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Laura Beretta

Publications and source records attributed to Laura Beretta.

17 recordsLinked to original sources
PubMed2006

Modification of host lipid raft proteome upon hepatitis C virus replication.

Hepatitis C virus (HCV) replication complex resides in detergent-insoluble subcellular domains or lipid rafts. We used two proteomics approaches to characterize the protein content of lipid rafts isolated from Huh7 cells and its modification upon HCV replication. Using two-dimensional electrophoresis and mass spectrometry, we identified approximately 100 protein spots in the isolated lipid rafts; among them, 39 were reproducibly modified in HCV replicon cell lines as compared with control cell lines. We also used stable isotope labeling by amino acids in cell culture (SILAC) combined with one-dimensional electrophoresis separation and mass spectrometry. Using this approach, we identified 1036 individual proteins based on peptides selected with at least 95% confidence; among them, 413 proteins were identified with at least two peptides. Quantification analysis identified 150 proteins modified by at least 2.5-fold (110 up-regulated and 40 down-regulated) in HCV-replicating cells compared with controls. Protein identifications and quantifications obtained by both proteomics approaches were largely concordant. Modulated proteins included a majority of proteins involved in vesicular and protein trafficking and in cell signaling. Remarkably for a large number of proteins, their up-regulation in lipid rafts of HCV replicon cells was due to their relocalization. By using small interfering RNAs directed to the modulated small GTPases Cdc42 and RhoA, we observed an increase in HCV replication, whereas reduction of syntaxin 7 expression resulted in decreased replication of HCV. Our findings indicate that protein subcellular relocalization occurs in HCV-containing cells that can directly affect HCV replication.

Cell Extracts↗
PubMed2006

The Human Liver Proteome Project (HLPP) workshop during the 4th HUPO World Congress.

The Human Liver Proteome Project (HLPP) is the first initiative of the Human Proteome Organisation (HUPO) to target a human organ/tissue and aims at the establishment of a biological atlas of the liver to advance our understanding of liver functions and to provide the tools to develop new diagnostics and therapeutics for liver diseases. The major achievements of HLPP during its pilot phase (2003-2005) were presented during a one-day workshop held on August 27, 2005, immediately prior to the 4th HUPO World Congress in Munich, Germany. These include (i) the participation of a large number of laboratories worldwide and the initiation of international research collaborations; (ii) the establishment of a management infrastructure and (iii) the development of standard operating procedures and the generation and collection of a first set of expression profile data. A summary of the workshop was subsequently presented by Professor Fuchu He, the Chair of this initiative, during the HUPO 4th Annual World Congress.

Congresses as Topic↗
PubMed2006

Cleavage of endoplasmic reticulum proteins in hepatocellular carcinoma: Detection of generated fragments in patient sera.

BACKGROUND & AIMS: In the past decade, there has been a rising incidence of hepatocellular carcinoma (HCC) and a progressive increase in HCC-related mortality in the United States and Western Europe. The poor survival of patients with HCC is largely related to the lack of reliable tools for early diagnosis. METHODS: We have applied proteomics tools to the comparative analysis of protein profiles between HCC and adjacent nontumor tissues as a means for discovering novel molecular markers. RESULTS: Forty-seven protein spots that showed reproducible variation were identified by mass spectrometry, corresponding to 23 distinct genes. A positive correlation between transcript and protein level variations was observed for only 7 out of the 23 genes. Proteolytic cleavage accounted for the discrepancies between messenger RNA and protein level changes for 7 genes including calreticulin, PDIA3, PDI, and GRP78. We detected a fragment of each of these 4 endoplasmic reticulum proteins in the culture supernatant of the PLC-PRF5 hepatoma cell line, suggesting that their cleavage leads to release of selected cleaved products in the extracellular compartment. We also detected calreticulin and PDIA3 cleavage products in sera of patients with HCC. A statistically highly significant difference in calreticulin and PDIA3 fragment serum levels between patients with HCC and healthy individuals was observed. Amounts of calreticulin and PDIA3 fragments were also significantly different between patients with HCC and at-risk patients (patients with chronic hepatitis or cirrhosis). CONCLUSIONS: Specific isoforms in general and cleavage products in particular should therefore be further evaluated as new markers for HCC.

