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The family of coronaviruses (CoVs) uses the autophagy machinery of host cells to promote their growth and replication; thus, this process stands out as a potential target to combat COVID-19. Considering the different roles of autophagy during viral infection, including SARS-CoV-2 infection, in this review, we discuss several clinically used drugs that have effects at different stages of autophagy. Among them, we mention (1) lysosomotropic agents, which can prevent CoVs infection by alkalinizing the acid pH in the endolysosomal system, such as chloroquine and hydroxychloroquine, azithromycin, artemisinins, two-pore channel modulators and imatinib; (2) protease inhibitors that can inhibit the proteolytic cleavage of the spike CoVs protein, which is necessary for viral entry into host cells, such as camostat mesylate, lopinavir, umifenovir and teicoplanin and (3) modulators of PI3K/AKT/mTOR signaling pathways, such as rapamycin, heparin, glucocorticoids, angiotensin-converting enzyme inhibitors (IECAs) and cannabidiol. Thus, this review aims to highlight and discuss autophagy-related drugs for COVID-19, from in vitro to in vivo studies. We identified specific compounds that may modulate autophagy and exhibit antiviral properties. We hope that research initiatives and efforts will identify novel or "off-label" drugs that can be used to effectively treat patients infected with SARS-CoV-2, reducing the risk of mortality.

Background/objectives: A dysregulated inflammatory profile plays an important role in coronavirus disease-2019 (COVID-19) pathogenesis. Moreover, the depletion of lymphocytes is typically associated with an unfavourable disease course. We studied the role and impact of p53 and deacetylase Sirtuin 1 (SIRT1) on lymph-monocyte homeostasis and their possible effect on T and B cell signalling., Methods: Gene expression analysis and flow cytometry were performed on peripheral blood mononuclear cells (PBMC) of 35 COVID-19 patients and 10 healthy donors (HD). Inflammatory cytokines, the frequency of Annexin+ cells among CD3+ T cells and CD19+ B cell subsets were quantified., Results: PBMC from COVID-19 patients had a higher p53 expression, and higher concentrations of plasma proinflammatory cytokines (IL1β, TNF-α, IL8, and IL6) than HD. Deacetylase Sirtuin 1 (SIRT1) expression was significantly decreased in COVID-19 patients and was negatively correlated with p53 (p = 0.003 and r = -0.48). A lower expression of IL-7R and B Cell linker (BLNK), key genes for lymphocyte homeostasis and function, was observed in COVID-19 than in HD. The reduction of IgK and IgL chains was seen in lymphopenic COVID-19 patients. A significant increase in both apoptotic B and T cells were observed. Inflammatory cytokines correlated positively with p53 (IL-1β: r = 0.5 and p = 0.05; IL-8: r = 0.5 and p = 0.05) and negatively with SIRT1 (IL1-β: r = -0.5 and p = 0.04; TNF-α: r = -0.4 and p = 0.04)., Conclusions: Collectively, our data indicate that the inflammatory environment, the dysregulated p53/SIRT1 axis and low expression of IL7R and BLNK may impact cell survival, B cell signalling and antibody production in COVID-19 patients. Further studies are required to define the functional impact of low BLNK/IL7R expression during severe acute respiratory syndrome coronavirus-2 infection., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

TG2 is a multifunctional enzyme involved in several cellular processes and has emerging as a potential regulator of gene expression. In this regard, we have recently shown that TG2 is able to activate HSF1, the master transcriptional regulator of the stress-responsive genes; however, its effect on the overall gene expression remains unclear. To address this point, we analyzed, by RNA-seq, the effect of TG2 on the overall transcriptome as well as we characterized the TG2 interactome in the nucleus. The data obtained from these omics approaches reveal that TG2 markedly influences the overall cellular transcriptome profile and specifically the Wnt and HSF1 pathways. In particular, its ablation leads to a drastic downregulation of many key members of these pathways. Interestingly, we found that key components of the Wnt/β-catenin pathway are also downregulated in cells lacking HSF1, thus confirming that TG2 regulates the HSF1 and this axis controls the Wnt signaling. Mechanistic studies revealed that TG2 can regulate the Wnt pathway by physically interacts with β-catenin and its nuclear interactome includes several proteins known to be involved in the regulation of the Wnt signaling. In order to verify whether this effect is playing a role in vivo, we ablated TG2 in Danio rerio. Our data show that the zebrafish lacking TG2 cannot complete the development and their death is associated with an evident downregulation of the Wnt pathway and a defective heat-shock response. Our findings show for the first time that TG2 is essential for the correct embryonal development of lower vertebrates, and its action is mediated by the Wnt/HSF1 axis.

SARS-CoV-2 is associated with a 3.4% mortality rate in patients with severe disease. The pathogenesis of severe cases remains unknown. We performed an in-depth prospective analysis of immune and inflammation markers in two patients with severe COVID-19 disease from presentation to convalescence. Peripheral blood from 18 SARS-CoV-2-infected patients, 9 with severe and 9 with mild COVID-19 disease, was obtained at admission and analyzed for T-cell activation profile, myeloid-derived suppressor cells (MDSCs) and cytokine profiles. MDSC functionality was tested in vitro. In four severe and in four mild patients, a longitudinal analysis was performed daily from the day of admission to the early convalescent phase. Early after admission severe patients showed neutrophilia, lymphopenia, increase in effector T cells, a persisting higher expression of CD95 on T cells, higher serum concentration of IL-6 and TGF-β, and a cytotoxic profile of NK and T cells compared with mild patients, suggesting a highly engaged immune response. Massive expansion of MDSCs was observed, up to 90% of total circulating mononuclear cells in patients with severe disease, and up to 25% in the patients with mild disease; the frequency decreasing with recovery. MDSCs suppressed T-cell functions, dampening excessive immune response. MDSCs decline at convalescent phase was associated to a reduction in TGF-β and to an increase of inflammatory cytokines in plasma samples. Substantial expansion of suppressor cells is seen in patients with severe COVID-19. Further studies are required to define their roles in reducing the excessive activation/inflammation, protection, influencing disease progression, potential to serve as biomarkers of disease severity, and new targets for immune and host-directed therapeutic approaches.

