Identification of Novel Compounds Inhibiting the Kinase Activity of the CDK5/p25 Complex via Direct Binding to p25.

Tamoxifen, the gold standard drug for endocrine therapy for breast cancer, modulates the phosphorylation status of the TAU protein in Alzheimer's disease by inhibiting CDK5 kinase activity. Its binding to p25 prevents CDK5/p25 complexation and hence a decrease of CDK5 activity. In breast tumors, this complex is involved in the proliferation and survival of cancer cells, as well as in the disease's prognosis. Still, the molecular stability of the CDK5/p25 complex following tamoxifen exposure in this cancer type has not yet been clearly deciphered. Here, we report the functional characterization of CDK5 and its p25 regulatory subunit in the absence and presence of tamoxifen. In addition, two novel inhibitors of the kinase activity of the CDK5/p25 complex are identified, both of which would reduce the risk of recurrence of estrogen receptor-positive (ER+) breast cancers and prevent drawbacks induced by tamoxifen exposure. Accordingly, 6His-CDK5 and 6His-p25 have been expressed and purified. Fluorescence anisotropy measurements have been used to assess that the two proteins do form an active complex, and thermodynamic parameters of their interaction were measured. It was also confirmed that tamoxifen directly binds to p25 and inhibits CDK5 kinase activity. Similar observations were obtained using 4-hydroxytamoxifen, an active metabolized form of tamoxifen. Two novel compounds have been identified here that harbor a benzofuran moiety and were shown to target directly p25, and their bindings resulted in decreased CDK5 kinase activity. This encouraging alternative opens the way to the ensuing chemical optimization of this scaffold. It also promises a more specific therapeutic approach that may both tackle the pathological signaling in breast cancer and provide a potential new drug for Alzheimer's disease.

Investigating the in Vitro Thermal Stability and Conformational Flexibility of Estrogen Receptors as Potential Key Factors of Their in Vivo Activity.

Among hormone-inducible transcription factors, estrogen receptors (ERs) play important roles in tissue growth and differentiation, via either direct or indirect binding, in the nucleus, to specific DNA targets called estrogen responsive elements (EREs), or through nongenomic pathways. In humans, two estrogen receptor isoforms (hERs), designated hERα and hERß, have been identified. These two hERs, encoded by genes located on distinct chromosomes, exhibit divergent tissue-specific functions and different subcellular distributions depending on their binding status, free or complexed to their cognate ligands. Because it is hypothesized that such distinct behaviors may arise from various conformational stabilities and flexibilities, the effect of salt concentration and temperature was studied on the free and estrogen-activated hERα and hERß. Our results show that the conformational stability of hERß is weakly modulated by salt concentration as opposed to hERα. In addition, we show that the estrogen-bound hERs exhibit a more constrained structure than the unliganded ones and that their conformational flexibility is more affected by diethylstilbestrol binding than that of estradiol, 4-hydroxytamoxifen, or raloxifen. In line with these results, conformational analysis and computational docking were performed on hERα and hERß, which confer molecular support of a diethylstilbestrol-induced restrained flexibility as compared to other ligands. We found that Trp383 in hERα and Trp335 in hERß can closely interact with the NR-box motif of the H12 helix and act as a gatekeeper of the agonist-bound versus antagonist-bound conformations. Altogether, our study contributes to an improved knowledge of the diverse physicochemical properties of full-length hERs, which will help in our understanding of their distinct cellular roles in various cellular contexts.

Rapid and qualitative fluorescence-based method for the assessment of PHA production in marine bacteria during batch culture.

The expansion of polyhydroxyalkanoates (PHAs) into the biodegradable polymers market is mainly prevented by their production process which is still complicated with a low efficiency, resulting in relatively expensive products. In this study, we developed a method that used the lipophilic fluorescent probe Nile Red (1 mg l(-1) solution in DMSO) directly into the culture broth to stain the PHA inclusions inside bacterial cells followed by detection of the emitted fluorescence by both microscopic and spectrometric techniques. Epifluorescence microscopy provides a rapid tool to distinguish producing from non-producing bacterial species and the relative fluorescence intensity (FI) determined at the maximum of emission spectra in the wavelength region of 560-710 nm (λ(ex): 543 nm), allows a fast assessment of the cultural conditions that may enhance PHA production yield. During two-step cultivation in 500-ml flasks with glucose as the sole carbon source, the method aimed to select bacterial strains efficient for PHA synthesis among a marine collection. Subsequently, the NR assay was used to determine the C0/N0 ratio of the producing media that may improve the polymer yield as well as to follow the time course of fermentation. Characterization by GC-MS and DSC confirmed the production of the P(3-HB) homopolymer.

