Photochemical multivariate curve resolution models for the investigation of photochromic systems under continuous irradiation.

We propose a multivariate curve resolution approach for the investigation of photochromic systems using UV-Visible spectroscopy. The incorporation of photochemical hard-models as constraints in multivariate curve resolution alternating least squares (MCR-ALS) allows extracting reaction quantum yields in situations where a complete knowledge of the system is not available. We apply this approach to the study of the photochromism of CMTE (cis-1,2-dicyano-1,2-bis(2,4,5-trimethyl-3-thienyl)ethene) under continuous monochromatic irradiation. The mechanism, involving 3 species and 2 reversible reactions, is written and translated into a kinetic constraint that can be applied to the concentration profiles within ALS. First, ambiguity of the solution obtained for photochemical model(s) is calculated and discussed for single set analysis. Multiset analysis is then proposed combining data obtained under different irradiation wavelengths to provide more reliable results. Finally, the photochemical reactivity of CMTE is widely unraveled, and some description of the mechanism observed under irradiation at 365 nm is given.

Comparative photophysical investigation of doubly-emissive photochromic-fluorescent diarylethenes.

Diarylethene molecules showing photochromism and fluorescence properties in both open and closed forms, associated with two different emission colors, are very promising for applications involving ratiometric emissive photoswitches. We report here a complete study on the competition between the multiple photophysical processes involved in the excited states for two sulfone derivatives of benzothiophene-based diarylethene molecules, only differing by the substituent groups on their reactive carbon (methyl for DAE-Me and ethyl for DAE-Et). Steady-state and time-resolved spectroscopy, combined with DFT and TD-DFT calculations, allow a complete determination of the kinetic constants leading to fluorescence and photoreaction pathways in different solvents, and enlighten the specific role of the substituent group in the photophysical properties due to a shielding effect against the solvation environment. The predominant role of the non-radiative deactivation processes in such a family of molecules is shown, and a tentative excited state mechanistic scheme is proposed based on femtosecond transient absorption experiments performed on the closed forms.

Photochromic-fluorescent-plasmonic nanomaterials: towards integrated three-component photoactive hybrid nanosystems.

Silica-coated gold nanorods functionalized with grafted fluorescent and photochromic derivatives were synthesized and characterized. Spectroscopic investigations demonstrated that cross-coupled interactions between plasmonic, photochromic, and fluorescence properties play a major role in such nanosystems, depending on the thickness of the silica spacer, leading to multi-signal photoswitchability.

Intramolecular electronic excitation energy transfer in donor/acceptor dyads studied by time and frequency resolved single molecule spectroscopy.

Electronic excitation energy transfer has been studied by single molecule spectroscopy in donor/acceptor dyads composed of a perylenediimide donor and a terrylenediimide acceptor linked by oligo(phenylene) bridges of two different lengths. For the shorter bridge (three phenylene units) energy is transferred almost quantitatively from the donor to the acceptor, while for the longer bridge (seven phenylene units) energy transfer is less efficient as indicated by the occurrence of donor and acceptor emission. To determine energy transfer rates and efficiencies at the single molecule level, several methods have been employed. These comprise time-correlated single photon counting techniques at room temperature and optical linewidth measurements at low temperature (1.4 K). For both types of measurement we obtain broad distributions of the rate constants of energy transfer. These distributions are simulated in the framework of Forster theory by properly taking into account static disorder and the flexibility of the dyads, as both effects can substantially contribute to the distributions of energy transfer times. The rate constants of energy transfer obtained from the calculated distributions are smaller on average than those extracted from the experimental distributions, whereby the discrepancy is larger for the shorter bridge. Furthermore, by plotting the experimentally determined transfer rates against the individual spectral overlaps, approximately linear dependencies are found being indicative of a Forster-type contribution to the energy transfer. For a given single molecule such a linear dependence could be followed by spectral diffusion induced fluctuations of the spectral overlap. The discrepancies between measured energy transfer rates and rates calculated by Forster theory are briefly discussed in light of recent results of quantum chemical calculations, which indicate that a bridge-mediated contribution is mainly responsible for the deviations from Forster theory. The availability of the inhomogeneous distributions of donor and acceptor electronic transition frequencies allows for comparing the energy transfer process at liquid helium and room temperature for the same set of molecules via simple simulations. It is found that on average the energy transfer is by a factor of approximately 3 faster at room temperature, which is due to an increase of spectral overlap.

Synergism between ERalpha transactivation function 1 (AF-1) and AF-2 mediated by steroid receptor coactivator protein-1: requirement for the AF-1 alpha-helical core and for a direct interaction between the N- and C-terminal domains.

