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ispartof: MOLECULAR THERAPY vol:30 issue:5 pages:1797-1800 ispartof: location:United States status: published

Human T-cell leukemia virus type-1 (HTLV-1) is the first pathogenic retrovirus discovered in human. Although HTLV-1-induced diseases are well characterized and linked to the encoded Tax-1 protein, there is currently no strategy to target Tax-1 functions with small molecules. Here, we report a comprehensive interaction map between Tax-1 and human PDZ domain-containing proteins (hPDZome), and we show that Tax-1 interacts with one-third of them. This includes proteins involved in cell cycle, cell-cell junction and cytoskeleton organization, as well as in membrane complexes assembly. Using nuclear magnetic resonance (NMR) spectroscopy, we have determined the structural basis of the interaction between the C-terminal PDZ binding motif (PBM) of Tax-1, and the PDZ domains of DLG1 and syntenin-1. Finally, we have used molecular modeling and mammalian cell-based assays to demonstrate that Tax-1/PDZ-domain interactions are amenable to small-molecule inhibition. Thus, our work provides a framework for the design of targeted therapies for HTLV-1-induced diseases. HighlightsO_LIcomprehensive interactome map of HTLV-1 Tax / human PDZ proteins C_LIO_LIbasis of Tax-1 PBM binding to human DLG1 and syntenin-1 PDZ domains". C_LIO_LIsignificance of inhibiting Tax-1 functions C_LIO_LIof the Tax-1 / PDZ interface C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/457680v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@6ba754org.highwire.dtl.DTLVardef@1b68124org.highwire.dtl.DTLVardef@d873c6org.highwire.dtl.DTLVardef@98f7ae_HPS_FORMAT_FIGEXP M_FIG C_FIG

Integration of multiomics and imaging identifies a glycosyltransferase enzyme regulating endoplasmic reticulum sizes., The endoplasmic reticulum (ER) is a central eukaryotic organelle with a tubular network made of hairpin proteins linked by hydrolysis of guanosine triphosphate nucleotides. Among posttranslational modifications initiated at the ER level, glycosylation is the most common reaction. However, our understanding of the impact of glycosylation on the ER structure remains unclear. Here, we show that exostosin-1 (EXT1) glycosyltransferase, an enzyme involved in N-glycosylation, is a key regulator of ER morphology and dynamics. We have integrated multiomics and superresolution imaging to characterize the broad effect of EXT1 inactivation, including the ER shape-dynamics-function relationships in mammalian cells. We have observed that inactivating EXT1 induces cell enlargement and enhances metabolic switches such as protein secretion. In particular, suppressing EXT1 in mouse thymocytes causes developmental dysfunctions associated with the ER network extension. Last, our data illuminate the physical and functional aspects of the ER proteome-glycome-lipidome structure axis, with implications in biotechnology and medicine.

SUMMARYWhile viral infections are known to hijack the transcription and translation of the host cell, the extent to which encoded viral proteins coordinate these perturbations remains unclear. Here we demonstrate that the oncoviral proteins Tax and HBZ interact with specific components of the spliceosome machinery, including the U2 auxiliary factor large subunit (U2AF2), and the complementary factor for APOBEC-1 (A1CF), respectively. Tax and HBZ perturb the splicing landscape in T-cells by altering cassette exons in opposing manners, with Tax inducing exon inclusion while HBZ induces exon exclusion. Among Tax- and HBZ-dependent splicing changes, we identify events that are also altered in Adult T cell leukemia (ATL) patients, and in well-known cancer census genes. Our interactome mapping approach, applicable to other viral oncogenes, has identified spliceosome perturbation as a novel mechanism coordinately used by Tax and HBZ to reprogram the transcriptome.HighlightsTax and HBZ interact with RNA-binding proteins as well as transcription factorsHTLV-1 encoded proteins Tax and HBZ alter the splicing landscape in T-cellsTax and HBZ expression affect alternative splicing of 33 and 63 cancer genes, respectivelyOpposing roles for Tax and HBZ in deregulation of gene expressionGraphical abstract

SUMMARYThe endoplasmic reticulum (ER) is a central eukaryotic organelle with a tubular network made of hairpin proteins linked by hydrolysis of GTP nucleotides. Among post-translational modifications initiated at the ER level, glycosylation is the most common reaction. However, our understanding of the impact of glycosylation on ER structure remains unclear. Here, we show that Exostosin-1 (EXT1) glycosyltransferase, an enzyme involved inN-glycosylation, is a key regulator of ER morphology and dynamics. We have integrated multi-omics data and super-resolution imaging to characterize the broad effect of EXT1 inactivation, including ER shape-dynamics-function relationships in mammalian cells. We have observed that, inactivating EXT1 induces cell enlargement and enhances metabolic switches such as protein secretion. In particular, suppressing EXT1 in mouse thymocytes causes developmental dysfunctions associated to ER network extension. Our findings suggest that EXT1 drives glycosylation reactions involving ER structural proteins and high-energy nucleotide sugars, which might also apply to other organelles.

© The Author(s) 2019., Complementary assays are required to comprehensively map complex biological entities such as genomes, proteomes and interactome networks. However, how various assays can be optimally combined to approach completeness while maintaining high precision often remains unclear. Here, we propose a framework for binary protein-protein interaction (PPI) mapping based on optimally combining assays and/or assay versions to maximize detection of true positive interactions, while avoiding detection of random protein pairs. We have engineered a novel NanoLuc two-hybrid (N2H) system that integrates 12 different versions, differing by protein expression systems and tagging configurations. The resulting union of N2H versions recovers as many PPIs as 10 distinct assays combined. Thus, to further improve PPI mapping, developing alternative versions of existing assays might be as productive as designing completely new assays. Our findings should be applicable to systematic mapping of other biological landscapes., This work was supported by a Claudia Adams Barr Award to S.G.C., a Fonds de la Recherche Scientifique (F.R.S.- FNRS)-Télévie Grant and a Wallonia-Brussels International (WBI)-World Excellence Fellowship to J.O., NHGRI grants U41HG001715 awarded to M.V., D.E.H. and M.A.C, and P50HG004233 awarded to M.V. We acknowledge the support of Labex IBEID (10- LABX-0062). M.V. is a Chercheur Qualifié Honoraire from the Fonds de la Recherche Scientifique (FRS-FNRS, Wallonia-Brussels Federation, Belgium).

Complementary assays are required to comprehensively map complex biological entities such as genomes, proteomes and interactome networks. However, how various assays can be optimally combined to approach completeness while maintaining high precision often remains unclear. Here, we propose the concept of an “assayome” for binary protein-protein interaction (PPI) mapping as an optimal combination of assays and/or assay versions that maximizes detection of true positive interactions, while avoiding detection of random protein pairs. We engineered a novel NanoLuc two-hybrid (N2H) system that integrates 12 different versions differing by protein expression systems and tagging configurations. The resulting N2H assayome recovers as many PPIs as 10 distinct assays combined. Thus, to further improve PPI mapping, developing alternative versions of existing assays might be as productive as designing completely new assays. Our assayome concept should be applicable to systematic mapping of other biological landscapes.