Data on the effects of low iron diet on serum lipid profile in HCV transgenic mouse model

Here, we presented new original data on the effects of iron depletion on the circulating lipid pro fi le in B6HCV mice, a murine model of HCV-related dyslipidemia. Male adult B6HCV mice were subjected to non-invasive iron depletion by low iron diet. Serum iron concentration was assessed for evaluating the effects of the dietary iron depletion. Concentrations of circulating triglycerides, total cholesterol, Low Density Lipoproteins (LDLs), High Density Lipoproteins (HDLs) were analyzed and reported by using stacked line charts. The present data indicated that low serum iron con- centration is associated to i) lower serum triglycerides con- centrations and ii) increased circulating LDLs. The presented ori- ginal data have not been published elsewhere

Pseudomonas aeruginosa LptE is crucial for LptD assembly, cell envelope integrity, antibiotic resistance and virulence

Lipopolysaccharide (LPS) is an essential structural component of the outer membrane (OM) of most Gram-negative bacteria. In the model organism Escherichia coli, LPS transport to the OM requires seven essential proteins (LptABCDEFG) that form a continuous bridge across the cell envelope. In Pseudomonas aeruginosa the recently-demonstrated essentiality of LptD and LptH, the P. aeruginosa LptA homologue, confirmed the crucial role of the Lpt system and, thus, of LPS in OM biogenesis in this species. Surprisingly, independent high-throughput transposon mutagenesis studies identified viable P. aeruginosa insertion mutants in the lptE gene, suggesting that it might be dispensable for bacterial growth. To test this hypothesis, we generated an lptE conditional mutant in P. aeruginosa PAO1. LptE depletion only slightly impairs P. aeruginosa growth in vitro. Conversely, LptE is important for cell envelope stability, antibiotic resistance and virulence in an insect model. Interestingly, the maturation and OM localization of LPS is only marginally affected in LptE-depleted cells, while the levels of the OM component LptD are strongly reduced. This suggests that P. aeruginosa LptE might not be directly involved in LPS transport, although it is clearly essential for the maturation and/or stability of LptD. While poor functionality of LptD caused by LptE depletion is somehow tolerated by P. aeruginosa, this has a high cost in terms of cell integrity, drug resistance and virulence, highlighting LptE function(s) as an interesting target to weaken P. aeruginosa defenses and reduce its infectivity

SILAC labeling coupled to shotgun proteomics analysis of membrane proteins of liver stem/hepatocyte allows to candidate the inhibition of TGF-beta pathway as causal to differentiation

International audienceDespite extensive research on hepatic cells precursors and their differentiated states, much remains to be learned about the mechanism underlying the self-renewal and differentiation. We apply the SILAC (stable isotope labeling by amino acids in cell culture) approach to quantitatively compare the membrane proteome of the resident liver stem cells (RLSCs) and their progeny spontaneously differentiated into epithelial/hepatocyte (RLSCdH). By means of nanoLC-MALDI-TOF/TOF approach, we identified and quantified 248 membrane proteins and 57 of them were found modulated during hepatocyte differentiation. Functional clustering of differentially expressed proteins by Ingenuity Pathway Analysis revealed that the most of membrane proteins found to be modulated are involved in cell-to-cell signaling/interaction pathways. Moreover, the upstream prediction analysis of proteins involved in cell-to-cell signaling and interaction unveiled that the activation of the mesenchymal to epithelial transition (MET), by the repression of TGFB1/Slug signaling, may be causal to hepatocyte differentiation. Taken together, this study increases the understanding of the underlying mechanisms modulating the complex biological processes of hepatic stem cell proliferation and differentiation