The current epidemic of coronavirus disease-19 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) calls for the development of inhibitors of viral replication. Here, we performed a bioinformatic analysis of published and purported SARS-CoV-2 antivirals including imatinib mesylate that we found to suppress SARS-CoV-2 replication on Vero E6 cells and that, according to the published literature on other coronaviruses is likely to act on-target, as a tyrosine kinase inhibitor. We identified a cluster of SARS-CoV-2 antivirals with characteristics of lysosomotropic agents, meaning that they are lipophilic weak bases capable of penetrating into cells. These agents include cepharentine, chloroquine, chlorpromazine, clemastine, cloperastine, emetine, hydroxychloroquine, haloperidol, ML240, PB28, ponatinib, siramesine, and zotatifin (eFT226) all of which are likely to inhibit SARS-CoV-2 replication by non-specific (off-target) effects, meaning that they probably do not act on their 'official' pharmacological targets, but rather interfere with viral replication through non-specific effects on acidophilic organelles including autophagosomes, endosomes, and lysosomes. Imatinib mesylate did not fall into this cluster. In conclusion, we propose a tentative classification of SARS-CoV-2 antivirals into specific (on-target) versus non-specific (off-target) agents based on their physicochemical characteristics.

Chronic HIV infection accelerates immune aging and is associated with abnormal hemato-lymphopoiesis, but the relationship between HIV-induced aging and Hematopoietic Progenitor Cells (HPC) function is not well-defined. In the context of aging, it has been demonstrated using a murine model that Per2 (Period circadian clock 2) is a negative regulator of HPC survival and lineage potential. A possible involvement of Per2 modulation on hematopoietic failure during HIV infection has not yet been investigated. The aim of this study was to analyze whether Per2 is differently expressed and regulated on HPC during HIV infection, possibly providing a therapeutic target to restore lymphoid potential in the HPC compartment. To this purpose, Per2 expression in circulating HPC was compared in 69 chronic HIV infected patients under successful ART and in matched 30 uninfected healthy donors (HD). HPC aging was assessed by measuring relative telomere length (RTL), and HPC functionality was evaluated by Colony Forming Cell (CFC) assay from both ex vivo HIV+ patients and in vitro Per2 overexpressing donors. Our results showed a lower RTL in HPC and a decrease of white progenitor colonies from HIV+ patients with lower CD4 respect to those with higher CD4 T cell count (<500 respect to >500 CD4 T cell/mmc). Interestingly, we found that the frequency of Per2-expressing HPC is higher in HIV+ patients than in HD and correlated to RTL of CFC derived cells, highlighting a relationship between low proliferative rate and Per2 expression. Indeed, the in vitro overexpression of Per2 resulted in a significant decrease of white progenitor colonies respect to control cells. Finally, we showed that the deacetylase Sirtuin 1, a negative regulator of Per2, was downregulated in HPC from HIV+ patients, and the peripheral blood treatment with resveratrol (Sirtuin 1 inducer) determined a decrease of Per2 expressing HPC. Altogether, these results suggest that during HIV infection, Per2 is involved in the regulation of HPC expansion and differentiation and its overexpression may impair the immune reconstitution. These data support the rationale to explore the role of this regulatory mechanism during aged-associated hemato-lymphopoiesis impairment in HIV infection., (Copyright © 2020 Bordoni, Tartaglia, Refolo, Sacchi, Grassi, Antinori, Fimia and Agrati.)

Skin melanoma is one of the most aggressive and difficult-to-treat human malignancies, characterized by poor survival rates, thus requiring urgent novel therapeutic approaches. Although metabolic reprogramming has represented so far, a cancer hallmark, accumulating data indicate a high plasticity of cancer cells in modulating cellular metabolism to adapt to a heterogeneous and continuously changing microenvironment, suggesting a novel therapeutic approach for dietary manipulation in cancer therapy. To this aim, we exposed melanoma cells to combined nutrient-restriction/sorafenib. Results indicate that cell death was efficiently induced, with apoptosis representing the prominent feature. In contrast, autophagy was blocked in the final stage by this treatment, similarly to chloroquine, which also enhanced melanoma cell sensitization to combined treatment. Energy stress was evidenced by associated treatment with mitochondrial dysfunction and glycolysis impairment, suggesting metabolic stress determining melanoma cell death. A reduction of tumor growth after cycles of intermittent fasting together with sorafenib treatment was also observed in vivo, reinforcing that the nutrient shortage can potentiate anti-melanoma therapy. Our findings showed that the restriction of nutrients by intermittent fasting potentiates the effects of sorafenib due to the modulation of cellular metabolism, suggesting that it is possible to harness the energy of cancer cells for the treatment of melanoma.

Autophagy, a main intracellular catabolic process, is induced in response to a variety of cellular stresses to promptly degrade harmful agents and to coordinate the activity of prosurvival and prodeath processes in order to determine the fate of the injured cells. While the main components of the autophagy machinery are well characterized, the molecular mechanisms that confer selectivity to this process both in terms of stress detection and cargo engulfment have only been partly elucidated. Here, we discuss the emerging role played by the E3 ubiquitin ligases of the TRIM family in regulating autophagy in physiological and pathological conditions, such as inflammation, infection, tumorigenesis, and muscle atrophy. TRIM proteins employ different strategies to regulate the activity of the core autophagy machinery, acting either as scaffold proteins or via ubiquitin-mediated mechanisms. Moreover, they confer high selectivity to the autophagy-mediated degradation as described for the innate immune response, where TRIM proteins mediate both the engulfment of pathogens within autophagosomes and modulate the immune response by controlling the stability of signaling regulators. Importantly, the elucidation of the molecular mechanisms underlying the regulation of autophagy by TRIMs is providing important insights into how selective types of autophagy are altered under pathological conditions, as recently shown in cancer and muscular dystrophy.

About 20% of total cancer cases are associated to infections. To date, seven human viruses have been directly linked to cancer development: high-risk human papillomaviruses (hrHPVs), Merkel cell polyomavirus (MCPyV), hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and human T-lymphotropic virus 1 (HTLV-1). These viruses impact on several molecular mechanisms in the host cells, often resulting in chronic inflammation, uncontrolled proliferation, and cell death inhibition, and mechanisms, which favor viral life cycle but may indirectly promote tumorigenesis. Recently, the ability of oncogenic viruses to alter autophagy, a catabolic process activated during the innate immune response to infections, is emerging as a key event for the onset of human cancers. Here, we summarize the current understanding of the molecular mechanisms by which human oncogenic viruses regulate autophagy and how this negative regulation impacts on cancer development. Finally, we highlight novel autophagy-related candidates for the treatment of virus-related cancers., (Copyright © 2020 Vescovo, Pagni, Piacentini, Fimia and Antonioli.)

In the last years, proteomics has represented a valuable approach to elucidate key aspects in the regulation of type I/III interferons (IFNs) and autophagy, two main processes involved in the response to viral infection, to unveil the molecular strategies that viruses have evolved to counteract these processes. Besides their main metabolic roles, mitochondria are well recognized as pivotal organelles in controlling signaling pathways essential to restrain viral infections. In particular, a major role in antiviral defense is played by mitochondrial antiviral signaling (MAVS) protein, an adaptor protein that coordinates the activation of IFN inducing pathways and autophagy at the mitochondrial level. Here, we provide an overview of how mass spectrometry-based studies of protein-protein interactions and post-translational modifications (PTMs) have fostered our understanding of the molecular mechanisms that control the mitochondria-mediated antiviral immunity., (Copyright © 2020 Refolo, Vescovo, Piacentini, Fimia and Ciccosanti.)

