Enzymatic Oxidation of Cholesterol: Properties and Functional Effects of Cholestenone in Cell Membranes

Abstract

Bacterial cholesterol oxidase is commonly used as an experimental tool to reduce cellular cholesterol content. That the treatment also generates the poorly degradable metabolite 4-cholesten-3-one (cholestenone) has received less attention. Here, we investigated the membrane partitioning of cholestenone using simulations and cell biological experiments and assessed the functional effects of cholestenone in human cells. Atomistic simulations predicted that cholestenone reduces membrane order, undergoes faster flip-flop and desorbs more readily from membranes than cholesterol. In primary human fibroblasts, cholestenone was released from membranes to physiological extracellular acceptors more avidly than cholesterol, but without acceptors it remained in cells over a day. To address the functional effects of cholestenone, we studied fibroblast migration during wound healing. When cells were either cholesterol oxidase treated or part of cellular cholesterol was exchanged for cholestenone with cyclodextrin, cell migration during 22 h was markedly inhibited. Instead, when a similar fraction of cholesterol was removed using cyclodextrin, cells replenished their cholesterol content in 3 h and migrated similarly to control cells. Thus, cholesterol oxidation produces long-term functional effects in cells and these are in part due to the generated membrane active cholestenone.

The aliphatic chain of cholesterol modulates bilayer interleaflet coupling and domain registration

“…S2b), an important indicator of condensation/ordering of lipids. [8-10] Furthermore, cholesterol preferences, order parameters, and heights of the saturated DPPC and the unsaturated DLiPC lipids are all comparable among the different sterol systems (Fig. 1d-f).…”

“…Cholesterol consists of a 3b-hydroxyl group, a rigid sterol ring, and a flexible aliphatic chain. Cholesterol's ability to enhance ordering of lipid hydrocarbon chains, or its condensing effect [5][6][7], was suggested to arise from the tilting of the sterol ring [8][9][10]. Other studies showed that the flat a-face of cholesterol preferentially interacts with saturated tails of high-melting lipids while the rough b-face has no preference for tail unsaturation [5,6,11,12].…”

“…Systematic experimental examination of cholesterol flip-flop rates can be complicated by the difficulties in accurate quantification of the degree of domain registration, and by the spontaneous oxidation of cholesterol and its analogues[27] that may have different flip-flop rates[10]. Molecular simulations enable direct modulation of cholesterol flip-flop rates via the use of artificial cholesterol models with varying aliphatic chain lengths, which can have different rates of flip-flop due to steric effects.…”

Mechanism of allosteric regulation of β2-adrenergic receptor by cholesterol

“…As compared to cholesterol, CHS is a more water-soluble cholesterol ester and is widely used in structural biology and biophysical studies as a cholesterol analogue (Zocher et al, 2012; Loll, 2014). Oxysterols, on the other hand, are derivatives of cholesterol with additional oxygen-containing substitutions at different positions of cholesterol (Olkkonen and Hynynen, 2009; Kulig et al, 2015a; Neuvonen et al, 2014). Due to the structural similarities with cholesterol, these analogues mimic cholesterol as to the effects on membrane properties (e.g., increasing bilayer order and thickness), although to different extents (Figure 2—figure supplement 6) (Kulig et al, 2015a, 2015b).…”

Using Sterol Substitution to Probe the Role of Membrane Domains in Membrane Functions

“…Neuvonen et al studied effect of cholesterol oxidation upon membrane properties and cellular functions by simulation and cellular experiments [56]. Cholesterol oxidase or MβCD-loaded with steroid was used to increase 4-cholesten-3-one and decrease cholesterol levels in human dermal fibroblasts.…”