Isabelle Muller

Optimisation of plant sterols incorporation in human keratinocyte plasma membrane and modulation of membrane fluidity.

The in vitro effects of plant sterols were investigated with regard to their uptake and membrane lipid fluidity in human keratinocytes. Among the different media tested to transport sterols (liposomes, micelles and organic solvents), the best results in terms of incorporation and viability were obtained by the use of the organic solvents dimethylsulfoxide and ethanol. After 48 h incubation exogenous sterol can account for about 30% of the total cell sterol content. The total sterol amount in plasma membranes increased 2-fold after incubation with cholesterol, whereas it was not altered when phytosterols were incorporated. The incorporation of cholesterol, sitosterol and stigmasterol led to an increase in the percent of unsaturated fatty acid C18:1 in the plasma membrane. The effect of this uptake on membrane fluidity was studied by means of fluorescence polarisation using DPH and TMA-DPH as fluorescent probes. Whereas cholesterol and sitosterol had no significant effect on the DPH fluorescence anisotropy (rs), the presence of stigmasterol induced a 12% decrease of rs reflecting an increase in membrane fluidity. We can conclude from this study that in the presence of sitosterol, the mean fluidity of the membrane is regulated whereas stigmasterol triggers a looseness of molecular packing of phospholipids acyl chains, in accordance with previous results obtained on purely lipid model membranes.

The uterine and vascular actions of estetrol delineate a distinctive profile of estrogen receptor α modulation, uncoupling nuclear and membrane activation

Estetrol (E4) is a natural estrogen with a long half‐life produced only by the human fetal liver during pregnancy. The crystal structures of the estrogen receptor α (ERα) ligand‐binding domain bound to 17β‐estradiol (E2) and E4 are very similar, as well as their capacity to activate the two activation functions AF‐1 and AF‐2 and to recruit the coactivator SRC3. In vivo administration of high doses of E4 stimulated uterine gene expression, epithelial proliferation, and prevented atheroma, three recognized nuclear ERα actions. However, E4 failed to promote endothelial NO synthase activation and acceleration of endothelial healing, two processes clearly dependent on membrane‐initiated steroid signaling (MISS). Furthermore, E4 antagonized E2 MISS‐dependent effects in endothelium but also in MCF‐7 breast cancer cell line. This profile of ERα activation by E4, uncoupling nuclear and membrane activation, characterizes E4 as a selective ER modulator which could have medical applications that should now be considered further.

Plant sterols: a neutron diffraction study of sitosterol and stigmasterol in soybean phosphatidylcholine membranes

Neutron scattering experiments have been performed on oriented Soybean phosphatidylcholine (SPC) bilayers, containing sitosterol or stigmasterol, two major sterols of plant plasma membranes. Sitosterol and stigmasterol were either protonated or deuterated on position C25 of the lateral chain. Incorporation of sitosterol leads to an increase of the hydrophobic thickness of SPC bilayers of 1.2 and 2 A when present, at 16 and 30 mol%, respectively. On the other hand, no change was observed when stigmasterol is present in the bilayer at its maximal solubility of 16 mol%. These results are in agreement with the fact that sitosterol is more efficient than stigmasterol to order acyl chains of SPC, as already shown with other biophysical techniques. In order to get more insight into the behavior of the lateral chains of the two sterols, the proton-deuterium contrast method was used in order to locate the (2)H25 atoms of the two sterols. For sitosterol, this atom was found close to the center of the bilayer at +/-(1.6+/-0.2 A), with a width, nu=2.5+/-0.5 A. For stigmasterol, the difference profile could be fitted in two different ways: either two possible locations are found at +/-(2.3+/-0.2 A) and +/-(10+/-0.2 A) with the same width, nu=2.5+/-0.5 A or only one broad distribution at +/-(6.1+/-0.3 A), nu=8.5+/-0.7 A. The results are discussed in terms of difference of dynamics for the lateral chain of the two sterols.

Ability of clionasterol and poriferasterol (24-epimers of sitosterol and stigmasterol) to regulate membrane lipid dynamics

Abstract Stigmasterol and sitosterol, which are the major sterols contained in plasma membranes of plant cells have been shown to possess a different ordering ability: stigmasterol is less efficient than sitosterol for regulating membrane permeability and fluidity. We have evaluated the activity of their C-24 epimers on model membranes systems by 2H-NMR. Clionasterol was found to be as efficient as sitosterol; in contrast, poriferasterol appeared to be less efficient than its epimer, stigmasterol. The modification of the configuration at C-24 has thus an influence on the sterol ordering ability in the case of a coexistence of the Δ12 double bond and the ethyl group, i.e. in the stigmasterol series.

Towards the classification of DYT6 dystonia mutants in the DNA-binding domain of THAP1

The transcription factor THAP1 (THanatos Associated Protein 1) has emerged recently as the cause of DYT6 primary dystonia, a type of rare, familial and mostly early-onset syndrome that leads to involuntary muscle contractions. Many of the mutations described in the DYT6 patients fall within the sequence-specific DNA-binding domain (THAP domain) of THAP1 and are believed to negatively affect DNA binding. Here, we have used an integrated approach combining spectroscopic (NMR, fluorescence, DSF) and calorimetric (ITC) methods to evaluate the effect of missense mutations, within the THAP domain, on the structure, stability and DNA binding. Our study demonstrates that none of the mutations investigated failed to bind DNA and some of them even bind DNA stronger than the wild-type protein. However, some mutations could alter DNA-binding specificity. Furthermore, the most striking effect is the decrease of stability observed for mutations at positions affecting the zinc coordination, the hydrophobic core or the C-terminal AVPTIF motif, with unfolding temperatures ranging from 46°C for the wild-type to below 37°C for two mutations. These findings suggest that reduction in population of folded protein under physiological conditions could also account for the disease.

X-ray structure determination of a chiral synthon, essential for the synthesis of 25-2H-stigmasterol

Synthesis of sterols with varying side chains, including deuterium labeled stigmasterol and sitosterol may be performed via the Wittig-Horner coupling of a 22 aldehyde derived from stigmasterol and a suitable sulfoxide derivative of the side chain. The X-ray structure determination of this synthon have been performed since it is a crucial step in order to know the absolute configuration of the chiral carbon atoms. Crystallographic data were as follows:a=7.437(2),b=10.103(4),c=10.274(3)Å, β=100.32(6)o,=759.4 Å3, space group P21 (No.4),Z=2,Dc=1.239 g cm−3.