Alternative splicing of hepatitis B virus: A novel virus/host interaction altering liver immunity

This work was supported by grants from Institut National de la Sante et de la Recherche Medicale (Inserm) – France, Universite Pierre et Marie Curie (UPMC) – France, Agence National de la Recherche sur le Sida et les Hepatites (ANRS) – France (n° N14015DR) and PHC-Tassili (11MDU826). MD was supported by ANRS (grant ASA14013DRA). YM was supported by French Ministry for Higher Education and Research and by the Ligue contre le Cancer (grant n° GB/MA/VSP-10504)

Alternative splicing of hepatitis B virus: A novel virus/host interaction altering liver immunity

Background & Aims: Hepatitis B virus (HBV) RNA can undergo alternative splicing, but the relevance of this post-transcriptional regulation remains elusive. The mechanism of HBV alternative splicing regulation and its impact on liver pathogenesis were investigated. Methods: HBV RNA-interacting proteins were identified by RNA pull-down, combined with mass spectrometry analysis. HBV splicing regulation was investigated in chemically and surgically induced liver damage, in whole HBV genome transgenic mice and in hepatoma cells. Viral and endogenous gene expression were quantified by quantitative reverse transcription polymerase chain reaction, Western blot and enzyme-linked immunosorbent assay. Resident liver immune cells were studied by fluorescence-activated cell sorting. Results: HBV pregenomic RNA-interacting proteins were identified and 15% were directly related to the splicing machinery. Expression of these splicing factors was modulated in HBV transgenic mice with liver injuries and contributed to an increase of the HBV spliced RNA encoding for HBV splicing-generated protein (HBSP). HBSP transgenic mice with chemically induced liver fibrosis exhibited attenuated hepatic damage. The protective effect of HBSP resulted from a decrease of inflammatory monocyte/macrophage recruitment through downregulation of C-C motif chemokine ligand 2 (CCL2) expression in hepatocytes. In human hepatoma cells, the ability of HBSP to control CCL2 expression was confirmed and maintained in a whole HBV context. Finally, viral spliced RNA detection related to a decrease of CCL2 expression in the livers of HBV chronic carriers underscored this mechanism. Conclusion: The microenvironment, modified by liver injury, increased HBSP RNA expression through splicing factor regulation, which in turn controlled hepatocyte chemokine synthesis. This feedback mechanism provides a novel insight into liver immunopathogenesis during HBV infection. Lay summary: Hepatitis B virus persists for decades in the liver of chronically infected patients. Immune escape is one of the main mechanisms developed by this virus to survive. Our study highlights how the crosstalk between virus and liver infected cells may contribute to this immune escape

Changes in lipid raft proteome upon TNF-α stimulation of cystic fibrosis cells

We have previously shown (i) that the cystic fibrosis transmembrane regulator (CFTR) locates to lipid raft-like microdomains of epithelial cells upon TNF-α proinflammatory stimulation; and (ii) that TNF-α increases the membrane localization and the channel function of F508del-mutated CFTR. In the present work, we hypothesized that CFTR mutations modify the proteome of lipid rafts in the same proinflammatory conditions. We prepared lipid rafts from HeLa cells transfected with either wild-type or F508del-CFTR and incubated for 10min with 100U/mL of TNF-α. Proteins were extracted, trypsin digested, and peptides analyzed by high resolution MS. Proteins were quantified by a stable isotope labeling with amino acids in cell culture approach. Out of the 22 proteins differentially recruited in lipid rafts after proinflammatory exposure, 17 were increased in F508del cells with respect to wild-type, including two G-protein coupled receptors, three anion transporters, and one cell surface mucin. In both HeLa and bronchial epithelial cells we confirmed that G-protein coupled receptor 5A relocates to lipid rafts along with F508del-CFTR after TNF-α treatment. These results could enlighten the cross-talk between CFTR and TNF-α and its impact on the cell response to proinflammatory challenge

Proteomics reveals that methylmalonyl-coa mutase modulates cell architecture and increases susceptibility to stress

Methylmalonic acidemia (MMA) is a rare inborn error of metabolism caused by deficiency of the methylmalonyl-CoA mutase (MUT) enzyme. Downstream MUT deficiency, methylmalonic acid accumulates together with toxic metabolites from propionyl-CoA and other compounds upstream of the block in the enzyme pathway. The presentation is with life-threatening acidosis, respiratory distress, brain disturbance, hyperammonemia, and ketosis. Survivors develop poorly understood multi-organ damage, notably to the brain and kidneys. The HEK 293 cell line was engineered by CRISPR/Cas9 technology to knock out the MUT gene (MUT-KO). Shotgun label-free quantitative proteomics and bioinformatics analyses revealed potential damaging biological processes in MUT-deficient cells. MUT-KO induced alteration of cellular architecture and morphology, and ROS overproduction. We found the alteration of proteins involved in cytoskeleton and cell adhesion organization, cell trafficking, mitochondrial, and oxidative processes, as validated by the regulation of VIM, EXT2, SDC2, FN1, GLUL, and CHD1. Additionally, a cell model of MUT-rescuing was developed in order to control the specificity of MUT-KO effects. Globally, the proteomic landscape of MUT-KO suggests the cell model to have an increased susceptibility to propionate-and H2O2-induced stress through an impairment of the mitochondrial functionality and unbalances in the oxidation-reduction processes

Dataset of differential lipid raft and global proteomes of SILAC-labeled cystic fibrosis cells upon TNF -α stimulation

