Hepatitis C virus relies on lipoproteins for its life cycle

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

C/EBP-β Regulates Endoplasmic Reticulum Stress–Triggered Cell Death in Mouse and Human Models

Endoplasmic reticulum (ER) stress elicits the unfolded protein response (UPR), initially aimed at coping with the stress, but triggering cell death upon further stress. ER stress induces the C/EBP-® variant Liver-enriched Activating Protein (LAP), followed by the dominant-negative variant, Liver Inhibitory Protein (LIP). However, the distinct role of LAP and LIP in ER stress is unknown. We found that the kinetics of the ER stress-induced expression of LIP overlapped with that of the cell death in mouse B16 melanoma cells. Furthermore, inducible over-expression of LIP augmented ER stress-triggered cell death whereas over-expression of LAP attenuated cell death. Similar results were obtained in human 293T cells. Limited vasculature in tumors triggers hypoxia, nutrient shortage and accumulation of toxic metabolites, all of which eliciting continuous ER stress. We found that LAP promoted and LIP inhibited B16 melanoma tumor progression without affecting angiogenesis or accelerating the cell cycle. Rather, LAP attenuated, whereas LIP augmented tumor ER stress. We therefore suggest that C/EBP-® regulates the transition from the protective to the death–promoting phase of the UPR. We further suggest that the over-expression of LAP observed in many solid tumors promotes tumor progression by attenuating ER stress–triggered tumor cell death

Autophagy in development and regeneration: role in tissue remodelling and cell survival

Morphogenetic events that occur during development and regeneration are energy demanding processes requiring profound rearrangements in cell architecture, which need to be coordinated in timely fashion with other cellular activities, such as proliferation, migration and differentiation. In the last 15 years, it has become evident that autophagy, an evolutionarily-conserved catabolic process that mediates the lysosomal turnover of organelles and macromolecules, is an essential "tool" to ensure remodelling events that occur at cellular and tissue levels. Indeed, studies in several model organisms have shown that the inactivation of autophagy genes has a significant impact on embryogenesis and tissue regeneration, leading to extensive cell death and persistence of unnecessary cell components. Interestingly, the increased understanding of the mechanisms that confers selectivity to the autophagic process has also contributed to identifying development-specific targets of autophagy across species. Moreover, alternative ways to deliver materials to the lysosome, such as microautophagy, are also emerging as key actors in these contexts, providing a more complete view of how the cell component repertoire is renovated. In this review, we discuss the role of different types of autophagy in development and regeneration of invertebrates and vertebrates, focusing in particular on its contribution in cnidarians, platyhelminthes, nematodes, insects, zebrafish and mammals

Iron overload down-regulates the expression of the HIV-1 Rev cofactor eIF5A in infected T lymphocytes

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

Beclin 1 A role in membrane dynamics and beyond

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Analysis of Secreted Proteins from Prepubertal Ovarian Tissues Exposed In Vitro to Cisplatin and LH

It is well known that secreted and exosomal proteins are associated with a broad range of physiological processes involving tissue homeostasis and differentiation. In the present paper, our purpose was to characterize the proteome of the culture medium in which the oocytes within the primordial/primary follicles underwent apoptosis induced by cisplatin (CIS) or were, for the most part, protected by LH against the drug. To this aim, prepubertal ovarian tissues were cultured under control and in the presence of CIS, LH, and CIS + LH. The culture media were harvested after 2, 12, and 24 h from chemotherapeutic drug treatment and analyzed by liquid chromatography-mass spectrometry (LC-MS). We found that apoptotic conditions generated by CIS in the cultured ovarian tissues and/or oocytes are reflected in distinct changes in the extracellular microenvironment in which they were cultured. These changes became evident mainly from 12 h onwards and were characterized by the inhibition or decreased release of a variety of compounds, such as the proteases Htra1 and Prss23, the antioxidants Prdx2 and Hbat1, the metabolic regulators Ldha and Pkm, and regulators of apoptotic pathways such as Tmsb4x. Altogether, these results confirm the biological relevance of the LH action on prepuberal ovaries and provide novel information about the proteins released by the ovarian tissues exposed to CIS and LH in the surrounding microenvironment. These data might represent a valuable resource for future studies aimed to clarify the effects and identify biomarkers of these compounds' action on the developing ovary

Anti-mycobacterial activity of labdane and halimane diterpenes obtained from Plectranthus ornatus Codd