Biomarkers, Tumor↗
PubMed2005

Activation of the N-Ras-PI3K-Akt-mTOR pathway by hepatitis C virus: control of cell survival and viral replication.

The hepatitis C virus (HCV) replication complex is localized within detergent-resistant membranes or lipid rafts. We analyzed the protein contents of detergent-resistant fractions isolated from Huh7 cells expressing a self-replicating full-length HCV-1b genome. Using two-dimensional gel electrophoresis followed by mass spectrometry, we identified N-Ras as one of the proteins in which expression was increased in the detergent-resistant fractions from HCV genomic replicon clones compared to control cells. N-Ras is an activator of the phosphatidylinositol-3-kinase (PI3K)-Akt pathway. We found that the activities of PI3K and Akt, as well as the activity of their downstream target, mTOR, in the HCV-replicating cells were increased. Both PI3K-Akt- and mTOR-dependent pathways have been shown to promote cell survival. In agreement with this, HCV replicon cells were resistant to serum starvation-induced apoptosis. We also characterized the role of this pathway in HCV replication. Reduction of N-Ras expression by transfection of N-Ras small interfering RNA (siRNA) resulted in increased replication of HCV. We observed a similar increase in HCV replication in cells treated with the PI3K inhibitor LY294002 and in cells transfected with mTOR siRNA. Taken together, these data suggest that increased N-Ras levels in subcellular sites of HCV replication and stimulation of the prosurvival PI3K-Akt pathway and mTOR by HCV not only protect cells against apoptosis but also contribute to the maintenance of steady-state levels of HCV replication. These effects may contribute to the establishment of persistent infection by HCV.

Apoptosis↗
PubMed2004

Global and distinct targets of IRF-5 and IRF-7 during innate response to viral infection.

The interferon regulatory factors (IRF) are transcriptional mediators of cellular response to viral invasion that play a critical role in the innate antiviral defense. Two of these factors, IRF-5 and IRF-7, play a critical role in the induction of interferon (IFNA) genes in infected cells; they are expressed constitutively in monocytes, B cells, and precursors of dendritic cells (pDC2) that are high producers of interferon alpha, and their expression can be further stimulated by type I interferon. The goal of the present study was to identify and analyze expression of cellular genes that are modulated by IRF-5 and IRF-7 during the innate response to viral infection. The transcription profiles of infected BJAB cells overexpressing IRF-5 or IRF-7 were determined by using oligonucleotide arrays with probe sets representing about 6800 human genes. This analysis shows that IRF-5 and IRF-7 activate a broad profile of heterologous genes encoding not only antiviral, inflammatory, and pro-apoptotic proteins but also proteins of other functional categories. The number of IRF-5- and IRF-7-modulated genes was significantly higher in infected than in uninfected cells, and the transcription signature was predominantly positive. Although IRF-5 and IRF-7 stimulated a large number of common genes, a distinct functional profile was associated with each of these IRFs. The noted difference was a broad antiviral and early inflammatory transcriptional profile in infected BJAB/IRF-5 cells, whereas the IRF-7-induced transcripts were enriched for the group of mitochondrial genes and genes affecting the DNA structure. Taken together, these data indicate that IRF-5 and IRF-7 act primarily as transcriptional activators and that IRF-5-and IRF-7-induced innate antiviral response results in a broad alteration of the transcriptional profile of cellular genes.

Blotting, Western↗
PubMed2004

Hepatitis C virus NS5A-regulated gene expression and signaling revealed via microarray and comparative promoter analyses.

