3-O-sulfated heparan sulfate interactors target synaptic adhesion molecules from neonatal mouse brain and inhibit neural activity and synaptogenesis in vitro.

Heparan sulfate (HS) chains, covalently linked to heparan sulfate proteoglycans (HSPG), promote synaptic development and functions by connecting various synaptic adhesion proteins (AP). HS binding to AP could vary according to modifications of HS chains by different sulfotransferases. 3-O-sulfotransferases (Hs3sts) produce rare 3-O-sulfated HSs (3S-HSs), of poorly known functions in the nervous system. Here, we showed that a peptide known to block herpes simplex virus by interfering with 3S-HSs in vitro and in vivo (i.e. G2 peptide), specifically inhibited neural activity, reduced evoked glutamate release, and impaired synaptic assembly in hippocampal cell cultures. A role for 3S-HSs in promoting synaptic assembly and neural activity is consistent with the synaptic interactome of G2 peptide, and with the detection of Hs3sts and their products in synapses of cultured neurons and in synaptosomes prepared from developing brains. Our study suggests that 3S-HSs acting as receptors for herpesviruses might be important regulators of neuronal and synaptic development in vertebrates.

Tspan15 Is a New Stemness-Related Marker in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the second cause of cancer-related deaths worldwide. A clearer understanding of the molecular mechanisms underlying tumor growth and invasiveness remains crucial for developing new therapies. Here, the expression of tetraspanins, a family of plasma membrane organizers involved in tumor progression, has been addressed. Integrative approaches combining transcriptomics and bioinformatics allow demonstrating the induced and heterogeneous expression of Tspan15 in HCC. Tspan15 positive tumors exhibit signatures related to hepatic progenitor cells as well as recurrence of cancer. Immunohistochemistry experiments confirm Tspan15 expression in the subset of HCC expressing stemness-related markers such as EpCAM and Cytokeratin-19. Functional networks reveal that most of these genes expressed in correlation to Tspan15 support cell proliferation. Furthermore, Tspan15 overexpression in the hepatoma cell line HepG2 significantly increases cell proliferation. A quantitative proteomic analysis of the secretome reveals a higher abundance of the protein connective tissue growth factor (CTGF), a pleiotropic matricellular signaling protein. Proteomic profiling of Tspan15 complexes allows identifying numerous membrane proteins including several growth factor receptors. Finally, Tspan15 increases ERK1/2 phosphorylation that directly controls CTGF expression and secretion. In conclusion, Tspan15 is a new stemness-related marker in HCC which exhibits high potential of tumor growth and recurrence.

Hepatitis C virus core protein targets 4E-BP1 expression and phosphorylation and potentiates Myc-induced liver carcinogenesis in transgenic mice.

Hepatitis C virus (HCV) is a leading cause of liver diseases including the development of hepatocellular carcinoma (HCC). Particularly, core protein has been involved in HCV-related liver pathologies. However, the impact of HCV core on signaling pathways supporting the genesis of HCC remains largely elusive. To decipher the host cell signaling pathways involved in the oncogenic potential of HCV core, a global quantitative phosphoproteomic approach was carried out. This study shed light on novel differentially phosphorylated proteins, in particular several components involved in translation. Among the eukaryotic initiation factors that govern the translational machinery, 4E-BP1 represents a master regulator of protein synthesis that is associated with the development and progression of cancers due to its ability to increase protein expression of oncogenic pathways. Enhanced levels of 4E-BP1 in non-modified and phosphorylated forms were validated in human hepatoma cells and in mouse primary hepatocytes expressing HCV core, in the livers of HCV core transgenic mice as well as in HCV-infected human primary hepatocytes. The contribution of HCV core in carcinogenesis and the status of 4E-BP1 expression and phosphorylation were studied in HCV core/Myc double transgenic mice. HCV core increased the levels of 4E-BP1 expression and phosphorylation and significantly accelerated the onset of Myc-induced tumorigenesis in these double transgenic mice. These results reveal a novel function of HCV core in liver carcinogenesis potentiation. They position 4E-BP1 as a tumor-specific target of HCV core and support the involvement of the 4E-BP1/eIF4E axis in hepatocarcinogenesis.

Characterization of hydrophobic peptides in the presence of detergent by photoionization mass spectrometry.

The characterization of membrane proteins is still challenging. The major issue is the high hydrophobicity of membrane proteins that necessitates the use of detergents for their extraction and solubilization. The very poor compatibility of mass spectrometry with detergents remains a tremendous obstacle in studies of membrane proteins. Here, we investigated the potential of atmospheric pressure photoionization (APPI) for mass spectrometry study of membrane proteins. This work was focused on the tetraspanin CD9 and the multidrug transporter BmrA. A set of peptides from CD9, exhibiting a broad range of hydropathicity, was investigated using APPI as compared to electrospray ionization (ESI). Mass spectrometry experiments revealed that the most hydrophobic peptides were hardly ionized by ESI whereas all peptides, including the highly hydrophobic one that corresponds to the full sequence of the first transmembrane domain of CD9, were easily ionized by APPI. The native protein BmrA purified in the presence of the non-ionic detergent beta-D-dodecyl maltoside (DDM) was digested in-solution using trypsin. The resulting peptides were investigated by flow injection analysis of the mixture followed by mass spectrometry. Upon ESI, only detergent ions were detected and the ionic signals from the peptides were totally suppressed. In contrast, APPI allowed many peptides distributed along the sequence of the protein to be detected. Furthermore, the parent ion corresponding to the first transmembrane domain of the protein BmrA was detected under APPI conditions. Careful examination of the APPI mass spectrum revealed a-, b-, c- and y- fragment ions generated by in-source fragmentation. Those fragment ions allowed unambiguous structural characterization of the transmembrane domain. In conclusion, APPI-MS appears as a versatile method allowing the ionization and fragmentation of hydrophobic peptides in the presence of detergent.