Host-directed therapies (HDTs) are emerging as a potential valid support in the treatment of drug-resistant tuberculosis (TB). Following our recent report indicating that genetic and pharmacological inhibition of transglutaminase 2 (TG2) restricts Mycobacterium tuberculosis ( Mtb ) replication in macrophages, we aimed to investigate the potentials of the TG2 inhibitors cystamine and cysteamine as HDTs against TB. We showed that both cysteamine and cystamine restricted Mtb replication in infected macrophages when provided at equimolar concentrations and did not exert any antibacterial activity when administered directly on Mtb cultures. Interestingly, infection of differentiated THP-1 mRFP-GFP-LC3B cells followed by the determination of the autophagic intermediates pH distribution (AIPD) showed that cystamine inhibited the autophagic flux while restricting Mtb replication. Moreover, both cystamine and cysteamine had a similar antimicrobial activity in primary macrophages infected with a panel of Mtb clinical strains belonging to different phylogeographic lineages. Evaluation of cysteamine and cystamine activity in the human ex vivo model of granuloma-like structures (GLS) further confirmed the ability of these drugs to restrict Mtb replication and to reduce the size of GLS. The antimicrobial activity of the TG2 inhibitors synergized with a second-line anti-TB drug as amikacin in human monocyte-derived macrophages and in the GLS model. Overall, the results of this study support the potential usefulness of the TG2-inhibitors cysteamine and cystamine as HDTs against TB., (Copyright © 2020 Palucci, Maulucci, De Maio, Sali, Romagnoli, Petrone, Fimia, Sanguinetti, Goletti, De Spirito, Piacentini and Delogu.)

The Ser/Thr protein kinase ULK1 is an upstream macroautophagy/autophagy regulator that is rapidly activated to ensure a proper adaptive response to stress conditions. Signaling pathways modulating ULK1 activity have been extensively characterized in response to nutrient/energy shortage, which mainly act by mediating ULK1 post-translational modifications, such as phosphorylation, acetylation and ubiquitination. Less characterized is how tissue-specific stress signals are able to activate ULK1 to induce autophagy. Our recent study has uncovered the E3 ubiquitin ligase TRIM32 as a novel ULK1 activator that regulates autophagy in muscle cells upon atrophy induction. TRIM32 is conveyed to ULK1 by the autophagy cofactor AMBRA1 to stimulate its kinase activity through unanchored K63-linked polyubiquitin chains. Notably, mutations in TRIM32 responsible for limb-girdle muscular dystrophy 2H disrupt its ability to bind ULK1 and to induce autophagy in muscle cells, resulting in a dysregulated activation of the atrophic process. In conclusion, we have identified a novel molecular mechanism by which autophagy is regulated in muscles, whose alteration is associated with the development of muscular dystrophy.

Interferon-γ inducible protein 10 (IP-10), is a potent chemoattractant that promotes migration of monocytes and activated T-cells to inflammation foci. IP-10 is elevated in serum of patients with chronic hepatitis C virus (HCV) and tuberculosis (TB) infections, although it remains to be determined the contribution of IP-10 in restricting Mycobacterium tuberculosis (Mtb) replication. Here, we investigated the impact of IP-10 on mycobacteria replication using the ex vivo model of human whole-blood (WB) assay. In particular, we compared the levels of IP-10 upon infection with different Mtb clinical strains and species of non-tuberculous mycobacteria (NTM) and evaluated how IP-10 may contain bacterial replication. Interestingly, we observed that the inhibition of the host enzyme dipeptidyl peptidase IV (DPP-IV), which inactivates IP-10 through cleavage of two amino acids at the chemokine N-terminus, restricted mycobacterial persistence in WB, supporting the critical role of full length IP-10 in mediating an anti-Mtb response. Addition of recombinant IP-10 expressed in eukaryotic cells enhanced the anti-mycobacterial activity in WB, although no differences were observed when IP-10 containing different proportions of cleaved and non-cleaved forms of the chemokine were added. Moreover, recombinant IP-10 did not exert a direct anti-mycobacterial effect. Our results underscore the clinical relevance of IP-10 in mycobacteria pathogenesis and support the potential outcomes that may derive by targeting the IP-10/CXCR3 pathway as host directed therapies for the treatment of Mtb or NTM infections., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Statins efficiently inhibit cholesterol synthesis by blocking 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase in the mevalonate pathway. However, the effect of statins on intracellular cholesterol is partially counterbalanced by a consequent increased uptake of extracellular lipid sources. Hepatitis C virus (HCV) infection induces intracellular accumulation of cholesterol by promoting both new synthesis and uptake of circulating lipoproteins, which is required for HCV replication and release. Hepatocytes respond to the increase in intracellular cholesterol levels by inducing lipophagy, a selective type of autophagy mediating the degradation of lipid deposits within lysosomes. In a cellular system of HCV replication based on HuH7 hepatoma cells, statin treatment was shown to be sufficient to decrease intracellular cholesterol, which is accompanied by reduced HCV replication and decreased lipophagy, and has no apparent impact on endocytosis-mediated cholesterol uptake. To understand whether these results were influenced by an altered response of cholesterol influx in hepatoma cells, we analyzed the effect of statins in non-transformed murine hepatocytes (MMHD3) harboring subgenomic HCV replicons. Notably, we found that total amount of cholesterol is increased in MMHD3 cells upon mevastatin treatment, which is associated with increased HCV replication and lipophagy. Conversely, mevastatin is able to reduce cholesterol amounts only when cells are grown in the presence of delipidated serum to prevent extracellular lipid uptake. Under this condition, HCV replication is reduced and autophagy flux is severely impaired. Altogether, these results indicate that both de novo synthesis and extracellular uptake have to be targeted in non-transformed hepatocytes in order to decrease intracellular cholesterol levels and consequently limit HCV replication.