Most individuals exposed to hepatitis C virus (HCV) become chronically infected and are predisposed to liver disease. The mechanisms underlying viral persistence and disease progression are unknown. A role for the HCV NS5A protein in viral replication and interferon resistance has been demonstrated. To identify mechanisms affected by NS5A, we analyzed the gene expression of Huh7 cells expressing NS5A and control cells using oligonucleotide microarrays. A set of 103 genes (43 up-regulated, 60 down-regulated) whose expression was modified by at least twofold was selected. These included genes involved in cell adhesion and motility, calcium homeostasis, lipid transport and metabolism, and genes regulating immune responses. The finding of modulated expression of genes related to the TGF-beta superfamily and liver fibrosis was observed. Interestingly, both the tumor necrosis factor and lymphotoxin beta receptors were down-regulated by NS5A. Similar data were obtained following expression of four NS5A mutants obtained from patients who were not responsive or were sensitive to interferon therapy. Through computational analysis, we determined that 39 of the 43 genes up-regulated by NS5A contained one or more nuclear factor kappaB (NF-kappaB) binding sites within their promoter region. Using the Gibbs sampling method, we also detected enrichment of NF-kappaB consensus binding sites in the upstream regions of the 43 coexpressed genes. Activation of NF-kappaB by NS5A was subsequently demonstrated in luciferase reporter assays. Adenovirus-mediated expression of IkappaBalpha reverted NS5A mediated up-regulation of gene expression. In conclusion, this study suggests a role of NS5A and NF-kappaB in HCV pathogenesis and related liver disease. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

Amino Acid Sequence↗
PubMed2004

Proteomics for hepatocellular carcinoma marker discovery.

Refinements of serological markers and screening of patients at high risk for developing hepatocellular carcinoma (HCC) may lead to better HCC detection, earlier intervention, and successful treatment, improving long-term outcomes. Proteomics promises the discovery of biomarkers for early HCC detection and diagnosis. Proteomic-based profiling uniquely allows delineation of global changes in expression patterns resulting from transcriptional and posttranscriptional control, posttranslational modifications, and shifts in proteins between cellular compartments. Approaches to that effect include direct serum protein profiling and comparative analysis of protein expression in normal, precancerous, and early-stage tumor tissues. Identification of panels of tumor antigens that elicit a humoral response also may contribute to the discovery of new markers for HCC screening and diagnosis. Today, 2-dimensional polyacrylamide gel electrophoresis, multidimensional liquid chromatography, mass spectrometry, and protein microarrays are among the proteomic tools available for biomarker and drug target discovery. We review these technologies and their application to the study of HCC. Our objective is to provide a framework for appreciating the promise, while at the same time understanding the challenges behind translating proteomics discovery into novel diagnostic tests.

Biomarkers, Tumor↗
PubMed2004

The future of proteomics in the study of alcoholism.

This article represents the proceedings of a workshop at the 2003 annual meeting of the Research Society on Alcoholism in Fort Lauderdale, FL. The workshop organizers/chairpersons were Chinnaswamy Kasinathan and Paul Manowitz. The presentations were (1) Introduction to the field of proteomics, by Kent Vrana; (2) Use of proteomics in the identification of urinary biomarkers for alcohol intake, by Chinnaswamy Kasinathan, Paul Thomas, and Paul Manowitz; (3) Proteomics screening illuminates ethanol-mediated induction of HDL proteins in macaques, by Kent Vrana, Randy Gooch, Travis Worst, Stephen Walker, Aaron Xu, Peter Pierre, Heather Green, and Kathleen Grant; and (4) Proteomics applied to the study of the liver, by Laura Beretta.

Alcoholism↗
PubMed2003

ERK and p38 inhibit the expression of 4E-BP1 repressor of translation through induction of Egr-1.

4E-BP1 plays a major role in translation by inhibiting cap-dependent translation initiation. Several reports have investigated the regulation of 4E-BP1 phosphorylation, which varies along with cell differentiation and upon various stimulations, but very little is known about the regulation of its expression. In a first part, we show that the expression of 4E-BP1 protein and transcript decreases in hematopoietic cell lines cultivated in the presence of phorbol 12-myristate 13-acetate (PMA). This decrease depends on the activation of the ERK/mitogen-activated protein kinases. 4E-BP1 expression also decreases when the p38/mitogen-activated protein kinase pathway is activated by granulocyte/macrophage colony-stimulating factor but to a lesser extent than with PMA. In a second part, we examine how 4e-bp1 promoter activity is regulated. PMA and granulocyte/macrophage colony-stimulating factor induce Egr-1 expression through ERK and p38 activation, respectively. Using a dominant negative mutant of Egr, ZnEgr, we show that this transcription factor is responsible for the inhibition of 4e-bp1 promoter activity. In a third part we show that histidine decarboxylase, whose activity and expression are inversely correlated with 4E-BP1 expression, is a potential target for the translational machinery. These data (i) are the first evidence of a new role of ERK and p38 on the translational machinery and (ii) demonstrate that 4E-BP1 is a new target for Egr-1.