ADAR1 restricts LINE-1 retrotransposition

Abstract Adenosine deaminases acting on RNA (ADARs) are involved in RNA editing that converts adenosines to inosines in double-stranded RNAs. ADAR1 was demonstrated to be functional on different viruses exerting either antiviral or proviral effects. Concerning HIV-1, several studies showed that ADAR1 favors viral replication. The aim of this study was to investigate the composition of the ADAR1 ribonucleoprotein complex during HIV-1 expression. By using a dual-tag affinity purification procedure in cells expressing HIV-1 followed by mass spectrometry analysis, we identified 14 non-ribosomal ADAR1-interacting proteins, most of which are novel. A significant fraction of these proteins were previously demonstrated to be associated to the Long INterspersed Element 1 (LINE1 or L1) ribonucleoparticles and to regulate the life cycle of L1 retrotransposons that continuously re-enter host-genome.Hence, we investigated the function of ADAR1 in the regulation of L1 activity.By using different cell-culture based retrotransposition assays in HeLa cells, we demonstrated a novel function of ADAR1 as suppressor of L1 retrotransposition. Apparently, this inhibitory mechanism does not occur through ADAR1 editing activity. Furthermore, we showed that ADAR1 binds the basal L1 RNP complex. Overall, these data support the role of ADAR1 as regulator of L1 life cycle

Ferritin heavy chain Is the host factor responsible for HCV-Induced inhibition of apoB-100 production and is required for efficient viral infection

Hepatic fat export occurs by apolipoprotein B-100-containing lipoprotein production, whereas impaired production leads to liver steatosis. Hepatitis C virus (HCV) infection is associated to dysregulation of apoB-100 secretion and steatosis; however, the molecular mechanism by which HCV affects the apoB-100 secretion is not understood. Here, combining quantitative proteomics and computational biology, we propose ferritin heavy chain (Fth) as being the cellular determinant of apoB-100 production inhibition. By means of molecular analyses, we found that HCV nonstructural proteins and NS5A appear to be sufficient for inducing Fth up-regulation. Fth in turn was found to inhibit apoB-100 secretion leading to increased intracellular degradation via proteasome. Notably, intracellular Fth down-regulation by siRNA restores apoB-100 secretion. The inverse correlation between ferritin and plasma apoB-100 concentrations was also found in JFH-1 HCV cell culture systems (HCVcc) and HCV-infected patients. Finally, Fth expression was found to be required for robust HCV infection. These observations provide a further molecular explanation for the onset of liver steatosis and allow for hypothesizing on new therapeutic and antiviral strategies

Overexpression of the vitronectin v10 subunit in patients with nonalcoholic steatohepatitis: Implications for noninvasive diagnosis of NASH

Nonalcoholic steatohepatitis (NASH) is the critical stage of nonalcoholic fatty liver disease (NAFLD). The persistence of necroinflammatory lesions and fibrogenesis in NASH is the leading cause of liver cirrhosis and, ultimately, hepatocellular carcinoma. To date, the histological examination of liver biopsies, albeit invasive, remains the means to distinguish NASH from simple steatosis (NAFL). Therefore, a noninvasive diagnosis by serum biomarkers is eagerly needed. Here, by a proteomic approach, we analysed the soluble low-molecular-weight protein fragments flushed out from the liver tissue of NAFL and NASH patients. On the basis of the assumption that steatohepatitis leads to the remodelling of the liver extracellular matrix (ECM), NASH-specific fragments were in silico analysed for their involvement in the ECM molecular composition. The 10 kDa C-terminal fragment of the ECM prote in vitro nectin (VTN) was then selected as a promising circulating biomarker in discriminating NASH. The analysis of sera of patients provided these major findings: the circulating VTN fragment (i) is overexpressed in NASH patients and positively correlates with the NASH activity score (NAS); (ii) originates from the disulfide bond reduction between the V10 and the V65 subunits. In conclusion, V10 determination in the serum could represent a reliable tool for the noninvasive discrimination of NASH from simple steatosi

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

MiR675-5p acts on HIF-1α to sustain hypoxic responses: A new therapeutic strategy for glioma

