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. The cholesterol molecule has achieved evolutionary perfection to fulfill its different functions in membrane organization. Here, we review basic approaches to explore the interaction of cholesterol with proteins, with a particular focus on the high diversity of fluorescent and photoreactive cholesterol prob…

Photo-DHEA--a functional photoreactive dehydroepiandrosterone (DHEA) analog.

Abstract The steroid hormone dehydroepiandrosterone (DHEA) has beneficial effects on vascular function, survival of neurons, and fatty acid metabolism. However, a specific receptor for DHEA has not been identified to date. Here, we describe the synthesis of a photoreactive DHEA derivative (Photo-DHEA). In Photo-DHEA, typical characteristics of DHEA are conserved: (i) a “planar” tetracyclic ring system with a Δ 5 double bond, (ii) a 3β-hydroxyl group, and (iii) a keto group at C17. In cell-based assays, Photo-DHEA showed the same properties as DHEA. We conclude that Photo-DHEA is suitable for radioiodination to yield a tool for the identification of the elusive DHEA receptor.

Specific and Nonspecific Regulation of GPCR Function by Cholesterol

P4–256: PACAP peptides as new α–secretase activators

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. Similar to Triton X-100 and CHAPS, CHAPSTEROL maintain…

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. However, our current understanding on the membrane organization, intracellular distribution and trafficking of cholesterol is rather poor. This is mainly due to inherent difficulties to label and track this small lipid. In this review, we describe different approaches to detect cholesterol in …

DHEA-Bodipy–a functional fluorescent DHEA analog for live cell imaging

International audience; The androgen dehydroepiandrosterone (DHEA) has been reported to protect neuronal cells against dysfunction and apoptosis. Several signaling pathways involved in these effects have been described but little is known about the intracellular trafficking of DHEA. We describe design, synthesis and characterization of DHEA-Bodipy, a novel fluorescent DHEA analog. DHEA-Bodipy proved to be a functional DHEA derivative: DHEA-Bodipy (i) induced estrogen receptor α-mediated gene activation, (ii) protected PC12 rat pheochromocytoma cells against serum deprivation-induced apoptosis, and (iii) induced stress fibers and focal adhesion contacts in SH-SY5Y human neuroblastoma cells. …

Perturbed interactions of mutant proteolipid protein/DM20 with cholesterol and lipid rafts in oligodendroglia: implications for dysmyelination in spastic paraplegia.

Missense mutations in the humanPLP1gene lead to dysmyelinating diseases with a broad range of clinical severity, ranging from severe Pelizaeus–Merzbacher disease (PMD) to milder spastic paraplegia type 2 (SPG-2). The molecular pathology has been generally attributed to endoplasmic reticulum (ER) retention of misfolded proteolipid protein (PLP) (and its splice isoform DM20) and induction of the unfolded protein response. As opposed to previous studies of heterologous expression systems, we have analyzed PLP/DM20 trafficking in oligodendroglial cells, thereby revealing differences between PMD and SPG-2-associated PLP/DM20 isoforms. PLPA242Vand DM20A242V(jimpy-msdin mice), associated with seve…

The neuropeptide PACAP promotes ?‐secretase pathway for processing Alzheimer amyloid precursor protein

SPECIFIC AIMSProteolytic cleavage of the amyloid precursor protein (APP) by α-secretase within the Aβ sequence precludes formation of amyloidogenic peptides and leads to a release of soluble APPsα,...