The transcriptional activity of ERalpha (or NR3A1) after binding of ligand is mediated through synergistic action between activation functions (AFs) AF-1 and AF-2 and the transcriptional machinery. This is functionally achieved by bridging coactivators such as CEBP binding protein/p300 and members of the p160 subfamily such as steroid receptor coactivator protein-1 (SRC-1). We previously identified a conserved potential alpha-helical structure within the AF-1 functional core, and by evaluating point mutants of human ERalpha (hERalpha) within this region, we show that in transfection experiments this structure is required for synergism between SRC-1 and hERalpha. We report that the transcriptional synergism between AF-1 mutants and SRC-1 was abolished in AF-1-sensitive cells such as HepG2, whereas it was reduced by 50% in CHO-K1 cells, which have a mixed context that is sensitive to both the AF-1 and AF-2 regions of hERalpha. Glutathione-S-transferase pulldown assays demonstrate that the AF-1 core is able and sufficient for the hERalpha N-terminal region to interact with SRC-1. Interestingly, an enhancement of this recruitment in the presence of the hERalpha ligand-binding domain was observed, which was found to be dependent on a direct interaction between the N-terminal B domain and the ligand-binding domain. Another functional consequence of this physical interaction, which is promoted by both partial and full agonists of hERalpha, was an increase in the phosphorylation state of the N-terminal domain. Binding of 4-hydroxytamoxifen (OHT) to the hERalpha C-terminal region induced a functional AF-1 conformation in vitro through this N- and C-terminal interaction. The involvement of an SRC-1-mediated pathway in transactivation mediated by hERalpha AF-1 was further substantiated by transfection experiments using the OHTresponsive human C3 promoter, which showed that OHT-induced hERalpha AF-1 activity was enhanced by SRC-1 and required the AF-1 alpha-helical structure. In conclusion, we demonstrate that the synergism between AF-1 and AF-2 is mediated in part by a cooperative recruitment of SRC-1 by both the AF-1 alpha-helical core and AF-2 regions and that it is stabilized by a direct interaction between the B and C-terminal domains. This interaction of SRC-1 with the AF-1 alpha-helical core is essential for both E2- and OHT-induced ERalpha activity.

COUP-TFI (chicken ovalbumin upstream promoter-transcription factor I) regulates cell migration and axogenesis in differentiating P19 embryonal carcinoma cells.

The developmental expression patterns of the nuclear orphan receptors COUP-TFs (chicken ovalbumin upstream promoter-transcription factors) have been correlated to neurogenesis in several animal species. Nevertheless, the role of COUP-TFs in neurogenesis remains unknown. We have studied the functional involvement of COUP-TFI in retinoic acid (RA)-induced neuronal differentiation of P19 embryonal carcinoma cells through two complementary approaches: 1) deregulated expression of COUP-TFI, and 2) inactivation of endogenous COUP-TFs by means of a dominant-negative COUP-TFI mutant. Low levels of wild-type (wt)COUP-TFI transgene expression did not inhibit neural cell fate and primarily enhanced neuron outgrowth from RA-treated P19 aggregates. In contrast, high COUP-TFI expression impeded the neuronal differentiation of P19 cells induced with RA, resulting in cell cultures lacking neurons. This morphological effect was correlated to an elevated level of E-cadherin mRNA. The dominant-negative COUP-TFI mutant induced cell packing after RA treatment and inhibited neurite extension and neuron outgrowth from aggregates. A RGD peptide interference assay indicated that endogenous COUP-TFs could favor migration of neurons through an integrin-dependent mechanism. Accordingly, vitronectin mRNA levels were shown to be up-regulated by COUP-TFI by RT-PCR analysis, and COUP-TFI stimulated the mouse vitronectin promoter activity in transient transfection assays. Taken together, these data indicate that COUP-TFI is not simply a global repressor of retinoid functions, but shows a high selectivity for regulating genes involved in cellular adhesion and migration processes that are particularly important for neuronal differentiation.

Function of N-terminal transactivation domain of the estrogen receptor requires a potential alpha-helical structure and is negatively regulated by the A domain.