The limited availability of rapid and reliable flow cytometry-based assays for ex vivo quantification of autophagy has hampered their clinical applications for studies of diseases pathogenesis or for the implementation of autophagy-targeting therapies. To this aim, we modified and improved the protocol of a commercial kit developed for quantifying the microtubule-associated protein 1A/1B light chain 3B (LC3), the most reliable marker for autophagosomes currently available. The protocol modifications were set up measuring the autophagic flux in neoplastic (THP-1 cells) and primary cells (peripheral blood mononuclear cells; PBMC) of healthy donors. Moreover, PBMC of active tuberculosis (TB) patients were stimulated with the Mycobacterium tuberculosis purified protein derivatives or infected with live Mycobacterium bovis bacillus Calmette-Guerin (BCG). We found that the baseline median fluorescent intensity (MFI) of THP-1 cells changed depending on the time of sample acquisition to the flow cytometer. To solve this problem, a fixation step was introduced in different stages of the assay's protocol, obtaining more reproducible and sensitive results when a post-LC3 staining fixation was performed, in either THP1 or PBMC. Furthermore, since we found that results are influenced by the type and the dose of the lysosome inhibitor used, the best dose of Chloroquine for LC3 accumulation were set up in either THP-1 cells or PBMC. Finally, applying these experimental settings, we measured the autophagic flux in CD14+ cells from active TB patients' PBMC upon BCG infection. In conclusion, our data indicate that the protocol modifications here described in this work improve the stability and accuracy of a flow cytometry-based assay for the evaluation of autophagy, thus assuring more standardised cell analyses.

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.

Objectives: Biomarkers for tuberculosis (TB) diagnosis and clinical management are needed to defeat TB. In chronic hepatitis, patients not responding to interferon/ribavirin treatment had high levels of an antagonist form of IP-10. Recently, antagonist IP-10 has been shown to be involved also in TB pathogenesis. Here, we investigated IP-10 agonist/antagonist forms as potential inflammatory biomarkers to support TB diagnosis and monitoring., Methods: Total IP-10 and its agonist/antagonist forms were measured by SIMOA digital ELISA in urine obtained from patients with active TB at baseline and after treatment. Healthy donors (HD) and patients with pneumonia were enrolled as controls., Results: Patients with active TB had significantly higher levels of total and agonist IP-10 at baseline compared to HD; conversely, no differences were observed between IP-10 levels in active TB vs pneumonia. Moreover, in active TB a decline of total urine IP-10 was observed at therapy completion; agonist/antagonist forms reflected this decline although their differences were not statistically significant., Conclusions: We showed for the first time that agonist/antagonist IP-10 forms are measurable in urine. IP-10 levels associate with TB and pneumonia disease, suggesting their association with acute inflammation. Further studies are needed to assess their role to monitor TB treatment efficacy., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Hepatitis C virus (HCV) is highly efficient in establishing a chronic infection, having evolved multiple strategies to suppress the host antiviral responses. The HCV nonstructural 5A (NS5A) protein, in addition to its role in viral replication and assembly, has long been known to hamper the interferon (IFN) response. However, the mechanism of this inhibitory activity of NS5A remains partly characterized. In a functional proteomic screening carried out in HCV replicon cells, we identified the mitochondrial protein LRPPRC as an NS5A binding factor. Notably, we found that downregulation of LRPPRC expression results in a significant inhibition of HCV infection, which is associated with an increased activation of the IFN response. Moreover, we showed that LRPPRC acts as a negative regulator of the mitochondrial-mediated antiviral immunity, by interacting with mitochondrial antiviral signaling protein (MAVS) and inhibiting its association with TRAF3 and TRAF6. Finally, we demonstrated that NS5A is able to interfere with MAVS activity in a LRPPRC-dependent manner. Conclusion: Overall, our results indicate that NS5A contributes to the inhibition of innate immune pathways during HCV infection by exploiting the ability of LRPPRC to inhibit MAVS-regulated antiviral signaling., (© 2018 by the American Association for the Study of Liver Diseases.)

Transglutaminase type 2 (TG2) is a multifunctional enzyme that plays a key role in mitochondria homeostasis under stressful cellular conditions. TG2 interactome analysis reveals an enzyme interaction with GRP75 (glucose-regulated protein 75). GRP75 localizes in mitochondria-associated membranes (MAMs) and acts as a bridging molecule between the two organelles by assembling the IP3R-GRP75-VDAC complex, which is involved in the transport of Ca 2+ from the endoplasmic reticulum (ER) to mitochondria. We demonstrate that the TG2 and GRP75 interaction occurs in MAMs. The absence of the TG2-GRP75 interaction leads to an increase of the interaction between IP3R-3 and GRP75; a decrease of the number of ER-mitochondria contact sites; an impairment of the ER-mitochondrial Ca 2+ flux; and an altered profile of the MAM proteome. These findings indicate TG2 is a key regulatory element of the MAMs., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Regulatory T cells (T reg ) are necessary to maintain immunological tolerance and are key players in the control of autoimmune disease susceptibility. Expression of the transcription factor FOXP3 is essential for differentiation of T reg cells and indispensable for their suppressive function. However, there is still a lack of knowledge about the mechanisms underlying its regulation. Here, we demonstrate that pro-autophagy protein AMBRA1 is also a key modulator of T cells, regulating the complex network that leads to human T reg differentiation and maintenance. Indeed, through its ability to interact with the phosphatase PP2A, AMBRA1 promotes the stability of the transcriptional activator FOXO3, which, in turn, triggers FOXP3 transcription. Furthermore, we found that AMBRA1 plays a significant role in vivo by regulating T reg cell induction in mouse models of both tumor growth and multiple sclerosis, thus highlighting the role of AMBRA1 in the control of immune homeostasis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Aim: Confirm the use of Plectanthus spp. plants in traditional medicine, particularly as anti-inflammatory and anti-infective agents., Materials & Methods: Compounds previously isolated from Plectranthus spp. were studied for their anti-inflammatory activity using the SNAP assay and RAW 264.7 cells, by the quantification of nitric oxide. An halimane diterpene and its derivatives were tested in infected macrophages with M. tuberculosis H37Rv, using CFU counts assay, at their minimum inhibitory concentration values. Results: The isolated compounds tested at noncytotoxic concentrations, did not reveal nitric oxide scavenging in the S-nitroso-N-acetylpenicillamine and the cellular assays. On the other hand, promising results were obtained regarding one semisynthetic halimane derivative (11R*,13E)-halima-5,13-diene-11,15-diol), previously prepared (2.1 × 10 5  CFU/mL), with an effect similar to the antitubercular drugs ethambutol (2.0 × 10 5  CFU/mL) and isoniazid (1.2 × 10 5  CFU/mL)., Conclusion: The present report demonstrates the relevance of Plectranthus spp. in medicinal chemistry drug development for TB and other infective respiratory complaints. Also, this work suggests that further studies involving other inflammatory mediators are needed to validate the anti-inflammatory use of these medicinal plants.

