In uninfected PBMC, PHA stimulation induced an increase in BLM mRNA and protein expression, while WRN expression remained virtually unchanged. When PBMC were infected in vitro with a lymphotropic HIV-1 strain, the level of BLM mRNA showed a peak at 24 h of infection, followed by a decline to uninfected culture levels. A similar result failed to be seen using an R5-tropic HIV-1 strain. In accordance with mRNA expression, in HIV-infected cultures PBMC were stained more frequently and more intensely by a BLM-specific antibody as compared to uninfected cultures, staining peaking at 24. Conversely, WRN expression was not modulated by HIV-1. The proportion of cells showing BLM up-regulation, established by immunocytochemical staining, was much greater than the proportion of productively infected PBMC, as established by proviral DNA measurement. This result indicates that BLM up-regulation is probably a result of an indirect bystander cell effect. Activation of the BLM gene in infected PBMC suggests that premature ageing could be a further immunopathogenetic mechanism involved in HIV-induced immunodeficiency, and points to a possible new candidate target for innovative therapeutic intervention.

"Tissue" transglutaminase expression in HIV-infected cells: an enzyme with an antiviral effect?

The cytopathic effect of HIV has been shown to be associated with the induction of apoptosis and the inhibition of proliferation of T cells. However, the cellular and molecular mechanisms at the basis of the dramatic immune cell loss caused by HIV in patients suffering from acquired immunodeficient syndrome (AIDS), are not yet fully established. We demonstrated that "tissue" transglutaminase (tTG) gene expression is induced in the immune system of seropositive individuals (peripheral blood mononuclear cells and lymph nodes). tTG is a multifunctional protein involved in a variety of fundamentally important cellular functions, in addition to cell death by apoptosis. The presence of high tTG levels in immune-competent cells of HIV+ persons might exert an important role in HIV-infection by influencing viral production. We propose that, in addition to its multiple functions, tTG might interfere with HIV replication by altering the viral mRNA trafficking between the nucleus and the cytoplasm. This effect might be due to its specific interaction with eIF5A, a cellular partner of HIV Rev protein, which is essential for HIV replication in immune-competent cells. Given the presence of high tTG levels in HIV+ individuals, it would be of interest to pursue the potential role of this multifunctional protein in the development of strategies aimed at the pharmacologic regulation of HIV production.