Cholesterol–Protein Interaction: Methods and Cholesterol Reporter Molecules

Abstract

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. Here, methods are described that are used to explore the binding and/or interaction of proteins to cholesterol. For this purpose, a variety of cholesterol probes bearing radio-, spin-, photoaffinity- or fluorescent labels are currently available. Examples of proven cholesterol binding molecules are polyene compounds, cholesterol-dependent cytolysins, enzymes accepting cholesterol as substrate, and proteins with cholesterol binding motifs. Main topics of this report are the localization of candidate membrane proteins in cholesterol-rich microdomains, the issue of specificity of cholesterol- protein interactions, and applications of the various cholesterol probes for these studies.

Cholesterol through the Looking Glass

“…The alternative view is that sterols induce a change in the physical properties of the membrane bilayer in the environs of the sensing proteins. Earlier studies on the specificity of the Scap conformational change were also consistent with this latter model, in that sterols that could induce the conformational change (5) most altered membrane fluidity (19), and this effect could be mimicked by particular amphiphiles known to influence membrane properties (6). Moreover, studies of the SREBP system in Drosophila cells indicated that phosphatidylethanolamine, rather than sterols, are sensed by Scap in flies, and it was suggested that cholesterol in mammals and phosphatidylethanolamine in flies may both be sensed by Scap through similar perturbations of the ER membrane (20).…”

Cholesterol lowering drug may influence cellular immune response by altering MHC II function

“…The effect of cholesterol may be due to specifi c interaction between MHC II and cholesterol or due to an increase in the membrane rigidity. It has been shown that function of the cholecystokinin receptor is related to the membrane fl uidity ( 41 ), whereas function of the oxytocin receptor is not related to membrane fl uidity but there is a specifi c requirement of cholesterol ( 16 ). We showed that liposomal cholesterol analog (4-cholestene-3-one) treatment increased the membrane rigidity but failed to restore peptide-MHC II complex formation and binding of conformation-specifi c mAbs ( Figs.…”

“…Structure activity analysis shows that cholesterol's effects are due to specifi c sterol-protein interactions, as shown in the case of a number of membrane bound receptors, such as those for cholecystokinin (type B), oxytocin, and nicotinic acetylcholine ( 16 ). Refi ned structure of the nicotinic acetylcholine receptor has been shown to have internal sites capable of forming adducts with cholesterol and resulting in stabilization of the protein structure ( 17 ).…”

Multiple Cholesterol Recognition/Interaction Amino Acid Consensus (CRAC) Motifs in Cytosolic C Tail of Slo1 Subunit Determine Cholesterol Sensitivity of Ca2+- and Voltage-gated K+ (BK) Channels

“…A wide variety of structurally unrelated domains that participate in CLR recognition have been identified in both cytosolic and transmembrane proteins (2,3,5,39,40). However, we initially focused on the CRAC motif because 1) it is defined by a relatively lax sequence (-(L/V)X 1-5 YX 1-5 (R/K)) (4), and 2) a CRAC domain in a cytosolic region participates in CLR sensing by another ionotropic receptor, the mitochondrial translocator protein TSPO (formerly known as peripheral benzodiazepine receptor) (41).…”