Abstract
In the present work, we have attempted a comprehensive analysis of cytosolic and microsomal proteomes to elucidate the signaling pathways impaired in human hepatoma (Huh7) cells upon herpes simplex virus type 1 (HSV-1; Cgal(+)) infection. Using a combination of differential in-gel electrophoresis and nano liquid chromatography/tandem mass spectrometry, 18 spots corresponding to 16 unique deregulated cellular proteins were unambiguously identified, which were involved in the regulation of essential processes such as apoptosis, mRNA processing, cellular structure and integrity, signal transduction, and endoplasmic-reticulum-associated degradation pathway. Based on our proteomic data and additional functional studies target proteins were identified indicating a late activation of apoptotic pathways in Huh7 cells upon HSV-1 Cgal(+) infection. Additionally to changes on RuvB-like 2 and Bif-1, down-regulation of Erlin-2 suggests stimulation of Ca(2+)-dependent apoptosis. Moreover, activation of the mitochondrial apoptotic pathway results from a time-dependent multi-factorial impairment as inferred from the stepwise characterization of constitutive pro- and anti-apoptotic factors. Activation of serine-threonine protein phosphatase 2A (PP2A) was also found in Huh7 cells upon HSV-1 Cgal(+) infection. In addition, PP2A activation paralleled dephosphorylation and inactivation of downstream mitogen-activated protein (MAP) kinase pathway (MEK(1/2), ERK(1/2)) critical to cell survival and activation of proapoptotic Bad by dephosphorylation of Ser-112. Taken together, our results provide novel molecular information that contributes to define in detail the apoptotic mechanisms triggered by HSV-1 Cgal(+) in the host cell and lead to the implication of PP2A in the transduction of cell death signals and cell survival pathway arrest.
Highlights
In the present work, we have attempted a comprehensive analysis of cytosolic and microsomal proteomes to elucidate the signaling pathways impaired in human hepatoma (Huh7) cells upon herpes simplex virus type 1 (HSV-1; Cgal؉) infection
Based on the proteomic information and additional functional experiments, we have identified deregulation of central intermediates targeted by HSV-1 Cgalϩ resulting in the impairment of apoptosis and cell survival pathways in human hepatoma cells
Cytosolic and Microsomal Differentially Expressed Proteins in HSV-1 Cgalϩ- infected Huh7 Cells—Cellular pathways interfered with HSV-1 Cgalϩ were investigated using a combination of difference gel electrophoresis (DIGE) and mass spectrometry
Summary
Human hepatoma; HSV-1, herpes simplex virus type-1; DIGE, differential in-gel electrophoresis; PP2A, protein phosphatase 2A; MAP, mitogen-activated protein; LCMS/MS, liquid chromatography/tandem mass spectrometry; PBS, phosphate-buffered saline; hepatocellular carcinoma; X-gal, 5-bromo-4-chloro-3-indolyl-- D-galactopyranoside; WT, wild-type; FAK, focal adhesion kinase; PI3K, phosphatidylinositol 3-kinase; hpi, hours post-infection; MEK, mitogen-activated ERK kinase; ERK, extracellular signal-related kinase. In contrast to other viruses [9], the cytolytic capacity of HSV-1 in murine cells facilitates the evaluation of the toxicity and safety of newly designed vectors in murine syngenic cancer models. Antiherpetic drugs, such as acyclovir or forscanet, are available and provide a safety mechanism to shut off viral replication in case of undesired local or systemic infection. Our present study was aimed at identifying differentially expressed proteins in human hepatoma (Huh7) cells at different time points after HSV-1 Cgalϩ infection by an organellar 2-DE proteomic study using a combination of DIGE and nanoLC-ESI-MS/MS. Based on the proteomic information and additional functional experiments, we have identified deregulation of central intermediates targeted by HSV-1 Cgalϩ resulting in the impairment of apoptosis and cell survival pathways in human hepatoma cells
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