When endoplasmic reticulum (ER) homeostasis is perturbed, an adaptive mechanism is triggered and named the unfolded protein response (UPR). Thus far, three known UPR signaling branches (IRE-1, PERK, and ATF-6) mediate the reestablishment of ER functions but can also lead to apoptosis if ER stress is not alleviated. However, the understanding of the molecular mechanisms integrating the UPR to other ER functions, such as membrane traffic or endomembrane signaling, remains incomplete. We consequently sought to identify new regulators of UPR-dependent transcriptional mechanisms and focused on a family of proteins known to mediate, among other, ER-related functions: the small GTP-binding proteins of the RAS superfamily. To this end, we used transgenic UPR reporter Caenorhabditis elegans strains as a model to specifically silence small-GTPase expression. We show that the Rho subfamily member CRP-1 is an essential component of UPR-induced transcriptional events through its physical and genetic interactions with the AAA ؉ ATPase CDC-48. In addition, we describe a novel signaling module involving CRP-1 and CDC-48 which may directly link the UPR to DNA remodeling and transcription control.The endoplasmic reticulum (ER) is an organelle found in eukaryotic cells, which is mainly involved in calcium sequestration, lipid biosynthesis and translation, folding, and transport of secretory proteins (9). These functions require specialized and integrated molecular machines (7). Most of the proteins distributed in organelles of the secretory pathway, expressed at the cell surface or secreted, transit through the ER before reaching their final destination. Indeed, polypeptide chains, translated on ER membrane-bound ribosomes, are first translocated into the ER lumen via the translocon and then processed through the ER folding machineries, which include a chaperone component (e.g., BiP or GRP94), a posttranslational modification machinery (e.g., N glycosylation with the oligosaccharyl transferase complex, S-S bound formation with the protein disulfide isomerases), and a quality control component (e.g., calnexin, UDP-glucose:glycoprotein glucosyltransferase). Proteins which do not acquire a correct conformation are retained in the ER by ER-specific quality control mechanisms and are consequently granted further folding attempts. If this fails again, terminally misfolded proteins are degraded via the ER-associated degradation (ERAD) machinery (9).Under basal conditions, these integrated mechanisms maintain the ER protein load in equilibrium with ER's folding and export capacities. However, if one of those components is dysfunctional, the entire chain reaction is perturbed and ER homeostasis is disrupted. This leads to an increased amount of improperly folded proteins, which accumulate within the ER. As a mechanism for adaption to this phenomenon, cells have evolved the unfolded protein response (UPR), which aims at restoring ER homeostasis (38, 41) by (i) attenuating protein translation, (ii) increasing ER folding capacity, (iii) incre...
IntroductIon Large-scale technologies used within the life science industry for drug discovery have recently proven useful in academic research laboratories, facilitating chemical genomics and the investigation of specific biological functions. these technologies, including homogeneous luminescence (e.g., alphascreen ® and aequorin), fluorescence intensity (fi) or polarization (fP), time-resolved fluorescence (trf), laser scanning cytometry, or microscopy-based approaches, have become enabling tools to characterize complex functions of biological targets.in the present study, we used alphascreen ® to investigate cell signaling pathways 1 and in particular those emanating from the endoplasmic reticulum (er) under stress conditions. the er ensures proper protein folding and export to later compartments of the secretory pathway. this is achieved through complex machineries, including protein synthesis, translocation/ folding, quality control, er-associated degradation (erad), and export.2 in addition to these functional attributes, the er has evolved a highly conserved adaptive signaling pathway, referred to as the unfolded protein response (uPr), whose activation occurs upon accumulation of improperly folded proteins in the er. 3 uPr signaling is mediated by 3 er resident transmembrane proteins-the PKr-like er kinase (PerK), the activating transcription factor 6 (atf6), and the inositol requiring enzyme 1 alpha (ire1α).3,4 our work has focused on ire1α, which is a transmembrane sensor of er stress. the luminal domain of ire1α contains binding sites for the chaperone BiP, whereas the cytosolic region has 2 main catalytic elements: a serine/threonine kinase and an endoribonuclease domain.4-6 under basal conditions, ire1α is thought to exist as a monomer, and in response to the accumulation of misfolded proteins in the er, ire1 proteins oligomerize, resulting in its trans-autophosphorylation and triggering of its endoribonuclease activity. 4 however, structural studies in Saccharomyces cerevisiae showed that the luminal domain of ire1 exists as a dimer/oligomer and suggested that it could potentially bind directly to unfolded peptides similar to Mhc class i molecules.7 this was confirmed by structural studies on the cytosolic domain of ire1 in S. cerevisiae, which revealed a dimer conformation that is subjected to conformational changes upon er stress, leading to activation of its kinase and rnase activities.8 recently, the cytosolic domain of ire1 was crystallized in an oligomeric form, which was promoted by the presence of the kinase inhibitor sunitinib.9,10 this highly ordered molecular structure was found assay technologies that were originally developed for high-throughput screening (hts) have recently proven useful in drug discovery for activities located upstream (target identification and validation) and downstream (adMet) of hts. here the authors investigated and characterized the biological properties of a novel target, ire1α, a bifunctional kinase/rnase stress sensor of the endoplasmic reticulum (er). they...
