Reversible translocation of acyl-CoA:cholesterol acyltransferase (ACAT) between the endoplasmic reticulum and vesicular structures.

The enzyme acyl-CoA:cholesterol acyltransferase (ACAT) is normally localized in the endoplasmic reticulum (ER) where it can esterify cholesterol for storage in lipid droplets and/or the formation of lipoproteins. Here, we report that ACAT can translocate from the ER into vesicular structures in response to different ACAT inhibitors. The translocation was fast (within minutes), reversible and occurred in different cell types.

OXTR-Related Markers in Clinical Depression: a Longitudinal Case-Control Psychotherapy Study.

We investigated stability and change of plasma and urinary oxytocin as well as OXTR DNA methylation patterns through psychotherapy. Furthermore, we explored the potential impact of inpatient psychotherapy on oxytocin-related biomarkers and vice versa by differentiating patients who remitted from depression versus non-remitters. Blood and urine samples were taken from 85 premenopausal women (aged 19-52), 43 clinically depressed patients from a psychosomatic inpatient unit, and 42 healthy control subjects matched for age and education at two points of time.

Sodium functions as a negative allosteric modulator of the oxytocin receptor.

The oxytocin receptor, a class A G protein coupled receptor (GPCR), is essentially involved in the physiology of reproduction. Two parameters are crucially important to support high-affinity agonist binding of the receptor: Mg and cholesterol, both acting as positive modulators. Using displacement assays with a high-affinity fluorescent antagonist (OTAN-A647), we now show that sodium functions as a negative allosteric modulator of the oxytocin receptor.

Synthesis and characterization of a novel rhodamine labeled cholesterol reporter.

We introduce the novel fluorescent cholesterol probe RChol in which a sulforhodamine group is linked to the sixth carbon atom of the steroid backbone of cholesterol. The same position has recently been selected to generate the fluorescent reporter 6-dansyl-cholestanol (DChol) and the photoreactive 6-azi-cholestanol. In comparison with DChol, RChol is brighter, much more photostable, and requires less energy for excitation, i.

Interaction of G protein coupled receptors and cholesterol.

G protein coupled receptors (GPCRs) form the largest receptor superfamily in eukaryotic cells. Owing to their seven transmembrane helices, large parts of these proteins are embedded in the cholesterol-rich plasma membrane bilayer. Thus, GPCRs are always in proximity to cholesterol.

Eimeria bovis infection modulates endothelial host cell cholesterol metabolism for successful replication.

During first merogony Eimeria bovis forms large macromeronts in endothelial host cells containing >120 000 merozoites I. During multiplication, large amounts of cholesterol are indispensable for the enormous offspring membrane production. Cholesterol auxotrophy was proven for other apicomplexan parasites.

Melittin modulates keratinocyte function through P2 receptor-dependent ADAM activation.

Melittin, the major component of the bee venom, is an amphipathic, cationic peptide with a wide spectrum of biological properties that is being considered as an anti-inflammatory and anti-cancer agent. It modulates multiple cellular functions but the underlying mechanisms are not clearly understood. Here, we report that melittin activates disintegrin-like metalloproteases (ADAMs) and that downstream events likely contribute to the biological effects evoked by the peptide.

Specification of the cholesterol interaction with the oxytocin receptor using a chimeric receptor approach.

The oxytocin receptor specifically requires cholesterol to maintain and stabilize its high-affinity agonist binding. Here, we applied a receptor chimeric approach to coarsely localize the cholesterol binding domain of the oxytocin receptor. During these studies, we identified the specific dependence on cholesterol as a common property of the oxytocin-vasopressin receptor family.

Unsaturated fatty acids drive disintegrin and metalloproteinase (ADAM)-dependent cell adhesion, proliferation, and migration by modulating membrane fluidity.

The disintegrin-metalloproteinases ADAM10 and ADAM17 mediate the release of several cell signaling molecules and cell adhesion molecules such as vascular endothelial cadherin or L-selectin affecting endothelial permeability and leukocyte transmigration. Dysregulation of ADAM activity may contribute to the pathogenesis of vascular diseases, but the mechanisms underlying the control of ADAM functions are still incompletely understood. Atherosclerosis is characterized by lipid plaque formation and local accumulation of unsaturated free fatty acids (FFA).