Adaptor Proteins, Signal Transducing↗
PubMed2002

An altered cellular response to interferon and up-regulation of interleukin-8 induced by the hepatitis C viral protein NS5A uncovered by microarray analysis.

There is evidence for an inhibition of interferon-alpha antiviral activity by the hepatitis C viral protein, NS5A. To identify the mechanisms through which NS5A blocks interferon activity, we compared the gene expression profile of interferon-treated Huh7 cells, stably expressing NS5A with control, using microarrays. Following interferon treatment, 50 genes were up-regulated by at least twofold in control clones, whereas induction of 9 of the 50 genes was significantly reduced in NS5A-expressing clones. The strongest effect of NS5A on interferon response was observed for the OAS-p69 gene. Remarkably, Huh7 cells expressing NS5A showed an up-regulation of interleukin-8. Up-regulation of interleukin-8 was also observed upon transient expression of NS5A mutants isolated from patients responsive or resistant to interferon therapy. Addition of interleukin-8 to Huh7 cells inhibited the antiviral activity of interferon and, similarly to NS5A, reduced the induction by interferon-alpha of selective genes including OAS-p69. Our findings provide a mechanism for NS5A-mediated interferon resistance.

Gene Expression Profiling↗
PubMed2002

Global and specific translational control by rapamycin in T cells uncovered by microarrays and proteomics.

Rapamycin has been shown to affect translation. We have utilized two complementary approaches to identify genes that are predominantly affected by rapamycin in Jurkat T cells. One was to compare levels of polysome-bound and total RNA using oligonucleotide microarrays complementary to 6,300 human genes. Another was to determine protein synthesis levels using two-dimensional PAGE. Analysis of expression changes at the polysome-bound RNA levels showed that translation of most of the expressed genes was partially reduced following rapamycin treatment. However, translation of 136 genes (6% of the expressed genes) was totally inhibited. This group included genes encoding RNA-binding proteins and several proteasome subunit members. Translation of a set of 159 genes (7%) was largely unaffected by rapamycin treatment. These genes included transcription factors, kinases, phosphatases, and members of the RAS superfamily. Analysis of [(35)S]methionine-labeled proteins from the same cell populations using two-dimensional PAGE showed that the integrated intensity of 111 of 830 protein spots changed in rapamycin-treated cells by at least 3-fold (70 increased, 41 decreased). We identified 22 affected protein spots representing protein products of 16 genes. The combined microarray and proteomic approach has uncovered novel genes affected by rapamycin that may be involved in its immunosuppressive effect and other genes that are not affected at the level of translation in a context of general inhibition of cap-dependent translation.

Blotting, Western↗
PubMed2002

A distinct repertoire of autoantibodies in hepatocellular carcinoma identified by proteomic analysis.

Chronic infections with hepatitis B (HBV) and hepatitis C (HCV) viruses are major risk factors for hepatocellular carcinoma (HCC). We have utilized a proteomic approach to determine whether a distinct repertoire of autoantibodies can be identified in HCC. Sera from 37 patients with HCC and 31 subjects chronically infected with HBV or HCV without HCC were investigated. Sera from 116 patients with other cancers, three patients with systemic lupus erythematosus, and 24 healthy subjects were utilized as controls. We report the identification of eight proteins, for each of which autoantibodies were detected in sera from more than 10% of patients with HCC but not in sera from healthy individuals (p < 0.05). Autoantibodies to four of these proteins were detected at a comparable frequency in sera from patients with chronic hepatitis. The other four proteins, which consisted of calreticulin isoforms, cytokeratin 8, nucleoside diphosphate kinase A, and F(1)-ATP synthase beta-subunit, induced autoantibodies among patients with HCC, independently of their HBV/HCV status. Calreticulin, and a novel truncated form of calreticulin (Crt32) we have identified, most commonly elicited autoantibodies among patients with HCC (27%). We conclude that a distinct repertoire of autoantibodies is associated with HCC that may have utility in early diagnosis of HCC among high risk subjects with chronic hepatitis.

Adult↗
PubMed2002

CD6: expression during development, apoptosis and selection of human and mouse thymocytes.