Hypoxia is a common feature in solid tumours. In glioma, it is considered the major driving force for tumour angiogenesis and correlates with enhanced resistance to conventional therapies, increased invasiveness and a poor prognosis for patients. Here we describe, for the first time, that miR675-5p, embedded in hypoxia-induced long non-coding RNA H19, plays a mandatory role in establishing a hypoxic response and in promoting hypoxia-mediated angiogenesis. We demonstrated, in vitro and in vivo, that miR675-5p over expression in normoxia is sufficient to induce a hypoxic moreover, miR675-5p depletion in low oxygen conditions, drastically abolishes hypoxic responses including angiogenesis. In addition, our data indicate an interaction of miR675-5p, HIF-1α mRNA and the RNA Binding Protein HuR in hypoxia-induced responses. We suggest the modulation of miR675-5p as a new therapeutic option to promote or abolish hypoxia induced angiogenesis

The RNA-dependent RNA polymerase essential for post-transcriptional gene silencing in Neurospora crassa interacts with replication protein A

Post-transcriptional gene silencing (PTGS) pathways play a role in genome defence and have been extensively studied, yet how repetitive elements in the genome are identified is still unclear. It has been suggested that they may produce aberrant transcripts (aRNA) that are converted by an RNA-dependent RNA polymerase (RdRP) into double-stranded RNA (dsRNA), the essential intermediate of PTGS. However, how RdRP enzymes recognize aberrant transcripts remains a key question. Here we show that in Neurospora crassa the RdRP QDE-1 interacts with Replication Protein A (RPA), part of the DNA replication machinery. We show that both QDE-1 and RPA are nuclear proteins and that QDE-1 is specifically recruited onto the repetitive transgenic loci. We speculate that this localization of QDE-1 could allow the in situ production of dsRNA using transgenic nascent transcripts as templates, as in other systems. Supporting a link between the two proteins, we found that the accumulation of short interfering RNAs (siRNAs), the hallmark of silencing, is dependent on an ongoing DNA synthesis. The interaction between QDE-1 and RPA is important since it should guide further studies aimed at understanding the specificity of the RdRP and it provides for the first time a potential link between a PTGS component and the DNA replication machinery

Hepatitis C virus production requires apolipoprotein A-I and affects its association with nascent low-density lipoproteins

Background/aims The life cycle of hepatitis C virus (HCV) is intimately linked to the lipid metabolism of the host. In particular, HCV exploits the metabolic machinery of the lipoproteins in several steps of its life cycle such as circulation in the bloodstream, cell attachment and entry, assembly and release of viral particles. However, the details of how HCV interacts with and influences the metabolism of the host lipoproteins are not well understood. A study was undertaken to investigate whether HCV directly affects the protein composition of host circulating lipoproteins. Methods A proteomic analysis of circulating very low-, low- and high-density lipoproteins (VLDL, LDL and HDL), isolated from either in-treatment naive HCV-infected patients or healthy donors (HD), was performed using two-dimensional gel electrophoresis and tandem mass spectrometry (MALDI-TOF/TOF). The results obtained were further investigated using in vitro models of HCV infection and replication. Results A decreased level of apolipoprotein A-I (apoA-I) was found in the LDL fractions of HCV-infected patients. This result was confirmed by western blot and ELISA analysis. HCV cellular models (JFH1 HCV cell culture system (HCVcc) and HCV subgenomic replicons) showed that the decreased apoA-I/LDL association originates from hepatic biogenesis rather than lipoprotein catabolism occurring in the circulation, and is not due to a downregulation of the apoA-I protein concentration. The sole non-structural viral proteins were sufficient to impair the apoA-I/LDL association. Functional evidence was obtained for involvement of apoA-I in the viral life cycle such as RNA replication and virion production. The specific siRNA-mediated downregulation of apoA-I led to a reduction in both HCV RNA and viral particle levels in culture. Conclusions This study shows that HCV induces lipoprotein structural modification and that its replication and production are linked to the host lipoprotein metabolism, suggesting apoA-I as a new possible target for antiviral therapy