Cystic fibrosis (CF) is a genetic disease due to mutations in the cystic fibrosis transmembrane regulator (CFTR), F508del-CFTR being the most frequent. Lipid raft-like microdomains (LRM) are regions of the plasma membrane that present a high cholesterol content and are insoluble to non-ionic detergents. LRM are essential functional and structural platforms that play an important role in the inflammatory response. CFTR is a known modulator of inflammation in LRM. Here we provide mass spectrometry data on the global impact of CFTR mutation and TNF-a stimulation on the LRM proteome. We used the Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) approach to quantify and identify 332 proteins in LRM upon TNF-a stimulation in CF cells and 1381 for the global proteome. We report two detailed tables containing lists of proteins obtained by mass spectrometry and the immunofluorescence validation results for one of these proteins, the G-protein coupled receptor 5A. These results are associated with the article "Changes in lipid raft proteome upon TNF-α stimulation of cystic fibrosis cells" (Chhuon et al., in press [1])

Disruption of pathways regulated by Integrator complex in Galloway–Mowat syndrome due to WDR73 mutations

International audienceAbstract Several studies have reported WDR73 mutations to be causative of Galloway–Mowat syndrome, a rare disorder characterised by the association of neurological defects and renal-glomerular disease. In this study, we demonstrate interaction of WDR73 with the INTS9 and INTS11 components of Integrator, a large multiprotein complex with various roles in RNA metabolism and transcriptional control. We implicate WDR73 in two Integrator-regulated cellular pathways; namely, the processing of uridylate-rich small nuclear RNAs (UsnRNA), and mediating the transcriptional response to epidermal growth factor stimulation. We also show that WDR73 suppression leads to altered expression of genes encoding cell cycle regulatory proteins. Altogether, our results suggest that a range of cellular pathways are perturbed by WDR73 loss-of-function, and support the consensus that proper regulation of UsnRNA maturation, transcription initiation and cell cycle control are all critical in maintaining the health of post-mitotic cells such as glomerular podocytes and neurons, and preventing degenerative disease

Dataset of differential lipid raft and global proteomes of SILAC-labeled cystic fibrosis cells upon TNF -α stimulation

Cystic fibrosis (CF) is a genetic disease due to mutations in the cystic fibrosis transmembrane regulator (CFTR), F508del-CFTR being the most frequent. Lipid raft-like microdomains (LRM) are regions of the plasma membrane that present a high cholesterol content and are insoluble to non-ionic detergents. LRM are essential functional and structural platforms that play an important role in the inflammatory response. CFTR is a known modulator of inflammation in LRM. Here we provide mass spectrometry data on the global impact of CFTR mutation and TNF-a stimulation on the LRM proteome. We used the Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) approach to quantify and identify 332 proteins in LRM upon TNF-a stimulation in CF cells and 1381 for the global proteome. We report two detailed tables containing lists of proteins obtained by mass spectrometry and the immunofluorescence validation results for one of these proteins, the G-protein coupled receptor 5A. These results are associated with the article “Changes in lipid raft proteome upon TNF-α stimulation of cystic fibrosis cells” (Chhuon et al., in press [1])

Threonine eliminylation by bacterial phosphothreonine lyases rapidly causes cross-linking of mitogen-activated protein kinase (MAPK) in live cells

Old long-lived proteins contain dehydroalanine (Dha) and dehydrobutyrine (Dhb), two amino acids engendered by dehydration of serines and threonines, respectively. Although these residues have a suspected role in protein cross-linking and aggregation, their direct implication has yet to be determined. Here, we have taken advantage of the ability of the enteropathogen Shigella to convert the phosphothreonine residue of the pT-X-pY consensus sequence of ERK and p38 into Dhb and followed the impact of dehydration on the fate of the two MAPKs. To that end, we have generated the first antibodies recognizing Dhb-modified proteins and allowing tracing them as they form. We showed that Dhb modifications accumulate in a long-lasting manner in Shigella-infected cells, causing subsequent formation of covalent cross-links of MAPKs. Moreover, the Dhb signal correlates precisely with the activation of the Shigella type III secretion apparatus, thus evidencing injectisome activity. This observation is the first to document a causal link between Dhb formation and protein cross-linking in live cells. Detection of eliminylation is a new avenue to phosphoproteome regulation in eukaryotes that will be instrumental for the development of type III secretion inhibitors

Downregulation of the Glial GLT1 glutamate transporter and purkinje cell dysfunction in a mouse model of myotonic dystrophy

Brain function is compromised in myotonic dystrophy type 1 (DM1), but the underlying mechanisms are not fully understood. To gain insight into the cellular and molecular pathways primarily affected, we studied a mouse model of DM1 and brains of adult patients. We found pronounced RNA toxicity in the Bergmann glia of the cerebellum, in association with abnormal Purkinje cell firing and fine motor incoordination in DM1 mice. A global proteomics approach revealed downregulation of the GLT1 glutamate transporter in DM1 mice and human patients, which we found to be the result of MBNL1 inactivation. GLT1 downregulation in DM1 astrocytes increases glutamate neurotoxicity and is detrimental to neurons. Finally, we demonstrated that the upregulation of GLT1 corrected Purkinje cell firing and motor incoordination in DM1 mice. Our findings show that glial defects are critical in DM1 brain pathophysiology and open promising therapeutic perspectives through the modulation of glutamate levels