Cholesterol-induced conformational changes in the oxytocin receptor.

Recent studies suggest that cholesterol binding is widespread among GPCRs (G-protein-coupled receptors). In the present study, we analysed putative cholesterol-induced changes in the OTR [OT (oxytocin) receptor], a prototype of cholesterol-interacting GPCRs. For this purpose, we have created recombinant OTRs that are able to bind two small-sized fluorescence-labelled ligands simultaneously.

Probes for studying cholesterol binding and cell biology.

Cholesterol is a multifunctional lipid in eukaryotic cells. It regulates the physical state of the phospholipid bilayer, is crucially involved in the formation of membrane microdomains, affects the activity of many membrane proteins, and is the precursor for steroid hormones and bile acids. Thus, cholesterol plays a profound role in the physiology and pathophysiology of eukaryotic cells.

Depletion of calcium stores contributes to progesterone-induced attenuation of calcium signaling of G protein-coupled receptors.

Progesterone non-genomically attenuates the calcium signaling of the human oxytocin receptor and several other Galpha(q) protein-coupled receptors. High progesterone concentrations are found in the endometrium during pregnancy opposing the responsiveness of the underlying myometrium to labor-inducing hormones. Here, we demonstrate that within minutes, progesterone inhibits oxytocin- and bradykinin-induced contractions of rat uteri, calcium responses induced by platelet-activating factor in the human endometrial cell line MFE-280, and oxytocin-induced calcium signals in PHM1-31 immortalized pregnant human myometrial cells.

Cholesterol-protein interaction: methods and cholesterol reporter molecules.

Cholesterol is a major constituent of the plasma membrane in eukaryotic cells. It regulates the physical state of the phospholipid bilayer and is crucially involved in the formation of membrane microdomains. Cholesterol also affects the activity of several membrane proteins, and is the precursor for steroid hormones and bile acids.

Oxidative stress resistance in hippocampal cells is associated with altered membrane fluidity and enhanced nonamyloidogenic cleavage of endogenous amyloid precursor protein.

Reactive oxygen species (ROS) have important roles as signaling molecules in the regulation of a variety of biological processes. On the other hand, chronic oxidative stress exerted by ROS is widely considered a causative factor in aging. Therefore, cells need to be able to adapt to a chronic oxidative challenge and do so to a certain cell-type-specific extent.

Adaptation of neuronal cells to chronic oxidative stress is associated with altered cholesterol and sphingolipid homeostasis and lysosomal function.

Chronic oxidative stress has been causally linked to several neurodegenerative disorders. As sensitivity for oxidative stress greatly differs between brain regions and neuronal cell types, specific cellular mechanisms of adaptation to chronic oxidative stress should exist. Our objective was to identify molecular mechanisms of adaptation of neuronal cells after applying chronic sublethal oxidative stress.

Orientation and dynamics of a novel fluorescent cholesterol analogue in membranes of varying phase.

Fluorescent analogues of cholesterol offer a powerful approach for monitoring cholesterol behavior in membranes because of their high sensitivity, suitable time resolution, and multiplicity of measurable parameters. In this work, we have monitored the orientation and dynamics of a novel fluorescent cholesterol probe, 6-dansylcholestanol (DChol), in membranes of different phase type utilizing sensitive fluorescence techniques including the red-edge excitation shift (REES) approach. Our results show that fluorescence emission maximum, anisotropy, and lifetime of DChol are dependent on the phase of the membrane.

Oxytocin receptors: ligand binding, signalling and cholesterol dependence.

The G protein coupled oxytocin receptor (OTR) reveals some specific molecular and physiological characteristics. Ligand-receptor interaction has been analysed by photoaffinity labelling, site-directed mutagenesis, the construction of receptor chimeras and molecular modelling. Major results of these studies will be summarized.

Cholesterol interaction with the related steroidogenic acute regulatory lipid-transfer (START) domains of StAR (STARD1) and MLN64 (STARD3).