We investigated the temperature dependence of the poly(9,9-dioctylfluorene) beta phase photoluminescence (PL) spectra in spin coated thin films from tetrahydrofuran solutions. As the temperature increases from 18 to 300 K a continuous blueshift of the 0-0 PL peak of about 25 meV and an increase of the peak full width at half maximum (FWHM) of about 49 meV are observed. We show that the PL spectra temperature dependence is not due to a temperature dependent average conjugation length, as often assumed, but instead it can be quantitatively explained in the frame of a thermal quasiequilibrium model for excitons in an inhomogeneously broadened excited states distribution. We demonstrate that the emission blueshift and broadening are mainly due to the increase of the excitons' temperature with the sample one. This effect is partially compensated by an increasing efficiency of the exciton energy migration. The interplay between these two processes quantitatively explains the observed temperature dependence of the PL peak energy and of its FWHM. On the contrary we show that the PL spectra are almost independent of the absorption blueshift with temperature.
The physical processes leading to solvent swelling induced glassy-to -phase transition in poly͑9,9-dio-ctylfluorene͒ thin films are investigated in real time by photoluminescence and confocal spectroscopy. We show that the vapor solvent swelling induced -phase formation takes place in much shorter times ͑few minutes͒ than the one usually employed in literature ͑several hours͒. Moreover, we show that the swelling is faster if the solvent mainly interacts with the PF8 aromatic rings ͑toluene͒ than with the octyl chains ͑iso-octane͒. On the contrary, no swelling is caused by nonsolvents such as n-butylic alcohol. Finally, we demonstrate that the -phase formation is due to athermal ͑simultaneous͒ nucleation followed by diffusion controlled one dimensional crystallization.The great interest in organic conjugated compounds is related to their potential applications in low cost electronics, optoelectronics, and photonics. Among several families of polymers showing electroluminescence, 1 lasing, 2 and field effect mobility, 3 polyfluorene has emerged as an attractive polymer in blue-light emitting diodes, 4,5 gain media in lasers 6 and optical amplifiers, 7 photovoltaic devices, 8 and thin film transistors. 9 In addition to its excellent properties for device applications, poly͑9,9-dioctylfluorene͒ ͑PF8͒ is a very interesting system for basic photophysics studies, due to the strong morphology dependence of its optical properties. Two distinct phases are usually present in spin coated PF8 thin films, usually named as glassy and -phases, with different electronic and optical properties. This feature, ascribed to a different chain geometry 10 of the two phases, allowed the investigation in thin PF8 films of the chain geometry dependence of the triplet 11 and polaron formation 12 and of the optical gain, 13 without the need for chemical modification of the molecules. Finally, as the -phase photoluminescence ͑PL͒ is at lower energy than the glassy-phase one, the -phase presence also modifies the relaxation processes in the film, due to the introduction of efficient glassy-→-phase Förster transfer ͑FRET͒, that significantly affects the emission spectra. 14,15 Moreover, the -phase PL shows unusually narrow emission features ͓about 18 meV at 25 K ͑Ref. 16͔͒ and an extremely well resolved vibronic structure that allowed the investigation of the origin of the PL linewidth temperature dependence 17 and of the vibronic coupling. 14 Concerning the -phase formation, the -phase has been observed in as-deposited samples obtained by spin coating from high boiling point solvents, 15,18 in Langmuir-Blodgett films, 19 and in spin coated PF8-PMMA blends. 20 Moreover, the -phase can be obtained in glassy-phase films by postdeposition process such as thermal treatment 16 or exposure to solvent vapors for several hours. 14 The investigation of the -phase formation kinetics during postdeposition thermal cycling allowed to ascribe 16 the -phase formation to simultaneous ͑athermal͒ nucleation, followed by one dimensional growth and cr...
We studied the microscopic dependence of poly(9,9-dioctylfluorene) photoluminescence (PL) on the deposition conditions. We show that in films spin coated from chloroform phase separation of β and glassy phases is present, with micrometric β phase clusters covering about 6% of the sample surface. The exposure to toluene vapors leads to the disappearance of the β phase clusters, but increases the β phase content in the films due to swelling induced polyfluorene chain planarization. The deposition from toluene solution leads to nonuniform PL intensity, dominated by the β phase emission, attributed to an interplay between aggregation during the solvent evaporation and solvent swelling induced chain planarization.
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