Proteomic Signatures in Thapsigargin-Treated Hepatoma Cells

Thapsigargin, an inhibitor of the endoplasmic reticulum (ER) calcium transporters, generates Ca2+-store depletion within the ER and simultaneously increases Ca2+ level in the cytosol. Perturbation of Ca2+ homeostasis leads cells to cope with stressful conditions, including ER stress, which affect the folding of newly synthesized proteins and induce the accumulation of unfolded polypeptides and eventually apoptosis, via activation of the unfolded protein response pathway. In the present work, we analyzed the proteome changes in human hepatoma cells following acute treatment with thapsigargin. We highlighted a peculiar pattern of protein expression, marked by altered expression of calcium-dependent proteins, and of proteins involved in secretory pathways or in cell survival. For specific deregulated proteins, the thapsigargin-induced proteomic signature was compared by Western blotting to that resulting from the treatment of hepatoma cells with reducing agents or with proteasome inhibitors, to elicit endoplasmic reticulum stress by additional means and to reveal novel, potential targets of the unfolded protein response pathway

Proteomic Signatures in Thapsigargin-Treated Hepatoma Cells

Thapsigargin, an inhibitor of the endoplasmic reticulum (ER) calcium transporters, generates Ca2+-store depletion within the ER and simultaneously increases Ca2+ level in the cytosol. Perturbation of Ca2+ homeostasis leads cells to cope with stressful conditions, including ER stress, which affect the folding of newly synthesized proteins and induce the accumulation of unfolded polypeptides and eventually apoptosis, via activation of the unfolded protein response pathway. In the present work, we analyzed the proteome changes in human hepatoma cells following acute treatment with thapsigargin. We highlighted a peculiar pattern of protein expression, marked by altered expression of calcium-dependent proteins, and of proteins involved in secretory pathways or in cell survival. For specific deregulated proteins, the thapsigargin-induced proteomic signature was compared by Western blotting to that resulting from the treatment of hepatoma cells with reducing agents or with proteasome inhibitors, to elicit endoplasmic reticulum stress by additional means and to reveal novel, potential targets of the unfolded protein response pathway

Proteomic Signatures in Thapsigargin-Treated Hepatoma Cells

Thapsigargin, an inhibitor of the endoplasmic reticulum (ER) calcium transporters, generates Ca2+-store depletion within the ER and simultaneously increases Ca2+ level in the cytosol. Perturbation of Ca2+ homeostasis leads cells to cope with stressful conditions, including ER stress, which affect the folding of newly synthesized proteins and induce the accumulation of unfolded polypeptides and eventually apoptosis, via activation of the unfolded protein response pathway. In the present work, we analyzed the proteome changes in human hepatoma cells following acute treatment with thapsigargin. We highlighted a peculiar pattern of protein expression, marked by altered expression of calcium-dependent proteins, and of proteins involved in secretory pathways or in cell survival. For specific deregulated proteins, the thapsigargin-induced proteomic signature was compared by Western blotting to that resulting from the treatment of hepatoma cells with reducing agents or with proteasome inhibitors, to elicit endoplasmic reticulum stress by additional means and to reveal novel, potential targets of the unfolded protein response pathway

Structural basis of antiviral activity of peptides from MPER of FIV gp36 - Fig 3

Confocal microscopy images of MLEVs in presence of NBD labelled C8 (left) and C6a (right) peptides. MLEVs vary in DOPC/DOPG composition moving from DOPC/DOPG 100:0 M/M (top) to DOPC/DOPG 0:100 M/M (bottom). Images were acquired on a laser scanning confocal microscope (LSM 510; Carl Zeiss MicroImaging) equipped with a plan Apo 63X, NA 1.4 oil immersion objective lens. For each field, both fluorescent and transmitted light images were acquired on separate photomultipliers and were analysed with Zeiss LSM 510 4.0 SP2 software.</p

Confocal microscopy images of MLVs in presence of NBD labelled C8, C6a and C6b peptides.

MLVs are composed of DOPC/DOPG 90:10 M/M. Images were acquired on a laser scanning confocal microscope (LSM 510; Carl Zeiss MicroImaging) equipped with a plan Apo 63X, NA 1.4 oil immersion objective lens. For each field, both fluorescent and transmitted light images were acquired on separate photomultipliers and were analysed with Zeiss LSM 510 4.0 SP2 software.</p

Pharmacophore model of anti-FIV fusion inhibitors.

Overlapping of C8 (light blue) and C6a NMR representative models. Molecular surfaces (grey) surround the carboxylic moiety of 772D side chain and indolyl rings of 770W and 776W in C8, and 773W and 776W in C6a.</p

Low energy NMR models calculated for C8, C6a and C6b peptides in DPC/SDS 90:10 molar ratio.

The calculation was carried out using standard CYANA protocol of simulated annealing in torsion angle space.</p

Molecular surface calculated as propensities of the different portions of the molecules to interact according to the specific surface properties.

Molecular surfaces of Trp residues are coloured in green-white: they account for the propensity of the peptides to engage hydrophobic interactions. Molecular surfaces of Asp residues are coloured in blue: they account for the propensity of the peptides to engage polar/electrostatic interactions.</p

Graphic representation of the gp36 MPER domain of FIV coat glycoprotein.

The fragments relative to C8, C6a and C6b peptide sequences are highlighted.</p

Processed far UV CD spectra of C8, C6a and C6b peptides in DPC/SDS 90:10 M/M.

The CD spectra were acquired using a JASCO 810 spectropolarimeter at room temperature with a cell path length of 1 mm. The measurement range spans from 190 to 260 nm.</p