Comprehensive Interactome Mapping of Nuclear Receptors Using Proximity Biotinylation.

Nuclear receptors, including hormone receptors, perform their cellular activities by modulating their protein-protein interactions. They engage with specific ligands and translocate to the nucleus, where they bind the DNA and activate extensive transcriptional programs. Therefore, gaining a comprehensive overview of the protein-protein interactions they establish requires methods that function effectively throughout the cell with fast dynamics and high reproducibility. Focusing on estrogen receptor alpha (ESR1), the founding member of the nuclear receptor family, this chapter describes a new lentiviral system that allows the expression of TurboID-hemagglutinin (HA)-2 × Strep tagged proteins in mammalian cells to perform fast proximity biotinylation assays. Key validation steps for these reagents and their use in interactome mapping experiments in two distinct breast cancer cell lines are described. Our protocol enabled the quantification of ESR1 interactome generated by cellular contexts that were hormone-sensitive or not.

Human papillomavirus E7 oncoprotein targets RNF168 to hijack the host DNA damage response.

High-risk human papillomaviruses (HR-HPVs) promote cervical cancer as well as a subset of anogenital and head and neck cancers. Due to their limited coding capacity, HPVs hijack the host cell's DNA replication and repair machineries to replicate their own genomes. How this host-pathogen interaction contributes to genomic instability is unknown. Here, we report that HPV-infected cancer cells express high levels of RNF168, an E3 ubiquitin ligase that is critical for proper DNA repair following DNA double-strand breaks, and accumulate high numbers of 53BP1 nuclear bodies, a marker of genomic instability induced by replication stress. We describe a mechanism by which HPV E7 subverts the function of RNF168 at DNA double-strand breaks, providing a rationale for increased homology-directed recombination in E6/E7-expressing cervical cancer cells. By targeting a new regulatory domain of RNF168, E7 binds directly to the E3 ligase without affecting its enzymatic activity. As RNF168 knockdown impairs viral genome amplification in differentiated keratinocytes, we propose that E7 hijacks the E3 ligase to promote the viral replicative cycle. This study reveals a mechanism by which tumor viruses reshape the cellular response to DNA damage by manipulating RNF168-dependent ubiquitin signaling. Importantly, our findings reveal a pathway by which HPV may promote the genomic instability that drives oncogenesis.

Fine mapping of Co-x, an anthracnose resistance gene to a highly virulent strain of Colletotrichum lindemuthianum in common bean.

KEY MESSAGE: The Co - x anthracnose R gene of common bean was fine-mapped into a 58 kb region at one end of chromosome 1, where no canonical NB-LRR-encoding genes are present in G19833 genome sequence. Anthracnose, caused by the phytopathogenic fungus Colletotrichum lindemuthianum, is one of the most damaging diseases of common bean, Phaseolus vulgaris. Various resistance (R) genes, named Co-, conferring race-specific resistance to different strains of C. lindemuthianum have been identified. The Andean cultivar JaloEEP558 was reported to carry Co-x on chromosome 1, conferring resistance to the highly virulent strain 100. To fine map Co-x, 181 recombinant inbred lines derived from the cross between JaloEEP558 and BAT93 were genotyped with polymerase chain reaction (PCR)-based markers developed using the genome sequence of the Andean genotype G19833. Analysis of RILs carrying key recombination events positioned Co-x at one end of chromosome 1 to a 58 kb region of the G19833 genome sequence. Annotation of this target region revealed eight genes: three phosphoinositide-specific phospholipases C (PI-PLC), one zinc finger protein and four kinases, suggesting that Co-x is not a classical nucleotide-binding leucine-rich encoding gene. In addition, we identified and characterized the seven members of common bean PI-PLC gene family distributed into two clusters located at the ends of chromosomes 1 and 8. Co-x is not a member of Co-1 allelic series since these two genes are separated by at least 190 kb. Comparative analysis between soybean and common bean revealed that the Co-x syntenic region, located at one end of Glycine max chromosome 18, carries Rhg1, a major QTL contributing to soybean cyst nematode resistance. The PCR-based markers generated in this study should be useful in marker-assisted selection for pyramiding Co-x with other R genes.

Crosstalks between myo-inositol metabolism, programmed cell death and basal immunity in Arabidopsis.