Heat-shock factor 1 (HSF1) is the master transcription factor that regulates the response to proteotoxic stress by controlling the transcription of many stress-responsive genes including the heat-shock proteins. Here, we show a novel molecular mechanism controlling the activation of HSF1. We demonstrate that transglutaminase type 2 (TG2), dependent on its protein disulphide isomerase activity, triggers the trimerization and activation of HSF1 regulating adaptation to stress and proteostasis impairment. In particular, we find that TG2 loss of function correlates with a defect in the nuclear translocation of HSF1 and in its DNA-binding ability to the HSP70 promoter. We show that the inhibition of TG2 restores the unbalance in HSF1-HSP70 pathway in cystic fibrosis (CF), a human disorder characterized by deregulation of proteostasis. The absence of TG2 leads to an increase of about 40% in CFTR function in a new experimental CF mouse model lacking TG2. Altogether, these results indicate that TG2 plays a key role in the regulation of cellular proteostasis under stressful cellular conditions through the modulation of the heat-shock response., (© 2018 The Authors.)

Autophagy is a catabolic process needed for maintaining cell viability and homeostasis in response to numerous stress conditions. Emerging evidence indicates that the ubiquitin system has a major role in this process. TRIMs, an E3 ligase protein family, contribute to selective autophagy acting as receptors and regulators of the autophagy proteins recognizing endogenous or exogenous targets through intermediary autophagic tags, such as ubiquitin. Here we report that TRIM50 fosters the initiation phase of starvation-induced autophagy and associates with Beclin1, a central component of autophagy initiation complex. We show that TRIM50, via the RING domain, ubiquitinates Beclin 1 in a K63-dependent manner enhancing its binding with ULK1 and autophagy activity. Finally, we found that the Lys-372 residue of TRIM50, critical for its own acetylation, is necessary for its E3 ligase activity that governs Beclin1 ubiquitination. Our study expands the roles of TRIMs in regulating selective autophagy, revealing an acetylation-ubiquitination dependent control for autophagy modulation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), has infected over 1.7 billion people worldwide and causes 1.4 million deaths annually. Recently, genome sequence analysis has allowed the reconstruction of Mycobacterium tuberculosis complex (MTBC) evolution, with the identification of seven phylogeographic lineages: four referred to as evolutionarily "ancient", and three "modern". The MTBC strains belonging to "modern" lineages appear to show enhanced virulence that may have warranted improved transmission in humans over ancient lineages through molecular mechanisms that remain to be fully characterized. To evaluate the impact of MTBC genetic diversity on the innate immune response, we analyzed intracellular bacterial replication, inflammatory cytokine levels, and autophagy response in human primary macrophages infected with MTBC clinical isolates belonging to the ancient lineages 1 and 5, and the modern lineage 4. We show that, when compared to ancient lineage 1 and 5, MTBC strains belonging to modern lineage 4 show a higher rate of replication, associated to a significant production of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) and induction of a functional autophagy process. Interestingly, we found that the increased autophagic flux observed in macrophages infected with modern MTBC is due to an autocrine activity of the proinflammatory cytokine IL-1β, since autophagosome maturation is blocked by an interleukin-1 receptor antagonist. Unexpectedly, IL-1β-induced autophagy is not disadvantageous for the survival of modern Mtb strains, which reside within Rab5-positive phagosomal vesicles and avoid autophagosome engulfment. Altogether, these results suggest that autophagy triggered by inflammatory cytokines is compatible with a high rate of intracellular bacilli replication and may therefore contribute to the increased pathogenicity of the modern MTBC lineages.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

Autophagy is a primordial eukaryotic pathway, which provides the immune system with multiple mechanisms for the elimination of invading pathogens including Mycobacterium tuberculosis (Mtb). As a consequence, Mtb has evolved different strategies to hijack the autophagy process. Given the crucial role of human primary dendritic cells (DC) in host immunity control, we characterized Mtb-DC interplay by studying the contribution of cellular microRNAs (miRNAs) in the post-transcriptional regulation of autophagy related genes. From the expression profile of de-regulated miRNAs obtained in Mtb-infected human DC, we identified 7 miRNAs whose expression was previously found to be altered in specimens of TB patients. Among them, gene ontology analysis showed that miR-155, miR-155* and miR-146a target mRNAs with a significant enrichment in biological processes linked to autophagy. Interestingly, miR-155 was significantly stimulated by live and virulent Mtb and enriched in polysome-associated RNA fraction, where actively translated mRNAs reside. The putative pair interaction among the E2 conjugating enzyme involved in LC3-lipidation and autophagosome formation-ATG3-and miR-155 arose by target prediction analysis, was confirmed by both luciferase reporter assay and Atg3 immunoblotting analysis of miR-155-transfected DC, which showed also a consistent Atg3 protein and LC3 lipidated form reduction. Late in infection, when miR-155 expression peaked, both the level of Atg3 and the number of LC3 puncta per cell (autophagosomes) decreased dramatically. In accordance, miR-155 silencing rescued autophagosome number in Mtb infected DC and enhanced autolysosome fusion, thereby supporting a previously unidentified role of the miR-155 as inhibitor of ATG3 expression. Taken together, our findings suggest how Mtb can manipulate cellular miRNA expression to regulate Atg3 for its own survival, and highlight the importance to develop novel therapeutic strategies against tuberculosis that would boost autophagy.

The accumulation of abnormal α-synuclein is the major histopathological feature of Lewy body disease and multiple system atrophy (MSA), which are referred to as synucleinopathies. Cytoplasmic degradation systems, such as the autophagy-lysosome and proteasome pathways, are involved in their pathogenesis. Autophagy is tightly regulated by several upstream proteins including UNC-51-like kinase 1/2, beclin1, vacuolar protein sorting-associated protein 34 and autophagy/beclin1 regulator 1 (AMBRA1). Recently, we revealed that both cortical and brainstem-type Lewy bodies were immunopositive for several upstream proteins of autophagy. Therefore, we conducted the present study to elucidate the role of upstream proteins of autophagy in the pathogenesis of MSA. Pathological and biochemical analyses using human brain samples revealed that AMBRA1 is a component of the pathological hallmarks of MSA and upstream proteins of autophagy are impaired in the MSA brain. In vitro and in vivo analyses revealed a ninefold stronger affinity of AMBRA1 with α-synuclein phosphorylated at serine 129 compared with non-phosphorylated α-synuclein. Furthermore, a weak but significant correlation between AMBRA1 overexpression and reduction of abnormal α-synuclein was observed. Silencing AMBRA1 function caused aggregates of α-synuclein in the cytoplasm of mouse primary cultured neurons, which was simulated by the treatment of Bafilomycin, an autophagy inhibitor. Our results demonstrated for the first time that AMBRA1 is a novel hub binding protein of α-synuclein and plays a central role in the pathogenesis of MSA through the degradative dynamics of α-synuclein. These results raise the possibility that molecular modulation targeting AMBRA1 can be a promising candidate for the treatment of synucleinopathies., (© 2016 International Society of Neuropathology.)