The steroidogenic acute regulatory (StAR)-related lipid transfer (START) domains are found in a wide range of proteins involved in intracellular trafficking of cholesterol and other lipids. Among the START proteins are the StAR protein itself (STARD1) and the closely related MLN64 protein (STARD3), which both function in cholesterol movement. We compared the cholesterol-binding properties of these two START domain proteins.

Cholesterol reporter molecules.

Cholesterol is a major constituent of the membranes in most eukaryotic cells where it fulfills multiple functions. Cholesterol regulates the physical state of the phospholipid bilayer, affects the activity of several membrane proteins, and is the precursor for steroid hormones and bile acids. Cholesterol plays a crucial role in the formation of membrane microdomains such as "lipid rafts" and caveolae.

Antidepressants and antipsychotic drugs colocalize with 5-HT3 receptors in raft-like domains.

Despite different chemical structure and pharmacodynamic signaling pathways, a variety of antidepressants and antipsychotics inhibit ion fluxes through 5-HT3 receptors in a noncompetitive manner with the exception of the known competitive antagonists mirtazapine and clozapine. To further investigate the mechanisms underlying the noncompetitive inhibition of the serotonin-evoked cation current, we quantified the concentrations of different types of antidepressants and antipsychotics in fractions of sucrose flotation gradients isolated from HEK293 (human embryonic kidney 293) cells stably transfected with the 5-HT3A receptor and of N1E-115 neuroblastoma cells in relation to the localization of the 5-HT3 receptor protein within the cell membrane. Western blots revealed a localization of the 5-HT3 receptor protein exclusively in the low buoyant density (LBD) fractions compatible with a localization within raft-like domains.

Binding domains of the oxytocin receptor for the selective oxytocin receptor antagonist barusiban in comparison to the agonists oxytocin and carbetocin.

We have analyzed binding domains of the oxytocin receptor for barusiban, a highly selective oxytocin receptor antagonist, in comparison to the combined vasopressin V1A/oxytocin receptor antagonist atosiban and the agonists oxytocin and carbetocin. For this purpose, chimeric 'gain-in function' oxytocin/vasopressin V2 receptors were expressed in COS-7 cells. These recombinant receptors have been produced by transfer of domains from the oxytocin receptor into the related vasopressin V2 receptor and have already been successfully employed for the identification of ligand binding domains at the oxytocin receptor (Postina, R.

CHAPSTEROL. A novel cholesterol-based detergent.

Design, synthesis and characterization of CHAPSTEROL, a novel cholesterol-based detergent developed for functional solubilization of cholesterol-dependent membrane proteins are described. To validate CHAPSTEROL, we employed the oxytocin receptor, a G protein-coupled receptor requiring cholesterol for its high-affinity binding state. Using the photoactivatable cholesterol analogue [3H]6,6-azocholestan-3beta-ol[3alphaH], we demonstrate that solubilization by CHAPSTEROL leads to an enrichment of cholesterol-binding proteins whereas the widely used bile acid derivative CHAPSO leads to a significant depletion of cholesterol-binding proteins.

Transport of plasma membrane-derived cholesterol and the function of Niemann-Pick C1 Protein.

To visualize the intracellular transport of plasma membrane-derived cholesterol under physiological and pathophysiological conditions, a novel fluorescent cholesterol analog, 6-dansyl cholestanol (DChol), has been synthesized. We present several lines of evidence that DChol mimics cholesterol. The cholesterol probe could be efficiently incorporated into the plasma membrane via cyclodextrin-donor complexes.

Cholesterol and steroid hormones: modulators of oxytocin receptor function.

The function and physiological regulation of the oxytocin-receptor system is strongly steroid-dependent. This is, unexpectedly, only partially reflected by the promoter sequences in the oxytocin receptor and favors the idea that posttranscriptional mechanisms may also play a significant role for the physiological regulation of the oxytocin-receptor system. Our data indicate that cholesterol acts as an allosteric modulator of the oxytocin receptor and stabilizes both membrane-associated and solubilized OT receptors in a high-affinity state for agonists and antagonists.