HRas signal transduction promotes hepatitis C virus cell entry by triggering assembly of the host tetraspanin receptor complex.

Hepatitis C virus (HCV) entry is dependent on coreceptor complex formation between the tetraspanin superfamily member CD81 and the tight junction protein claudin-1 (CLDN1) on the host cell membrane. The receptor tyrosine kinase EGFR acts as a cofactor for HCV entry by promoting CD81-CLDN1 complex formation via unknown mechanisms. We identify the GTPase HRas, activated downstream of EGFR signaling, as a key host signal transducer for EGFR-mediated HCV entry. Proteomic analysis revealed that HRas associates with tetraspanin CD81, CLDN1, and the previously unrecognized HCV entry cofactors integrin ß1 and Ras-related protein Rap2B in hepatocyte membranes. HRas signaling is required for lateral membrane diffusion of CD81, which enables tetraspanin receptor complex assembly. HRas was also found to be relevant for entry of other viruses, including influenza. Our data demonstrate that viruses exploit HRas signaling for cellular entry by compartmentalization of entry factors and receptor trafficking.

Infrared spectral signatures of CDCP1-induced effects in colon carcinoma cells.

Metastasis is the major cause of death by cancer. Indeed, metastatic colonies can reactivate and become life threatening, sometimes months or years after the initial diagnosis and surgery of the primary tumor. Therefore, there is a crucial need to develop methods for diagnosis of tumor cells that exhibit high metastatic potential. Here, we addressed the capability of vibrational spectroscopy for investigating the effects induced by CDCP1 expression in colon carcinoma cells. This transmembrane protein has been suggested to play a key role in metastasis by its pleiotropic function. We focused on a cellular model constituted by the cell lines SW480 and SW620 derived respectively from the primary tumor and a lymph node metastasis of the same patient. Induced CDCP1 expression in SW480 led to marked changes in cell morphology. Whereas SW480 form a cell layer, the SW480/CDCP1 cells exhibited reduced cell-to-cell contact. On collagen I, SW480 was more spread and filopodia were observed. In contrast, SW480/CDCP1 cells exhibited lower spreading with no formation of filopodia. Synchrotron Fourier transform infrared microspectroscopy experiments were performed on this cellular model. High quality spectroscopic information at sub-cellular resolution, provided by the use of the synchrotron source in infrared microspectroscopy, was recorded on numerous individual cells. Multivariate analysis of spectra recorded using principal component analysis indicated a highest intensity increase of the 970 and 1080 cm(-1) bands, and a modest intensity increase of the 1240 cm(-1) band in the SW480/CDCP1 cells. These bands were correlated with an increased content of phosphorylated proteins as confirmed by in situ labelling using a monoclonal antibody directed against phosphorylated tyrosines. Altogether, these results demonstrate that the vibrational technique used in this study exhibits the capability to characterize spectral signatures of CDCP1-induced effects in colon carcinoma cells. This study may open new avenues for rapid diagnosis of cells with a metastatic potential.

Melatonin protects PLPC liposomes and LDL towards radical-induced oxidation.

This study investigated the in vitro protective effects of melatonin against oxidation of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC) liposomes [(PLPC) = 250 µm] and low-density lipoproteins (LDL, 3 g/L total concentration) by hydroxyl radicals produced by water gamma radiolysis. Conjugated dienes (CD) and hydroperoxides from cholesteryl esters (CEOOH) and phospholipids (PCOOH) were measured as indices of lipid peroxidation. Protein (apoB) oxidation in LDL was assessed by carbonyl groups. Two LDL antioxidants (vitamin E and ß-carotene) were monitored as a function of the radiation dose. Three concentrations of melatonin were studied in PLPC liposomes, i.e., 20, 50 and 100 µm, and one in LDL, i.e., 100 µm. Melatonin consumption was also followed up in both lipid models upon irradiation, together with the residual PLPC concentration in liposomes. In PLPC liposomes, scavenging of lipid-derived peroxyl radicals was not the only phenomenon to explain the protective properties of melatonin towards lipid peroxidation. Indeed, melatonin also reacted with hydroxyl radicals generated in aqueous phase, which led us to suggest that hydroxyl radicals reacted relatively slowly with PLPC. Melatonin was efficient in lowering lipid peroxidation in LDL, as shown by the decrease in the formation of CDs and in hydroperoxides. Moreover, melatonin clearly slowed radio-induced apolipoprotein B carbonylation and protected α-tocopherol and ß-carotene in LDL.

Interaction between non-anionic phospholipids and cytochrome c induced by reactive oxygen species.

The oxidative interaction of cytochrome c (Cyt c) with liposomes of Palmitoyl Linoleyl Phosphatidyl Choline (PLPC) initiated by radio-induced free radicals was investigated. Results showed that the peroxidation of PLPC is decreased in the presence of Cyt c, meaning that this latter is the preferential target of hydroxyl radicals. In addition, when Cyt c was incubated with peroxidized PLPC, it was found to be able to decompose hydroperoxides of PLPC into hydroxides. The peroxidase activity of Cyt c proceeded via the opening of the tertiary structure of Cyt c, as suggested by the loss of the sixth coordination bond of the heme-iron. Even if it is known to preferentially interact with cardiolipin, this work shows that Cyt c is also able to interact with hydroperoxide species of non-anionic phospholipids.