Background: Changes in iron metabolism frequently accompany HIV-1 infection. However, while many clinical and in vitro studies report iron overload exacerbates the development of infection, many others have found no correlation. Therefore, the multi-faceted role of iron in HIV-1 infection remains enigmatic., Methods: RT-qPCR targeting the LTR region, gag , Tat and Rev were performed to measure the levels of viral RNAs in response to iron overload. Spike-in SILAC proteomics comparing i) iron-treated, ii) HIV-1-infected and iii) HIV-1-infected/iron treated T lymphocytes was performed to define modifications in the host cell proteome. Data from quantitative proteomics were integrated with the HIV-1 Human Interaction Database for assessing any viral cofactors modulated by iron overload in infected T lymphocytes., Results: Here, we demonstrate that the iron overload down-regulates HIV-1 gene expression by decreasing the levels of viral RNAs. In addition, we found that iron overload modulates the expression of many viral cofactors. Among them, the downregulation of the REV cofactor eIF5A may correlate with the iron-induced inhibition of HIV-1 gene expression. Therefore, we demonstrated that eiF5A downregulation by shRNA resulted in a significant decrease of Nef levels, thus hampering HIV-1 replication., Conclusions: Our study indicates that HIV-1 cofactors influenced by iron metabolism represent potential targets for antiretroviral therapy and suggests eIF5A as a selective target for drug development.

Over the past two decades, the molecular machinery that underlies autophagic responses has been characterized with ever increasing precision in multiple model organisms. Moreover, it has become clear that autophagy and autophagy-related processes have profound implications for human pathophysiology. However, considerable confusion persists about the use of appropriate terms to indicate specific types of autophagy and some components of the autophagy machinery, which may have detrimental effects on the expansion of the field. Driven by the overt recognition of such a potential obstacle, a panel of leading experts in the field attempts here to define several autophagy-related terms based on specific biochemical features. The ultimate objective of this collaborative exchange is to formulate recommendations that facilitate the dissemination of knowledge within and outside the field of autophagy research., (© 2017 The Authors.)

This study aims to determine the interaction (uptake and biological effects on cell viability and cell cycle progression) of glucose capped silver nanoparticles (AgNPs-G) on human epithelioid cervix carcinoma (HeLa) cells, in relation to amount, 2×10 3 or 2×10 4 NPs/cell, and exposure time, up to 48h. The spherical and well dispersed AgNPs (30±5nm) were obtained by using glucose as reducing agent in a green synthesis method that ensures to stabilize AgNPs avoiding cytotoxic soluble silver ions Ag + release. HeLa cells take up abundantly and rapidly AgNPs-G resulting toxic to cells in amount and incubation time dependent manner. HeLa cells were arrested at S and G2/M phases of the cell cycle and subG1 population increased when incubated with 2×10 4 AgNPs-G/cell. Mitotic index decreased accordingly. The dissolution experiments demonstrated that the observed effects were due only to AgNPs-G since glucose capping prevents Ag + release. The AgNPs-G influence on HeLa cells viability and cell cycle progression suggest that AgNPs-G, alone or in combination with chemotherapeutics, may be exploited for the development of novel antiproliferative treatment in cancer therapy. However, the possible influence of the cell cycle on cellular uptake of AgNPs-G and the mechanism of AgNPs entry in cells need further investigation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (© 2017 The Association for the Publication of the Journal of Internal Medicine.)

Perturbation of endoplasmic reticulum (ER) homeostasis results in a stress condition termed "ER stress" determining the activation of a finely regulated program defined as unfolded protein response (UPR) and whose primary aim is to restore this organelle's physiological activity. Several physiological and pathological stimuli deregulate normal ER activity causing UPR activation, such as hypoxia, glucose shortage, genome instability, and cytotoxic compounds administration. Some of these stimuli are frequently observed during uncontrolled proliferation of transformed cells, resulting in tumor core formation and stage progression. Therefore, it is not surprising that ER stress is usually induced during solid tumor development and stage progression, becoming an hallmark of such malignancies. Several UPR components are in fact deregulated in different tumor types, and accumulating data indicate their active involvement in tumor development/progression. However, although the UPR program is primarily a pro-survival process, sustained and/or prolonged stress may result in cell death induction. Therefore, understanding the mechanism(s) regulating the cell survival/death decision under ER stress condition may be crucial in order to specifically target tumor cells and possibly circumvent or overcome tumor resistance to therapies. In this review, we discuss the role played by the UPR program in tumor initiation, progression and resistance to therapy, highlighting the recent advances that have improved our understanding of the molecular mechanisms that regulate the survival/death switch.

Mitophagy is a highly specialized process to remove dysfunctional or superfluous mitochondria through the macroautophagy/autophagy pathway, aimed at protecting cells from the damage of disordered mitochondrial metabolism and apoptosis induction. PINK1, a neuroprotective protein mutated in autosomal recessive Parkinson disease, has been implicated in the activation of mitophagy by selectively accumulating on depolarized mitochondria, and promoting PARK2/Parkin translocation to them. While these steps have been characterized in depth, less is known about the process and site of autophagosome formation upon mitophagic stimuli. A previous study reported that, in starvation-induced autophagy, the proautophagic protein BECN1/Beclin1 (which we previously showed to interact with PINK1) relocalizes at specific regions of contact between the endoplasmic reticulum (ER) and mitochondria called mitochondria-associated membranes (MAM), from which the autophagosome originates. Here we show that, following mitophagic stimuli, autophagosomes also form at MAM; moreover, endogenous PINK1 and BECN1 were both found to relocalize at MAM, where they promoted the enhancement of ER-mitochondria contact sites and the formation of omegasomes, that represent autophagosome precursors. PARK2 was also enhanced at MAM following mitophagy induction. However, PINK1 silencing impaired BECN1 enrichment at MAM independently of PARK2, suggesting a novel role for PINK1 in regulating mitophagy. MAM have been recently implicated in many key cellular events. In this light, the observed prevalent localization of PINK1 at MAM may well explain other neuroprotective activities of this protein, such as modulation of mitochondrial calcium levels, mitochondrial dynamics, and apoptosis.

Melanoma is one of leading cause of tumor death worldwide. Anti-cancer strategy includes combination of different chemo-therapeutic agents as well as radiation; however these treatments have limited efficacy and induce significant toxic effects on healthy cells. One of most promising novel therapeutic approach to cancer therapy is the combination of anti-cancer drugs with calorie restriction. Here we investigated the effect Cisplatin (CDDP), one of the most potent chemotherapeutic agent used to treat tumors, in association with fasting in wild type and mutated BRAF V600E melanoma cell lines. Here we show that nutrient deprivation can consistently enhance the sensitivity of tumor cells to cell death induction by CDDP, also of those malignancies particularly resistant to any treatment, such as oncogenic BRAF melanomas. Mechanistic studies revealed that the combined therapy induced cell death is characterized by ROS accumulation and ATF4 in the absence of ER-stress. In addition, we show that autophagy is not involved in the enhanced sensitivity of melanoma cells to combined CDDP/EBSS-induced apoptosis. While, the exposure to 2-DG further enhanced the apoptotic rate observed in SK Mel 28 cells upon treatment with both CDDP and EBSS., (Copyright © 2016. Published by Elsevier Inc.)

