Nuclear Export of the Oncoprotein v-ErbA Is Mediated by Acquisition of a Viral Nuclear Export Sequence

v-ErbA, an oncogenic derivative of the thyroid hormone receptor α (TRα) carried by the avian erythroblastosis virus, contains several alterations including fusion of a portion of avian erythroblastosis virus Gag to its N terminus, N- and C-terminal deletions, and 13 amino acid substitutions. Nuclear export of v-ErbA occurs through a CRM1-mediated pathway. In contrast, nuclear export of TRα and another isoform, TRβ, is CRM1-independent. To determine which amino acid changes in v-ErbA confer CRM1-dependent nuclear export, we expressed a panel of green and yellow fluorescent protein-tagged mutant and chimeric proteins in mammalian cells. The sensitivity of subcellular trafficking of these mutants to leptomycin B (LMB), a specific inhibitor of CRM1, was assessed by fluorescence microscopy. Our data showed that a nuclear export sequence resides within a 70-amino acid domain in the C-terminal portion of the p10 region of Gag, and in vitro binding assays demonstrated that Gag interacts directly with CRM1. However, a panel of ligand-binding domain mutants of v-ErbA lacking the Gag sequence exhibited greater nuclear localization in the presence of LMB, suggesting that the various amino acid substitutions/deletions may cause a conformation shift, unmasking an additional CRM1-dependent nuclear export sequence. In contrast, the altered DNA-binding domain of the oncoprotein did not contribute to CRM1-dependent nuclear export. Heterokaryon experiments revealed that v-ErbA did not undergo nucleocytoplasmic shuttling when the CRM1 export pathway was blocked by LMB treatment, suggesting that the ability to follow the export pathway used by TRα has been lost by the oncoprotein during its evolution. Our findings thus point to the intriguing possibility that acquisition of altered nuclear export capabilities contributes to the oncogenic properties of v-ErbA

Oncogenic conversion of the thyroid hormone receptor by altered nuclear transport

Nuclear receptors (NRs) are transcription factors whose activity is modulated by ligand binding. These receptors are at the core of complex signaling pathways and act as integrators of many cellular signals. In the last decade our understanding of NRs has greatly evolved. In particular, regulation of NR subcellular dynamics has emerged as central to their activity. Research on the subcellular distribution of the thyroid hormone receptor (TR) has revealed new dimensions in the complexity of NR regulation, and points to the possibility that NR mislocalization plays a key role in oncogenesis. For many years, TR was thought to reside exclusively in the nucleus. It is now known that TR is a dynamic protein that shuttles between the nucleus and cytoplasm. TR is localized to the nucleus in a phosphorylated form, suggesting that compartment-specific phosphorylation mediates cross-talk between multiple cell signaling pathways. The oncoprotein v-ErbA, a viral-derived dominant negative variant of TR is actively exported to the cytoplasm by the CRM1 export receptor. Strikingly, the oncoprotein causes mislocalization of cellular TR and some of its coactivators by direct interaction. Here, we offer some perspectives on the role of subcellular trafficking in the oncogenic conversion of TR, and propose a new model for oncoprotein dominant negative activity

Etude de la distribution et du transit subcellulaire de l'oncoprotéine V-ERBA (implication dans ses propriétés carcinogéniques)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Multiparametric analysis of a high-content ultra-high-throughput screen identifies inhibitors of the intramembrane protease SPPL2a

The intramembrane protease SPPL2a (signal peptide peptidase-like 2a) is a potential drug target for the treatment of autoimmune diseases due to its essential role in B cells and dendritic cells. To screen a library of 1.4 million compounds for inhibitors of SPPL2a, we developed an imaging assay detecting nuclear translocation of the proteolytically released cytosolic substrate fragment. The state-of-the-art hit calling approach based on nuclear translocation resulted in numerous false positive hits, mainly interrupting intracellular protein trafficking. To filter the false positives we extracted 340 image-based readouts and developed a novel multiparametric analysis method which successfully triaged the primary hit list. The identified scaffolds were validated by demonstrating activity on endogenous SPPL2a and substrate CD74/p8 in B cells. The multiparametric analysis discovered diverse cellular phenotypes and provided profiles for the whole library. The principle of the presented imaging assay, the screening strategy and multiparametric analysis are potentially applicable in future screening campaigns

Development of a novel cytopathic effect-based phenotypic screening assay against Cryptosporidium

Cryptosporidiosis is a diarrheal disease predominantly caused by Cryptosporidium parvum (Cp) and Cryptosporidium hominis (Ch), apicomplexan parasites which infect the intestinal epithelial cells of their human hosts. The only approved drug for cryptosporidiosis is nitazoxanide, which shows limited efficacy in immunocompromised children, the most vulnerable patient population. Thus, new therapeutics and in vitro infection models are urgently needed to address the current unmet medical need. Toward this aim, we have developed novel cytopathic effect (CPE)-based Cp and Ch assays in human colonic tumor (HCT-8) cells and compared them to traditional imaging formats. Further model validation was achieved through screening a collection of FDA-approved drugs and confirming many previously known anti-Cryptosporidium hits as well as identifying a few novel candidates. Collectively, our data reveals this model to be a simple, functional, and homogeneous gain of signal format amenable to high throughput screening, opening new avenues for the discovery of novel anticryptosporidials

Discovery of 2-oxopiperazine dengue inhibitors by scaffold morphing of a phenotypic high-throughput screening hit

A series of 2-oxopiperazine derivatives were designed from the pyrrolopiperazinone cell-based screening hit 4 as a dengue virus inhibitor. Systematic investigation of the structure-activity relationship (SAR) around the piperazinone ring led to the identification of compound (S)-29, which exhibited potent anti-dengue activity in the cell-based assay across all four dengue serotypes with EC50 < 0.1 μM. Cross-resistant analysis confirmed that the virus NS4B protein remained the target of the new oxopiperazine analogs obtained via scaffold morphing from the HTS hit 4