Transcriptional activation by the estrogen receptor (NR3A1, or ER) requires specific ligand-inducible activation functions located in the amino (AF-1) and the carboxyl (AF-2 and AF-2a) regions of the protein. Although several detailed reports of ER structure and function describe mechanisms whereby AF-2 activates transcription, less precise data exist for AF-1. We recently reported that the rainbow trout and human estrogen receptors (rtERs and hERs, respectively), two evolutionary distant proteins, exhibit comparable AF-1 activities while sharing only 20% homology in their N-terminal region. These data suggested that the basic mechanisms whereby AF-1 and the ER N-terminal region activate transactivation might be evolutionary conserved. Therefore, a comparative approach between rtER and hER could provide more detailed information on AF-1 function. Transactivation analysis of truncated receptors and Gal4DBD (DNA binding domain of the Gal4 factor) fusion proteins in Saccharomyces cerevisiae defined a minimal region of 11 amino acids, located at the beginning of the B domain, necessary for AF-1 activity in rtER. Hydrophobic cluster analysis (HCA) indicated the presence of a potential alpha-helix within this minimal region that is conserved during evolution. Both rtER and hER sequences corresponding to this potential alpha-helical structure were able to induce transcription when fused to the Gal4DBD, indicating that this region can transactivate in an autonomous manner. Furthermore, point mutations in this 11-amino acid region of the receptors markedly reduced their transcriptional activity either within the context of a whole ER or a Gal4DBD fusion protein. Data were confirmed in mammalian cells and, interestingly, ERs with an inverted alpha-helix were as active as their corresponding wild-type proteins, indicating a conserved role in AF-1 for these structures. Moreover, using two naturally occurring rtER N-terminal variants possessing or not the A domain (rtER(L) and rtER(S), respectively), together with A domain-truncated hER and chimeric rtER/hER receptors, we demonstrated that the A domain of the ER plays an inhibitory role in ligand-independent activity of the receptor. In vitro and in vivo protein-protein interaction assays using both rtER and hER demonstrated that this repression is likely to be mediated by a ligand-sensitive direct interaction between the A domain and the C-terminal region of the ER.

Identification of a new isoform of the human estrogen receptor-alpha (hER-alpha) that is encoded by distinct transcripts and that is able to repress hER-alpha activation function 1.

A new isoform of the human estrogen receptor-alpha (hER-alpha) has been identified and characterized. This 46 kDa isoform (hERalpha46) lacks the N-terminal 173 amino acids present in the previously characterized 66 kDa isoform (hERalpha66). hERalpha46 is encoded by a new class of hER-alpha transcript that lacks the first coding exon (exon 1A) of the ER-alpha gene. We demonstrated that these Delta1A hER-alpha transcripts originate from the E and F hER-alpha promoters and are produced by the splicing of exon 1E directly to exon 2. Functional analysis of hERalpha46 showed that, in a cell context sensitive to the transactivation function AF-2, this receptor is an effective ligand-inducible transcription factor. In contrast, hERalpha46 is a powerful inhibitor of hERalpha66 in a cell context where the transactivating function of AF-1 predominates over AF-2. The mechanisms by which the AF-1 dominant-negative action is exerted may involve heterodimeri zation of the two receptor isoforms and/or direct competition for the ER-alpha DNA-binding site. hERalpha66/hERalpha46 ratios change with the cell growth status of the breast carcinoma cell line MCF7, suggesting a role of hERalpha46 in cellular proliferation.

Interplay between liganded and orphan nuclear receptors controls reproductive pathways.

Nuclear receptors are transcription factors that belong to an evolutionary ancient superfamily. These proteins, which are even present in primitive metazoans, are implicated in all levels of cell fate: proliferation, differentiation, and apoptosis. Some of these nuclear receptors behave as ligand-inducible transcription factors, as they have acquired during evolution the ability to bind ligands. This is the case for some proteins that recognize small hydrophobic signaling molecules, and particularly the estrogen receptor (ER or NR3A1), which regulates the target gene's transcription rate under estrogen binding. It is now known that the ER alone regulates the transcription of many genes, such as those implicated in reproductive functions. However, this ER-mediated signaling pathway could be modulated by other transcription factors. Our work has established that two other orphan nuclear receptors (SF-1 or NR5A1 and the COUP-TFs, NR2F1 and NR2F2) can enhance two ER-regulated genes implicated in salmonid reproductive functions: the ER gene itself, and the sGTHIIbeta gene. Moreover, some xenoestrogens could disturb these regulations. Therefore, our data contribute to the concept that interplay between nuclear receptors is an important event for the transcriptional regulation of genes controlling cellular functions.

Inhibition of rainbow trout (Oncorhynchus mykiss) estrogen receptor activity by cadmium.