NAADP (nicotinic acid adenine dinucleotide phosphate) has been proposed as a second messenger for glutamate in neuronal and glial cells via the activation of the lysosomal Ca2+ channels TPC1 and TPC2. However, the activities of glutamate that are mediated by NAADP remain unclear. In this study, we evaluated the effect of glutamate on autophagy in astrocytes at physiological, non-toxic concentration. We found that glutamate induces autophagy at similar extent as NAADP. By contrast, the NAADP antagonist NED-19 or SiRNA-mediated inhibition of TPC1/2 decreases autophagy induced by glutamate, confirming a role for NAADP in this pathway. The involvement of TPC1/2 in glutamate-induced autophagy was also confirmed in SHSY5Y neuroblastoma cells. Finally, we show that glutamate leads to a NAADP-dependent activation of AMPK, which is required for autophagy induction, while mTOR activity is not affected by this treatment. Taken together, our results indicate that glutamate stimulates autophagy via NAADP/TPC/AMPK axis, providing new insights of how Ca2+ signalling glutamate-mediated can control the cell metabolism in the central nervous system.

Autophagy is a major degradative process activated in a rapid and transient manner to cope with stress conditions. Whether autophagy is beneficial or detrimental depends upon the rate of induction and the appropriateness of the duration. Alterations in both autophagy initiation and termination predispose the cell to death, and affect the execution of other inducible processes such as inflammation. In this review we discuss how stress signaling pathways dynamically control the activity of the autophagy machinery by mediating post-translational modifications and regulatory protein interactions. In particular, we highlight the emerging role of TRIM and CULLIN families of ubiquitin ligases which play opposite roles in the autophagy response by promoting or inhibiting, respectively, the activity of the autophagy initiation complex., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (© 2017 Elsevier Inc. All rights reserved.)

Autophagy is an intracellular degradation pathway whose levels are tightly controlled to secure cell homeostasis. Unc-51-like kinase 1 (ULK1) is a conserved serine-threonine kinase that plays a central role in the initiation of autophagy. Here, we report that upon autophagy progression, ULK1 protein levels are specifically down-regulated by the E3 ligase NEDD4L, which ubiquitylates ULK1 for degradation by the proteasome. However, whereas ULK1 protein is degraded, ULK1 mRNA is actively transcribed. Upon reactivation of mTOR-dependent protein synthesis, basal levels of ULK1 are promptly restored, but the activity of newly synthesized ULK1 is inhibited by mTOR. This prepares the cell for a new possible round of autophagy stimulation. Our results thus place NEDD4L and ULK1 in a key position to control oscillatory activation of autophagy during prolonged stress to keep the levels of this process under a safe and physiological threshold., (© 2016 Nazio et al.)

Hepatitis C virus (HCV) is a major leading cause of hepatocellular carcinoma (HCC). HCV-induced hepatocarcinogenesis is a multistep process resulting from a combination of pathway alterations that are either caused directly by viral factors or immune mediated as a consequence of a chronic state of inflammation. Host genetic variation is now emerging as an additional element that contribute to increase the risk of developing HCC. The advent of direct-acting antiviral agents foresees a rapid decline of HCC rate in HCV patients. However, a full understanding of the HCV-mediated tumourigenic process is required to elucidate if pro-oncogenic signatures may persist after virus clearance, and to identify novel tools for HCC prevention and therapy. In this review, we summarize the current knowledge of the molecular mechanisms responsible for HCV-induced hepatocarcinogenesis., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Numerous studies are revealing a role of exosomes in intercellular communication, and growing evidence indicates an important function for these vesicles in the progression and pathogenesis of cancer and neurodegenerative diseases. However, the biogenesis process of exosomes is still unclear. Tissue transglutaminase (TG2) is a multifunctional enzyme with different subcellular localizations. Particularly, under stressful conditions, the enzyme has been also detected in the extracellular matrix, but the mechanism(s) by which TG2 is released outside the cells requires further investigation. Therefore, the goal of the present study was to determine whether exosomes might be a vehicle for TG2 to reach the extracellular space, and whether TG2 could be involved in exosomes biogenesis. To address this issue, we isolated and characterized exosomes derived from cells either expressing or not TG2, under stressful conditions (i.e. proteasome impairment or expressing a mutated form of huntingtin (mHtt) containing 84 polyglutamine repeats). Our results show that TG2 is present in the exosomes only upon proteasome blockade, a condition in which TG2 interacts with TSG101 and ALIX, two key proteins involved in exosome biogenesis. Interestingly, we found that TG2 favours the assembly of a protein complex including mHtt, ALIX, TSG101 and BAG3, a co-chaperone involved in the clearance of mHtt. The formation of this complex is paralleled by the selective recruitment of mHtt and BAG3 in the exosomes derived from TG2 proficient cells only. Overall, our data indicate that TG2 is an important player in the biogenesis of exosomes controlling the selectivity of their cargo under stressful cellular conditions. In addition, these vesicles represent the way by which cells can release TG2 into the extracellular space under proteostasis impairment., (Copyright © 2016. Published by Elsevier B.V.)

The ERCC8/CSA gene encodes a WD-40 repeat protein (CSA) that is part of a E3-ubiquitin ligase/COP9 signalosome complex. When mutated, CSA causes the Cockayne Syndrome group A (CS-A), a rare recessive progeroid disorder characterized by sun sensitivity and neurodevelopmental abnormalities. CS-A cells features include ROS hyperproduction, accumulation of oxidative genome damage, mitochondrial dysfunction and increased apoptosis that may contribute to the neurodegenerative process. In this study, we show that CSA localizes to mitochondria and specifically interacts with the mitochondrial fission protein dynamin-related protein (DRP1) that is hyperactivated when CSA is defective. Increased fission is not counterbalanced by increased mitophagy in CS-A cells thus leading to accumulation of fragmented mitochondria. However, when mitochondria are challenged with the mitochondrial toxin carbonyl cyanide m-chloro phenyl hydrazine, CS-A fibroblasts undergo mitophagy as efficiently as normal fibroblasts, suggesting that this process remains targetable to get rid of damaged mitochondria. Indeed, when basal mitophagy was potentiated by overexpressing Parkin in CSA deficient cells, a significant rescue of the dysfunctional mitochondrial phenotype was observed. Importantly, Parkin overexpression not only reactivates basal mitophagy, but plays also an anti-apoptotic role by significantly reducing the translocation of Bax at mitochondria in CS-A cells. These findings provide new mechanistic insights into the role of CSA in mitochondrial maintenance and might open new perspectives for therapeutic approaches., Competing Interests: CONFLICTS OF INTEREST The authors declare no competing financial interests.

