Analysis of EYA3 Phosphorylation by Src Kinase Identifies Residues Involved in Cell Proliferation

Eyes absent (EYA) are non-thiol-based protein tyrosine phosphatases (PTPs) that also have transcriptional co-activator functions. Their PTP activity is involved in various pathologies. Recently, we demonstrated that Src tyrosine kinase phosphorylates human EYA3 by controlling its subcellular localization. We also found EYA3′s ability to autodephosphorylate, while raising the question if the two opposing processes could be involved in maintaining a physiologically adequate level of phosphorylation. Using native and bottom-up mass spectrometry, we performed detailed mapping and characterization of human EYA3 Src-phosphorylation sites. Thirteen tyrosine residues with different phosphorylation and autodephosphorylation kinetics were detected. Among these, Y77, 96, 237, and 508 displayed an increased resistance to autodephosphorylation. Y77 and Y96 were found to have the highest impact on the overall EYA3 phosphorylation. Using cell cycle analysis, we showed that Y77, Y96, and Y237 are involved in HEK293T proliferation. Mutation of the three tyrosine residues abolished the pro-proliferative effect of EYA3 overexpression. We have also identified a Src-induced phosphorylation pattern of EYA3 in these cells. These findings suggest that EYA3′s tyrosine phosphorylation sites are non-equivalent with their phosphorylation levels being under the control of Src-kinase activity and of EYA3′s autodephosphorylation

Lettuce-produced hepatitis C virus E1E2 heterodimer triggers immune responses in mice and antibody production after oral vaccination

The hepatitis C virus (HCV) is a major etiologic agent for severe liver diseases ( e.g . cirrhosis, fibrosis and hepatocellular carcinoma). Approximately 140 million people have chronic HCV infections and about 500 000 die yearly from HCV-related liver pathologies. To date, there is no licensed vaccine available to prevent HCV infection and production of a HCV vaccine remains a major challenge. Here, we report the successful production of the HCV E1E2 heterodimer, an important vaccine candidate, in an edible crop (lettuce, Lactuca s ativa ) using Agrobacterium - mediated transient expression technology. The wild-type dimer (E1E2) and a variant without an N-glycosylation site in the E2 polypeptide (E1E2 Δ N6) were expressed, and appropriate N-glycosylation pattern and functionality of the E1E2 dimers were demonstrated. The humoral immune response induced by the HCV proteins was investigated in mice following oral administration of lettuce antigens with or without previous intramuscular prime with the mammalian HEK293T cell-expressed HCV dimer. Immunization by oral feeding only resulted in development of weak serum levels of anti-HCV IgM for both antigens; however, the E1E2 Δ N6 proteins produced higher amounts of secretory IgA, suggesting improved immunogenic properties of the N-glycosylation mutant. The mice group receiving the intramuscular injection followed by two oral boosts with the lettuce E1E2 dimer developed a systemic but also a mucosal immune response, as demonstrated by the presence of anti-HCV secretory IgA in faeces extracts. In summary, our study demonstrates the feasibility of producing complex viral antigens in lettuce, using plant transient expression technology, with great potential for future low-cost oral vaccine development.Lettuce-produced hepatitis C virus E1E2 heterodimer triggers immune responses in mice and antibody production after oral vaccinationpublishedVersio

Comparative Proteomics Reveals Novel Components at the Plasma Membrane of Differentiated HepaRG Cells and Different Distribution in Hepatocyte- and Biliary-Like Cells

<div><p>Hepatitis B virus (HBV) is a human pathogen causing severe liver disease and eventually death. Despite important progress in deciphering HBV internalization, the early virus-cell interactions leading to infection are not known. HepaRG is a human bipotent liver cell line bearing the unique ability to differentiate towards a mixture of hepatocyte- and biliary-like cells. In addition to expressing metabolic functions normally found in liver, differentiated HepaRG cells support HBV infection <i>in vitro,</i> thus resembling cultured primary hepatocytes more than other hepatoma cells. Therefore, extensive characterization of the plasma membrane proteome from HepaRG cells would allow the identification of new cellular factors potentially involved in infection. Here we analyzed the plasma membranes of non-differentiated and differentiated HepaRG cells using nanoliquid chromatography-tandem mass spectrometry to identify the differences between the proteomes and the changes that lead to differentiation of these cells. We followed up on differentially-regulated proteins in hepatocytes- and biliary-like cells, focusing on Cathepsins D and K, Cyclophilin A, Annexin 1/A1, PDI and PDI A4/ERp72. Major differences between the two proteomes were found, including differentially regulated proteins, protein-protein interactions and intracellular localizations following differentiation. The results advance our current understanding of HepaRG differentiation and the unique properties of these cells.</p></div

Validation of the plasma membrane (PM) purification.

<p>Equal amounts of proteins from total HepaRG cell lysate (TL) and purified PM fraction were loaded on SDS-PAGE and analyzed by Western blotting using organelle-specific Abs. The molecular weight markers are indicated. The representative blot of three independent experiments is shown.</p

Summary of the proteomic analysis of the PM from undifferentiated (ND) and differentiated (D) HepaRG cells.

<p>(A and B) SDS-PAGE resolved proteins were analyzed by LC-MS/MS as described. Venn diagrams show the number of proteins identified in PM from (ND) and (D) HepaRG cells in experiments 1 (A) and 2 (B).</p

Analysis of differentially expressed proteins identified at the PM of (ND) and (D) HepaRG cells.

<p>The PM fractions of (ND) and (D) cells were analyzed by Western blotting using Abs specific for proteins with significantly altered levels of expression following differentiation. The molecular weight markers are indicated.</p

Classification of the proteins identified at the PM of (ND) and (D) HepaRG cells.

<p>The mascot search results from experiments 1 and 2 were exported as.dat files and processed with the license-based Scaffold 4.0 software (www. proteomescience.com). The proteins identified in (ND) and (D) cells were classified according to their involvement in a biological process (A), subcellular localization (B) and molecular function (C).</p

Analysis of differentially expressed proteins in hepatocyte- and biliary-like HepaRG cells (II).

<p>HepaRG cells were analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071859#pone-0071859-g004" target="_blank">Figure 4</a>, except that expression of PDI (panel a), PDI A4 (panel b), Cathepsin D (panel C) and Cathepsin K (panel d) were evidenced using specific Abs. Where possible, albumin expression was also evidenced (panels b and d).</p