This study was conducted to determine if the cadmium-mediated inhibition of vitellogenesis observed in fish collected from contaminated areas or undergoing experimental exposure to cadmium correlated with modification in the transcriptional activity of the estrogen receptor. A recombinant yeast system expressing rainbow trout (Oncorhynchus mykiss) estradiol receptor or human estradiol receptor was used to evaluate the direct effect of cadmium exposure on estradiol receptor transcriptional activity. In recombinant yeast, cadmium reduced the estradiol-stimulated transcription of an estrogen-responsive reporter gene. In vitro-binding assays indicated that cadmium did not affect ligand binding to the receptor. Yeast one- and two-hybrid assays showed that estradiol-induced conformational changes and receptor dimerization were not affected by cadmium; conversely, DNA binding of the estradiol receptor to its cognate element was dramatically reduced in gel retardation assay. This study provides mechanistic data supporting the idea that cadmium is an important endocrine disrupter through a direct effect on estradiol receptor transcriptional activity and may affect a number of estrogen signaling pathways.

Two estrogen receptor (ER) isoforms with different estrogen dependencies are generated from the trout ER gene.

A characteristic of all estrogen receptors (ER) cloned from fish to date is the lack of the first 37-42 N-terminal amino acids specific to the A domain. Here we report the isolation and characterization from trout ovary of a full-length complementary DNA (cDNA) clone encoding an N-terminal variant form of the rainbow trout ER (rtER). Sequence analysis of open reading frame of this cDNA predicts a 622-amino acid protein. The C-terminal region of this protein, from amino acid position 45 to the end, was very similar to the previously reported rtER (referred to as the short form, or rtER(S)). In contrast, this novel rtER cDNA (referred to as the long form, or rtER(L)) contains an additional in-frame ATG initiator codon that adds 45 residues to the N-terminal region of the protein. This new N-terminal region may represent the A domain of ER found in tetrapod species. The first 227 bp of this new cDNA were similar to the 3'-end intronic sequence of the rtER gene intron 1. These data together with S1 nuclease, primer extension, and RT-PCR experiments demonstrate that the rtER(L) represents a second isoform of rtER that arises from an alternative promoter within the first intron of the gene. Transcripts encoding both rtER forms were expressed in the liver. In vitro translation of the rtER(L) cDNA produced 2 proteins with molecular masses of 71 and 65 kDa, whereas rtER(S) cDNA produced 1 65-kDa protein. Interestingly, Western blot analysis with a specific antibody against the C-terminal region of rtER revealed 2 receptor forms of 65 and 71 kDa in trout liver nuclear extracts, in agreement with the presence of the 2 distinct classes of rtER messenger RNA in this tissue. Functional analysis of both rtER isoforms revealed that although rtER(S) consistently exhibited a basal (estrogen-independent) trans-activation activity that could be further increased in the presence of estrogens, the novel isoform rtER(L) is characterized by a strict estrogen-dependent transcriptional activity. These data suggest that the additional 45 residues at the N-terminal region of rtER(L) clearly modify the hormone-independent trans-activation function of the receptor.

Synergism between a half-site and an imperfect estrogen-responsive element, and cooperation with COUP-TFI are required for estrogen receptor (ER) to achieve a maximal estrogen-stimulation of rainbow trout ER gene.

In all oviparous, liver represents one of the main E2-target tissues where estrogen receptor (ER) constitutes the key mediator of estrogen action. The rainbow trout estrogen receptor (rtER) gene expression is markedly up-regulated by estrogens and the sequences responsible for this autoregulation have been located in a 0.2 kb upstream transcription start site within - 40/- 248 enhancer region. Absence of interference with steroid hormone receptors and tissue-specific factors and a conserved basal transcriptional machinery between yeast and higher eukaryotes, make yeast a simple assay system that will enable determination of important cis-acting regulatory sequences within rtER gene promoter and identification of transcription factors implicated in the regulation of this gene. Deletion analysis allowed to show a synergistic effect between an imperfect estrogen-responsive element (ERE) and a consensus half-ERE to achieve a high hormone-dependent transcriptional activation of the rtER gene promoter in the presence of stably expressed rtER. As in mammalian cells, here we observed a positive regulation of the rtER gene promoter by the chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) through enhancing autoregulation. Using a point mutation COUP-TFI mutant unable to bind DNA demonstrates that enhancement of rtER gene autoregulation requires the interaction of COUP-TFI to the DNA. Moreover, this enhancement of transcriptional activation by COUP-TFI requires specifically the AF-1 transactivation function of ER and can be observed in the presence of E2 or 4-hydroxytamoxifen but not ICI 164384. Thus, this paper describes the reconstitution of a hormone-responsive transcription unit in yeast in which the regulation of rtER gene promoter could be enhanced by the participation of cis-elements and/or trans-acting factors, such as ER itself or COUP-TF.