Autophagy and apoptosis are 2 stress-response mechanisms that are closely interconnected. However, the molecular interplays between these 2 pathways remain to be clarified. Here we report that the crucial proautophagic factor AMBRA1 can act as a positive mediator of mitochondrial apoptosis. Indeed, we show that, in a proapoptotic positive feedback loop, the C-terminal part of AMBRA1, generated by CASP/CASPASE cleavage upon apoptosis induction, inhibits the antiapoptotic factor BCL2 by a direct binding through its BH3-like domain. The mitochondrial AMBRA1-BCL2 complex is thus at the crossroad between autophagy and cell death and may represent a novel target in development of therapeutic approaches in clinical diseases.

Hepatitis C virus (HCV) infects over 150 million people worldwide. In most cases, HCV infection becomes chronic causing liver disease ranging from fibrosis to cirrhosis and hepatocellular carcinoma. Viral persistence and pathogenesis are due to the ability of HCV to deregulate specific host processes, mainly lipid metabolism and innate immunity. In particular, HCV exploits the lipoprotein machineries for almost all steps of its life cycle. The aim of this review is to summarize current knowledge concerning the interplay between HCV and lipoprotein metabolism. We discuss the role played by members of lipoproteins in HCV entry, replication and virion production.

Background: Diabetic cardiomyopathy (DbCM) is indistinguishable from idiopathic dilated cardiomyopathy (IDCM) as specific histological and/or biochemical markers are unavailable., Methods and Results: Comparative histology, electron microscopy, morphometry for cell volume composition and myocardial fibrosis, reactive oxygen species (ROS), polymerase chain reaction for cardiotropic viruses, immunohistochemistry for nitrotyrosine, inducible nitric oxide synthase (iNOS), 8-hydroxydeoxyguanosine (8-OH-dG) and proteomics have been evaluated in endomyocardial biopsies from 9 patients (pts) (5 male and 4 female, mean age 61 ± 13 years) with DbCM (left ventricular end-diastolic diameter 65 ± 2.3mm; ejection fraction 27 ± 6) and type 2 diabetes mellitus and 9 pts with IDCM (mean age 60 ± 9 years) matched for sex, age and severity of left ventricular (LV) dysfunction. Controls were surgical biopsies from 9 pts with mitral stenosis and normal LV dimensions and function. No qualitative morphological changes were observed between DbCM and IDCM although mitochondrial damage and myofibrillolysis appeared more pronounced in DbCM. ROS were 5 times higher in DbCM than in IDCM and controls and were associated with higher expression of cytoplasm iNOS and nitrotyrosine and nuclear 8-OH-dG. Apoptosis was 14 times higher in DbCM than in IDCM and 41 times higher than in controls. Proteomic profile showed in DbCM a reduced expression of proteins related to beta-oxidation and detoxification pathway., Conclusions: DbCM is a distinctive ROS-mediated disorder with oxidative damage of myocyte's structural proteins and DNA causing cell dysfunction and death. Reduced expression of beta-oxidation proteins suggests a decline of energy production and of mitochondrial function., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Autophagy is a self-degradative physiological process by which the cell removes worn-out or damaged components. Constant at basal level it may become highly active in response to cellular stress. The type 2 transglutaminase (TG2), which accumulates under stressful cell conditions, plays an important role in the regulation of autophagy and cells lacking this enzyme display impaired autophagy/mitophagy and a consequent shift their metabolism to glycolysis. To further define the molecular partners of TG2 involved in these cellular processes, we analysed the TG2 interactome under normal and starved conditions discovering that TG2 interacts with various proteins belonging to different functional categories. Herein we show that TG2 interacts with pyruvate kinase M2 (PKM2), a rate limiting enzyme of glycolysis which is responsible for maintaining a glycolytic phenotype in malignant cells and displays non metabolic functions, including transcriptional co-activation and protein kinase activity. Interestingly, the ablation of PKM2 led to the decrease of intracellular TG2's transamidating activity paralleled by an increase of its tyrosine phosphorylation. Along with this, a significant decrease of ULK1 and Beclin1 was also recorded, thus suggesting a block in the upstream regulation of autophagosome formation. These data suggest that the PKM2/TG2 interplay plays an important role in the regulation of autophagy in particular under cellular stressful conditions such as those displayed by cancer cells.

Epithelial-to-mesenchymal transition (EMT) and the reverse process mesenchymal-to-epithelial transition (MET) are events involved in development, wound healing and stem cell behaviour and contribute pathologically to cancer progression. The identification of the molecular mechanisms underlying these phenotypic conversions in hepatocytes are fundamental to design specific therapeutic strategies aimed at optimising liver repair. The role of autophagy in EMT/MET processes of hepatocytes was investigated in liver-specific autophagy-deficient mice (Alb-Cre;ATG7(fl/fl)) and using the nontumorigenic immortalised hepatocytes cell line MMH. Autophagy deficiency in vivo reduces epithelial markers' expression and increases the levels of mesenchymal markers. These alterations are associated with an increased protein level of the EMT master regulator Snail, without transcriptional induction. Interestingly, we found that autophagy degrades Snail in a p62/SQSTM1 (Sequestosome-1)-dependent manner. Moreover, accordingly to a pro-epithelial function, we observed that autophagy stimulation strongly affects EMT progression, whereas it is necessary for MET. Finally, we found that the EMT induced by TGFβ affects the autophagy flux, indicating that these processes regulate each other. Overall, we found that autophagy regulates the phenotype plasticity of hepatocytes promoting their epithelial identity through the inhibition of the mesenchymal programme.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that, beyond its apoptotic function, is required for the normal expression of major respiratory chain complexes. Here we identified an AIF-interacting protein, CHCHD4, which is the central component of a redox-sensitive mitochondrial intermembrane space import machinery. Depletion or hypomorphic mutation of AIF caused a downregulation of CHCHD4 protein by diminishing its mitochondrial import. CHCHD4 depletion sufficed to induce a respiratory defect that mimicked that observed in AIF-deficient cells. CHCHD4 levels could be restored in AIF-deficient cells by enforcing its AIF-independent mitochondrial localization. This modified CHCHD4 protein reestablished respiratory function in AIF-deficient cells and enabled AIF-deficient embryoid bodies to undergo cavitation, a process of programmed cell death required for embryonic morphogenesis. These findings explain how AIF contributes to the biogenesis of respiratory chain complexes, and they establish an unexpected link between the vital function of AIF and the propensity of cells to undergo apoptosis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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.