Control of vitellogenin genes expression by sequences derived from transposable elements in rainbow trout.

In most of oviparous animals, vitellogenins (VTG) are the major egg yolk precursors. They are produced in the liver under the control of estrogens. In rainbow trout (Oncorhynchus mykiss), the vtg genes cluster contains an unusually large number of almost identical gene copies. In order to identify the regulatory elements in their promoters, we used a combination of reporter plasmids containing genomic sequences including putative estrogen response elements (EREs) and we performed transient transfection assays in MCF-7 and yeast cells. We found a functional ERE corresponding to the sequence GGGGCAnnnTAACCT (rtvtgERE), which differs from the consensus ERE (ERE(cs)) by three base pairs. This non-palindromic ERE is located in the env gene of a retrotransposon relic, 180 base pairs upstream of the transcriptional start site. Fluorescence anisotropy experiments confirmed that the purified human estrogen receptor alpha (hERalpha) can specifically bind to rtvtgERE. Furthermore, we observe that the stability of hERalpha-ERE(cs) and hERalpha-rtvtgERE complexes is similar with equilibrium dissociation constants of 3.0nM and 6.2nM respectively, under our experimental conditions. Additionally, this rtvtgERE sequence displays a high E2-responsiveness through ER activation in cellulo. In the rainbow trout, the functional ERE (rtvtgERE) lies within promoter sequences which are mostly composed of sequences derived from transposable elements (TEs), which therefore may have acted as an evolutionary buffer to secure the proper expression of these genes.

Threat of plastic ageing in marine environment. Adsorption/desorption of micropollutants.

Ageing of various plastics in marine environment was monitored after immersion of two synthetic (polyvinylchloride, PVC, and polyethylene terephthalate, PET) and one biodegradable (poly(butylene adipate co-terephtalate), PBAT) plastics for 502days in the bay of Lorient (Brittany, France). Data analysis indicates that aged PVC rapidly releases estrogenic compounds in seawater with a later adsorption of heavy metals; PET undergoes a low weakening of the surface whereas no estrogenic activity is detected; PBAT ages faster in marine environment than PVC. Aged PBAT exhibits heterogeneous surface with some cavities likely containing clay minerals from the chlorite group. Besides, this degraded material occasionally shows a high estrogenic activity. Overall, this study reports, for the first time, that some aged plastics, without being cytotoxic, can release estrogenic compounds in marine environment.

Investigation of the functional properties and subcellular localization of alpha human and rainbow trout estrogen receptors within a unique yeast cellular context.

Estrogens are steroid hormones that play a pivotal role in growth, differentiation and function of reproductive and non-reproductive tissues, mediated through estrogen receptors (ERs). Estrogens are involved in different genomic and non-genomic cell signaling pathways which involve well-defined subcellular ER localizations. Thus, ER activity results from complex interplays between intrinsic binding properties and specific subcellular localization. Since these two factors are deeply intricate, we carried out, in a unique yeast cell context, a comparative study to better understand structure/function/subcellular distribution relationships. This was carried out by comparing two ERs: the human ER α subtype (hERα) and the short form of the α isoform of the rainbow trout ER (rtERαS). Their distinct binding properties to agonist and antagonist ligands and subcellular localizations were characterized in Saccharomyces cerevisiae yeast cells. An unexpected partial agonistic effect of ICI 182-780 was observed for rtERαS. Concomitant to distinct binding properties, distinct subcellular localizations were observed before and after ligand stimulation. Due to the unique cell context, the link between ERs intrinsic binding properties and subcellular localizations is partly unveiled and issues are hypothesized based on the role of cytoplasmic transient complexes which play a role in the ER cytoplasmic/nuclear partition, which in turn is critical for the recruitment of co-regulators in the nucleus.