Biomedical and biopharmaceutical research : jornal de investigação biomédica e biofarmacêuticaOs produtos naturais são uma fonte única de compostos-tipofpara o desenvolvimento de fármacos em química medicinal. Vários diterpenos das espécies Plectranthus foram referidos com actividade tuberculostática interessante, sendo que o P. omatus Codd. é usado como anti-infeccioso em algumas regiões do Brasil. Em trabalhos anteriores, um diterpeno de esqueleto de halimano outro labdano foram isolados a partir de P. ornatus Codd. Neste trabalho, avaliou-se preliminarmente a sua actividade micobacteriana com uma estirpe não virulenta de Mycobacterium smegmatis. A citotoxicidade dos compostos foi testada medindo a libertação de lactato desidrogenase (LDH) não sendo encontrados efeitos citotóxicos consideráveis até 25 ug/mL. Posteriormente, o método de microdiluição foi utilizado para determinar a concentração mínima inibitória (CMI) de M. smegmatis. A CMI>99% para o esqueleto de halimano foi 100 ug/mL e >100 ug/mL para o esqueleto de labdano. De acordo com o nosso conhecimento, este é o primeiro estudo usando diterpenos de esqueleto de halimano e de labdano isolados a partir de P. omatus em ensaios de citotoxicidade em macrófagos, e num ensaio preliminar sobre a sua atividade anti-micobacteriana. Estudos futuros são sugeridos na estirpe virulenta de M. tugerculosis, particularmente para os diterpenos de esqueleto de halimano.Plectranthus spp. have been reported to have interesting tuberculostatic activity and P. ornatus Codd. has been used in some regions of Brazil as an anti-infective. Previously, diterpenes with halimane and labdane skeletons were isolated in large quantities from P. ornatus Codd. We assessed the anti-mycobacterial activity of these compounds, performing a preliminar assay with the non-virulent strain Mycobacterium smegmatis. The cytotoxicity of the diterpenes with halimane and labdane skeletons was tested with the lactate dehydrogenase assay, where no considerable cytotoxic effects were found up to 25 μg.mL-1. Subsequently, the microdilution method was used to determine the minimum inhibitory concentration (MIC) against M. smegmatis. The MIC that inhibited the growth of the non-virulent mycobacteria by ≥99% was 100 μg.mL-1 for the diterpene with halimane skeleton, whereas for the diterpene with a labdane skeleton was >100 μg.mL-1. To the best of our knowledge, this is the first report on diterpenes with halimane and labdane skeletons isolated from P. ornatus in macrophages cytotoxicity, and in a preliminar assay for anti-mycobacterial activity. Further studies are suggested on M. tuberculosis, particularly for the diterpenes with halimane skeleton

The STING/TBK1/IRF3/IFN type I pathway is defective in cystic fibrosis

Cystic fibrosis (CF) is a rare autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most common mutation is F508del-CFTR (ΔF) which leads the encoded ion channel towards misfolding and premature degradation. The disease is characterized by chronic bronchopulmonary obstruction, inflammation and airways colonization by bacteria, which are the major cause of morbidity and mortality. The STING pathway is the main signaling route activated in the presence of both self and pathogen DNA, leading to Type I Interferon (IFN I) production and the innate immune response. In this study, we show for the first time the relationship existing in CF between resistant and recurrent opportunistic infections by Pseudomonas aeruginosa and the innate immunity impairment. We demonstrate through ex vivo and in vivo experiments that the pathway is inadequately activated in ΔF condition and the use of direct STING agonists, as 2′,3′-cyclic GMP-AMP (2’, 3’ cGAMP), is able to restore the immune response against bacterial colonization. Indeed, upon treatment with the STING pathway agonists, we found a reduction of colony forming units (CFUs) consequent to IFN-β enhanced production in Pseudomonas aeruginosa infected bone marrow derived macrophages and lung tissues from mice affected by Cystic Fibrosis. Importantly, we also verified that the impairment detected in the primary PBMCs obtained from ΔF patients can be corrected by 2’, 3’ cGAMP. Our work indicates that the cGAS/STING pathway integrity is crucial in the Cystic Fibrosis response against pathogens and that the restoration of the pathway by 2’, 3’ cGAMP could be exploited as a possible new target for the symptomatic treatment of the disease

A Proteomic Approach for Comprehensively Screening Substrates of Protein Kinases Such as Rho-Kinase

BACKGROUND: Protein kinases are major components of signal transduction pathways in multiple cellular processes. Kinases directly interact with and phosphorylate downstream substrates, thus modulating their functions. Despite the importance of identifying substrates in order to more fully understand the signaling network of respective kinases, efficient methods to search for substrates remain poorly explored. METHODOLOGY/PRINCIPAL FINDINGS: We combined mass spectrometry and affinity column chromatography of the catalytic domain of protein kinases to screen potential substrates. Using the active catalytic fragment of Rho-kinase/ROCK/ROK as the model bait, we obtained about 300 interacting proteins from the rat brain cytosol fraction, which included the proteins previously reported as Rho-kinase substrates. Several novel interacting proteins, including doublecortin, were phosphorylated by Rho-kinase both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity

AMBRA1 regulates mitophagy by interacting with ATAD3A and promoting PINK1 stability

PINK1 accumulation at the outer mitochondrial membrane (OMM) is a key event required to signal depolarized mitochondria to the autophagy machinery. How this early step is, in turn, modulated by autophagy proteins remains less characterized. Here, we show that, upon mitochondrial depolarization, the proautophagic protein AMBRA1 is recruited to the OMM and interacts with PINK1 and ATAD3A, a transmembrane protein that mediates mitochondrial import and degradation of PINK1. Downregulation of AMBRA1 expression results in reduced levels of PINK1 due to its enhanced degradation by the mitochondrial protease LONP1, which leads to a decrease in PINK1-mediated ubiquitin phosphorylation and mitochondrial PRKN/PARKIN recruitment. Notably, ATAD3A silencing rescues defective PINK1 accumulation in AMBRA1-deficient cells upon mitochondrial damage. Overall, our findings underline an upstream contribution of AMBRA1 in the control of PINK1-PRKN mitophagy by interacting with ATAD3A and promoting PINK1 stability. This novel regulatory element may account for changes of PINK1 levels in neuropathological conditions.Abbreviations: ACTB/β-actin: actin beta; AMBRA1: autophagy and beclin 1 regulator 1; ATAD3A: ATPase family AAA domain containing 3A; BCL2L1/BCL-xL: BCL2 like 1; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; OMA1: OMA1 zinc metallopeptidase; OMM: outer mitochondrial membrane; PARL: presenilin associated rhomboid like; PARP: poly(ADP-ribose) polymerase; PD: Parkinson disease; PINK1: PTEN induced kinase 1; PRKN/PARKIN: parkin RBR E3 ubiquitin protein ligase; SDHA: succinate dehydrogenase complex flavoprotein subunit A; TOMM70: translocase of outer mitochondrial membrane 70