CD6, a 130-kDa surface glycoprotein, is expressed primarily on cells of T lineage. A co-stimulatory role for CD6 in mature T cells has been shown, but the function of CD6 during thymocyte development is unknown. Since CD6 ligands are expressed on thymic epithelium, their interactions with CD6 could be important in thymic selection. In this report we show that CD6 is developmentally regulated in human and mouse thymocytes, and further demonstrate that increase in the level of CD6 expression correlates with expression of the selection marker CD69. We also show that activation via CD2 induces CD6 expression on mature human thymocytes and on a subset of immature human thymocytes that are resistant to apoptosis. In human and mouse thymocytes that express heterogeneous TCR, CD6 increases occur as double-positive thymocytes are selected to a single-positive stage. In contrast, in thymocytes from TCR transgenic mice, CD6 is barely increased following selection, suggesting that as functional avidity increases, requirements for CD6-dependent co-stimulation decrease. Taken together, these results indicate that during thymic development CD6-dependent signals may contribute both to thymocyte survival, and to the overall functional avidity of selection in both man and mouse.

Animals↗
PubMed2002

Integrated genomic and proteomic analysis of signaling pathways in dendritic cell differentiation and maturation.

Dendritic cells (DCs) are antigen-presenting cells that play a major role in initiating primary immune responses. Their phenotypic and functional characteristics are intimately linked to their stage of maturation. The specific biochemical pathways and genes whose expression mediates differentiation of progenitors to DCs and their maturation are largely undefined. We recently utilized two approaches, DNA microarrays and proteomics, to analyze the expression profile of human CD14(+) blood monocytes and their derived DCs. Approximately 4% of the genes or proteins expressed were found to be regulated during DC differentiation. Most of these genes were not previously associated with DCs and included genes highly relevant to DC functions (genes involved in antigen presentation, cell adhesion and motility, lipid metabolism). Genes involved in specific signaling pathways, including IkappaBalpha, PPAR-gamma and C/EBPalpha as well as two members of the family of transcription factors, interferon regulatory factors (IRFs), were also modified. Modulation of IRF gene expression is of particular interest because of their functional roles in innate and adaptive immune responses. IRF-family members control the expression of proteins that include type-1 interferons, interleukin-12, interleukin-15, MHC molecules and adhesion molecules. They have also been found to play an important role in lymphocyte development. In contrast to DC differentiation, very few genes were modified at the transcript level during DC maturation as determined by microarray experiments. Further analysis suggested that DC maturation is largely controlled by posttranscriptional and posttranslational modifications. The use of proteomics is therefore necessary for a full comprehension of DC maturation process.

Animals↗
PubMed2002

Identification of tumor-associated antigens using proteomics.

In the post-genomic era, the identification of tumor-associated antigens that elicit a humoral response is allowed at the protein level using proteomics. Indeed, the screening of autoantibodies using 2-D Western blot experiments with sera from cancer patients, followed by the subsequent identification of the target protein by mass spectrometry and database search has permitted the exploitation of the B-cell repertoire of patients with cancer. Applied to several types of cancer, a proteomic-based approach has revealed a high frequency of autoantibodies in sera from patients. Several of the antigenic proteins identified may constitute novel cancer markers and may have clinical utility in diagnosis or in establishing prognosis. Furthermore, the approach has allowed to distinguish isoforms that may help to define epitopes. On the other hand, the analysis of the expression levels of some of the antigenic proteins has revealed differential expression in tumors as compared with healthy tissues that might explain antigenicity.

Antigens, Neoplasm↗
PubMedDate not supplied

Translational control in T lymphocytes.

Translational control is an important but relatively unappreciated mechanism that regulates levels of protein products. In addition to a global translational control that regulates the cell's response to external stimuli such as growth factors, cytokines, stress, and viral infections, selective translational control has recently been demonstrated to affect many genes related to growth and apoptotic processes. Translational infidelity has recently been suggested as a new mechanism of T cell dysregulation in SLE. This review discusses current data on translational control of T cell biology and the central aspect of translational control in the signalling pathway leading to T cell proliferation, apoptotic response, and cytokine production. The utility for global analysis by genomics to study translational control of T cell gene expression is also discussed.

Eukaryotic Initiation Factor-4E↗