BACKGROUND: Although it is a crucial cellular process required for both normal development and to face stress conditions, the control of programmed cell death in plants is not fully understood. We previously reported the isolation of ATXR5 and ATXR6, two PCNA-binding proteins that could be involved in the regulation of cell cycle or cell death. A yeast two-hybrid screen using ATXR5 as bait captured AtIPS1, an enzyme which catalyses the committed step of myo-inositol (MI) biosynthesis. atips1 mutants form spontaneous lesions on leaves, raising the possibility that MI metabolism may play a role in the control of PCD in plants. In this work, we have characterised atips1 mutants to gain insight regarding the role of MI in PCD regulation. METHODOLOGY/PRINCIPAL FINDINGS: - lesion formation in atips1 mutants depends of light intensity, is due to PCD as evidenced by TUNEL labelling of nuclei, and is regulated by phytohormones such as salicylic acid - MI and galactinol are the only metabolites whose accumulation is significantly reduced in the mutant, and supplementation of the mutant with these compounds is sufficient to prevent PCD - the transcriptome profile of the mutant is extremely similar to that of lesion mimic mutants such as cpr5, or wild-type plants infected with pathogens. CONCLUSION/SIGNIFICANCE: Taken together, our results provide strong evidence for the role of MI or MI derivatives in the regulation of PCD. Interestingly, there are three isoforms of IPS in Arabidopsis, but AtIPS1 is the only one harbouring a nuclear localisation sequence, suggesting that nuclear pools of MI may play a specific role in PCD regulation and opening new research prospects regarding the role of MI in the prevention of tumorigenesis. Nevertheless, the significance of the interaction between AtIPS1 and ATXR5 remains to be established.

Effects of PM2.5 components in the release of amphiregulin by human airway epithelial cells.

Exposure to ambient particulate matter (PM) is responsible for airway inflammation and tissue remodeling. Urban PM(2.5) (aerodynamic diameter <2.5microm) is a complex mixture rich in soots and containing hydrosoluble and organic components. We previously showed that the exposure of airway epithelial cells to PM(2.5) triggers the release of amphiregulin (AR), ligand of the epidermal growth factor receptor (EGFR) involved in proinflammatory and repair responses. The effect of Paris PM(2.5) organic and aqueous fractions in AR expression and secretion was investigated on the bronchial epithelial cell line 16HBE and normal human nasal epithelial (NHNE) cells. Both a macroarray specific for inflammation pathways and RT-PCR showed an AR upregulation in organic extract-treated 16HBE cells. AR release is induced in 16HBE and NHNE cells grown on plastic and exposed to native PM(2.5), organic extract and to a lesser extent washed PM(2.5) (deprived of its hydrosoluble content) and aqueous extract. Furthermore, as assessed by using NHNE cells grown on Transwell inserts, this secretion is polarized toward the basolateral side where the EGFR is expressed. To conclude, both PM(2.5) organic and hydrosoluble components are involved in the expression and secretion of AR; organic compounds exhibiting a strong effect when they are easily bioavailable.

Fine urban atmospheric particulate matter modulates inflammatory gene and protein expression in human bronchial epithelial cells.

Ambient particulate matter (PM) is known to induce inflammation in the respiratory tract of exposed subjects. The aim of the present study was to detect, in bronchial epithelial cells, candidate inflammatory genes exhibiting transcriptional modifications following urban PM2.5 exposure. Paris urban PM2.5 sampled either at a curbside or a background station in winter and in summer was tested in comparison with diesel exhaust particles (DEP) at 10 microg/cm2 on human bronchial epithelial (16-HBE) cells (18 h of exposure). The gene profiling study performed using a 375 cDNA cytokine expression array highlighted the differential expression of certain genes, three of which were selected as genes of interest: the IL-1 alpha cytokine, the GRO-alpha chemokine, and amphiregulin, a ligand of the EGF receptor. Their increased expression was confirmed by RT-PCR and/or by Northern blotting in bronchial epithelial cells. In the culture medium of particle-treated cultures, increased release of GRO-alpha and amphiregulin was shown. The particle component responsible for protein release varied for the two genes. The organic extract seemed to be mainly involved in amphiregulin expression and secretion, whereas both the aqueous and organic extracts induced GRO-alpha release. In conclusion, in bronchial epithelial cells, Paris PM2.5 increased mRNA and protein expression of GRO-alpha and AR involved in the chemoattraction process and bronchial remodeling, respectively.

Fine particulate matter induces amphiregulin secretion by bronchial epithelial cells.

Particulate matter (PM) is thought to be responsible for respiratory health problems. Epithelial cells exposed to particles release pro-inflammatory cytokines leading to inflammation of airways. However, the signaling cascades triggered by particles are poorly understood. We demonstrate that PM with an aerodynamic diameter < 2.5 microm (PM2.5) or diesel exhaust particles upregulate the expression of amphiregulin (AR), a ligand of the epidermal growth factor receptor (EGFR), in human bronchial epithelial cells. AR secretion was blocked by an inhibitor of the EGFR tyrosine kinase (AG1478), or a selective mitogen-activated protein (MAP) kinase/extracellular regulated kinase (Erk) inhibitor (PD98059), but not by the p38 MAP kinase inhibitor (SB203580). Thus, AR secretion is mediated through the activation of the EGFR and Erk MAP kinase pathway. In addition, AR secretion was inhibited by the antioxidant N-acetyl cysteine, but not by a neutralizing anti-EGFR, suggesting an EGFR transactivation via oxidative stress. AR may be involved in cytokine secretion, as AR can induce granulocyte macrophage-colony-stimulating factor (GM-CSF) release and a neutralizing anti-EGFR reduces the particle-induced GM-CSF release. This study indicates that PM2.5 induces the expression and secretion of AR, an EGFR ligand contributing to GM-CSF release, which may reflect an important mechanism for sustaining the proinflammatory response.